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  1. NF-κB activation impairs somatic cell reprogramming in ageing.

    Science.gov (United States)

    Soria-Valles, Clara; Osorio, Fernando G; Gutiérrez-Fernández, Ana; De Los Angeles, Alejandro; Bueno, Clara; Menéndez, Pablo; Martín-Subero, José I; Daley, George Q; Freije, José M P; López-Otín, Carlos

    2015-08-01

    Ageing constitutes a critical impediment to somatic cell reprogramming. We have explored the regulatory mechanisms that constitute age-associated barriers, through derivation of induced pluripotent stem cells (iPSCs) from individuals with premature or physiological ageing. We demonstrate that NF-κB activation blocks the generation of iPSCs in ageing. We also show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs the generation of iPSCs by eliciting the reprogramming repressor DOT1L, which reinforces senescence signals and downregulates pluripotency genes. Genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor-Guillermo progeria syndrome and Hutchinson-Gilford progeria syndrome patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo extends lifespan and ameliorates the accelerated ageing phenotype of progeroid mice, supporting the interest of studying age-associated molecular impairments to identify targets of rejuvenation strategies.

  2. Up-regulation of microRNA-1290 impairs cytokinesis and affects the reprogramming of colon cancer cells.

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    Wu, Jia; Ji, Xiaowei; Zhu, Linlin; Jiang, Qiaoli; Wen, Zhenzhen; Xu, Song; Shao, Wei; Cai, Jianting; Du, Qin; Zhu, Yongliang; Mao, Jianshan

    2013-02-28

    Abnormal cytokinesis increases the possibility of nuclear fusion in tumor cells. However, the role of microRNAs (miRNAs) in abnormal cytokinesis is unclear. Here, we found that miR-1290 was significantly up-regulated in clinical colon cancer tissues. Up-regulation of miR-1290 postponed cytokinesis and led to the formation of multinucleated cells. KIF13B was a target of miR-1290 that was involved in aberrant cytokinesis. Furthermore, enforced expression of miR-1290 activated the Wnt pathway and increased the reprogramming-related transcript factors c-Myc and Nanog. Our results suggest that up-regulation of miR-1290 in colon cancer cells impaired cytokinesis and affected reprogramming. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  3. Optimal ROS Signaling Is Critical for Nuclear Reprogramming

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    Gang Zhou

    2016-05-01

    Full Text Available Efficient nuclear reprogramming of somatic cells to pluripotency requires activation of innate immunity. Because innate immune activation triggers reactive oxygen species (ROS signaling, we sought to determine whether there was a role of ROS signaling in nuclear reprogramming. We examined ROS production during the reprogramming of doxycycline (dox-inducible mouse embryonic fibroblasts (MEFs carrying the Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc [OSKM] into induced pluripotent stem cells (iPSCs. ROS generation was substantially increased with the onset of reprogramming. Depletion of ROS via antioxidants or Nox inhibitors substantially decreased reprogramming efficiency. Similarly, both knockdown and knockout of p22phox—a critical subunit of the Nox (1–4 complex—decreased reprogramming efficiency. However, excessive ROS generation using genetic and pharmacological approaches also impaired reprogramming. Overall, our data indicate that ROS signaling is activated early with nuclear reprogramming, and optimal levels of ROS signaling are essential to induce pluripotency.

  4. Metabolic Reprogramming During Multidrug Resistance in Leukemias

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    Raphael Silveira Vidal

    2018-04-01

    Full Text Available Cancer outcome has improved since introduction of target therapy. However, treatment success is still impaired by the same drug resistance mechanism of classical chemotherapy, known as multidrug resistance (MDR phenotype. This phenotype promotes resistance to drugs with different structures and mechanism of action. Recent reports have shown that resistance acquisition is coupled to metabolic reprogramming. High-gene expression, increase of active transport, and conservation of redox status are one of the few examples that increase energy and substrate demands. It is not clear if the role of this metabolic shift in the MDR phenotype is related to its maintenance or to its induction. Apart from the nature of this relation, the metabolism may represent a new target to avoid or to block the mechanism that has been impairing treatment success. In this mini-review, we discuss the relation between metabolism and MDR resistance focusing on the multiple non-metabolic functions that enzymes of the glycolytic pathway are known to display, with emphasis with the diverse activities of glyceraldehyde-3-phosphate dehydrogenase.

  5. Reprogramming chromatin

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    Ehrensberger, Andreas Hasso; Svejstrup, Jesper Qualmann

    2012-01-01

    attributed to high kinetic barriers that affect all cells equally and can only be overcome by rare stochastic events. The barriers to reprogramming are likely to involve transformations of chromatin state because (i) inhibitors of chromatin-modifying enzymes can enhance the efficiency of reprogramming...... and (ii) knockdown or knock-out of chromatin-modifying enzymes can lower the efficiency of reprogramming. Here, we review the relationship between chromatin state transformations (chromatin reprogramming) and cellular reprogramming, with an emphasis on transcription factors, chromatin remodeling factors...

  6. Deterministic direct reprogramming of somatic cells to pluripotency.

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    Rais, Yoach; Zviran, Asaf; Geula, Shay; Gafni, Ohad; Chomsky, Elad; Viukov, Sergey; Mansour, Abed AlFatah; Caspi, Inbal; Krupalnik, Vladislav; Zerbib, Mirie; Maza, Itay; Mor, Nofar; Baran, Dror; Weinberger, Leehee; Jaitin, Diego A; Lara-Astiaso, David; Blecher-Gonen, Ronnie; Shipony, Zohar; Mukamel, Zohar; Hagai, Tzachi; Gilad, Shlomit; Amann-Zalcenstein, Daniela; Tanay, Amos; Amit, Ido; Novershtern, Noa; Hanna, Jacob H

    2013-10-03

    Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells). Our findings uncover a dichotomous molecular function for the reprogramming factors, serving to reactivate endogenous pluripotency networks while simultaneously directly recruiting the Mbd3/NuRD repressor complex that potently restrains the reactivation of OSKM downstream target genes. Subsequently, the latter interactions, which are largely depleted during early pre-implantation development in vivo, lead to a stochastic and protracted reprogramming trajectory towards pluripotency in vitro. The deterministic reprogramming approach devised here offers a novel platform for the dissection of molecular dynamics leading to establishing pluripotency at unprecedented flexibility and resolution.

  7. Joint Contribution of Left Dorsal Premotor Cortex and Supramarginal Gyrus to Rapid Action Reprogramming

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    Hartwigsen, Gesa; Siebner, Hartwig R

    2015-01-01

    human subjects performed a spatially-precued reaction time task. RESULTS: Relative to sham rTMS, effective online perturbation of left PMd significantly impaired both the response speed and accuracy in trials that were invalidly pre-cued and required the subject to reprogram the prepared action......BACKGROUND: The rapid adaptation of actions to changes in the environment is crucial for survival. We previously demonstrated a joint contribution of left dorsal premotor cortex (PMd) and left supramarginal gyrus (SMG) to action reprogramming. However, we did not probe the contribution of PMd...... to the speed and accuracy of action reprogramming and how the functional relevance of PMd changes in the presence of a dysfunctional SMG. OBJECTIVE: This study further dissociated the unique contribution of left PMd and SMG to action reprogramming. Specifically, we tested whether the critical contribution...

  8. Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

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    Page Grier P

    2009-04-01

    Full Text Available Abstract Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT. Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively. However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research.

  9. Nuclear reprogramming of somatic nucleus hybridized with embryonic stem cells by electrofusion.

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    Tada, Masako; Tada, Takashi

    2006-01-01

    Cell fusion is a powerful tool for understanding the molecular mechanisms of epigenetic reprogramming. In hybrid cells of somatic cells and pluripotential stem cells, including embryonic stem (ES) and embryonic germ cells, somatic nuclei acquire pluripotential competence. ES and embryonic germ cells retain intrinsic trans activity to induce epigenetic reprogramming. For generating hybrid cells, we have used the technique of electrofusion. Electrofusion is a highly effective, reproducible, and biomedically safe in vitro system. For successful cell fusion, two sequential steps of electric pulse stimulation are required for the alignment (pearl chain formation) of two different types of cells between electrodes in response to alternating current stimulation and for the fusion of cytoplasmic membranes by direct current stimulation. Optimal conditions for electrofusion with a pulse generator are introduced for ES and somatic cell fusion. Topics in the field of stem cell research include the successful production of cloned animals via the epigenetic reprogramming of somatic cells and contribution of spontaneous cell fusion to generating intrinsic plasticity of tissue stem cells. Cell fusion technology may make important contributions to the fields of epigenetic reprogramming and regenerative medicine.

  10. Discovery and progress of direct cardiac reprogramming.

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    Kojima, Hidenori; Ieda, Masaki

    2017-06-01

    Cardiac disease remains a major cause of death worldwide. Direct cardiac reprogramming has emerged as a promising approach for cardiac regenerative therapy. After the discovery of MyoD, a master regulator for skeletal muscle, other single cardiac reprogramming factors (master regulators) have been sought. Discovery of cardiac reprogramming factors was inspired by the finding that multiple, but not single, transcription factors were needed to generate induced pluripotent stem cells (iPSCs) from fibroblasts. We first reported a combination of cardiac-specific transcription factors, Gata4, Mef2c, and Tbx5 (GMT), that could convert mouse fibroblasts into cardiomyocyte-like cells, which were designated as induced cardiomyocyte-like cells (iCMs). Following our first report of cardiac reprogramming, many researchers, including ourselves, demonstrated an improvement in cardiac reprogramming efficiency, in vivo direct cardiac reprogramming for heart regeneration, and cardiac reprogramming in human cells. However, cardiac reprogramming in human cells and adult fibroblasts remains inefficient, and further efforts are needed. We believe that future research elucidating epigenetic barriers and molecular mechanisms of direct cardiac reprogramming will improve the reprogramming efficiency, and that this new technology has great potential for clinical applications.

  11. The cellular memory disc of reprogrammed cells.

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    Anjamrooz, Seyed Hadi

    2013-04-01

    The crucial facts underlying the low efficiency of cellular reprogramming are poorly understood. Cellular reprogramming occurs in nuclear transfer, induced pluripotent stem cell (iPSC) formation, cell fusion, and lineage-switching experiments. Despite these advances, there are three fundamental problems to be addressed: (1) the majority of cells cannot be reprogrammed, (2) the efficiency of reprogramming cells is usually low, and (3) the reprogrammed cells developed from a patient's own cells activate immune responses. These shortcomings present major obstacles for using reprogramming approaches in customised cell therapy. In this Perspective, the author synthesises past and present observations in the field of cellular reprogramming to propose a theoretical picture of the cellular memory disc. The current hypothesis is that all cells undergo an endogenous and exogenous holographic memorisation such that parts of the cellular memory dramatically decrease the efficiency of reprogramming cells, act like a barrier against reprogramming in the majority of cells, and activate immune responses. Accordingly, the focus of this review is mainly to describe the cellular memory disc (CMD). Based on the present theory, cellular memory includes three parts: a reprogramming-resistance memory (RRM), a switch-promoting memory (SPM) and a culture-induced memory (CIM). The cellular memory arises genetically, epigenetically and non-genetically and affects cellular behaviours. [corrected].

  12. Development Refractoriness of MLL-Rearranged Human B Cell Acute Leukemias to Reprogramming into Pluripotency

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    Alvaro Muñoz-López

    2016-10-01

    Full Text Available Induced pluripotent stem cells (iPSCs are a powerful tool for disease modeling. They are routinely generated from healthy donors and patients from multiple cell types at different developmental stages. However, reprogramming leukemias is an extremely inefficient process. Few studies generated iPSCs from primary chronic myeloid leukemias, but iPSC generation from acute myeloid or lymphoid leukemias (ALL has not been achieved. We attempted to generate iPSCs from different subtypes of B-ALL to address the developmental impact of leukemic fusion genes. OKSM(L-expressing mono/polycistronic-, retroviral/lentiviral/episomal-, and Sendai virus vector-based reprogramming strategies failed to render iPSCs in vitro and in vivo. Addition of transcriptomic-epigenetic reprogramming “boosters” also failed to generate iPSCs from B cell blasts and B-ALL lines, and when iPSCs emerged they lacked leukemic fusion genes, demonstrating non-leukemic myeloid origin. Conversely, MLL-AF4-overexpressing hematopoietic stem cells/B progenitors were successfully reprogrammed, indicating that B cell origin and leukemic fusion gene were not reprogramming barriers. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of MLL-rearranged B-ALL to reprogramming into pluripotency.

  13. Testicular Metabolic Reprogramming in Neonatal Streptozotocin-Induced Type 2 Diabetic Rats Impairs Glycolytic Flux and Promotes Glycogen Synthesis

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    Rato, L.; Alves, M. G.; Dias, T. R.; Cavaco, J. E.; Oliveira, Pedro F.

    2015-01-01

    Defects in testicular metabolism are directly implicated with male infertility, but most of the mechanisms associated with type 2 diabetes- (T2DM) induced male infertility remain unknown. We aimed to evaluate the effects of T2DM on testicular glucose metabolism by using a neonatal-streptozotocin- (n-STZ) T2DM animal model. Plasma and testicular hormonal levels were evaluated using specific kits. mRNA and protein expression levels were assessed by real-time PCR and Western Blot, respectively. Testicular metabolic profile was assessed by 1H-NMR spectroscopy. T2DM rats showed increased glycemic levels, impaired glucose tolerance and hyperinsulinemia. Both testicular and serum testosterone levels were decreased, whereas those of 17β-estradiol were not altered. Testicular glycolytic flux was not favored in testicles of T2DM rats, since, despite the increased expression of both glucose transporters 1 and 3 and the enzyme phosphofructokinase 1, lactate dehydrogenase activity was severely decreased contributing to lower testicular lactate content. However, T2DM enhanced testicular glycogen accumulation, by modulating the availability of the precursors for its synthesis. T2DM also affected the reproductive sperm parameters. Taken together these results indicate that T2DM is able to reprogram testicular metabolism by enhancing alternative metabolic pathways, particularly glycogen synthesis, and such alterations are associated with impaired sperm parameters. PMID:26064993

  14. Reprogramming cells with synthetic proteins

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    Xiaoxiao Yang

    2015-06-01

    Full Text Available Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors (TFs has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to "read" genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivocounterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongenetically by using drug-like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore, the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies.

  15. Reprogramming cells with synthetic proteins.

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    Yang, Xiaoxiao; Malik, Vikas; Jauch, Ralf

    2015-01-01

    Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors (TFs) has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to "read" genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivocounterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongenetically by using drug-like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore, the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies.

  16. Reprogramming cells with synthetic proteins

    Science.gov (United States)

    Yang, Xiaoxiao; Malik, Vikas; Jauch, Ralf

    2015-01-01

    Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors (TFs) has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to “read” genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivo counterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongenetically by using drug-like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore, the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies. PMID:25652623

  17. Boosters and barriers for direct cardiac reprogramming.

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    Talkhabi, Mahmood; Zonooz, Elmira Rezaei; Baharvand, Hossein

    2017-06-01

    Heart disease is currently the most significant cause of morbidity and mortality worldwide, which accounts for approximately 33% of all deaths. Recently, a promising and alchemy-like strategy has been developed called direct cardiac reprogramming, which directly converts somatic cells such as fibroblasts to cardiac lineage cells such as cardiomyocytes (CMs), termed induced CMs or iCMs. The first in vitro cardiac reprogramming study, mediated by cardiac transcription factors (TFs)-Gata4, Tbx5 and Mef2C-, was not enough efficient to produce an adequate number of fully reprogrammed, functional iCMs. As a result, numerous combinations of cardiac TFs exist for direct cardiac reprogramming of mouse and human fibroblasts. However, the efficiency of direct cardiac reprogramming remains low. Recently, a number of cellular and molecular mechanisms have been identified to increase the efficiency of direct cardiac reprogramming and the quality of iCMs. For example, microgrooved substrate, cardiogenic growth factors [VEGF, FGF, BMP4 and Activin A], and an appropriate stoichiometry of TFs boost the direct cardiac reprogramming. On the other hand, serum, TGFβ signaling, activators of epithelial to mesenchymal transition, and some epigenetic factors (Bmi1 and Ezh2) are barriers for direct cardiac reprogramming. Manipulating these mechanisms by the application of boosters and removing barriers can increase the efficiency of direct cardiac reprogramming and possibly make iCMs reliable for cell-based therapy or other potential applications. In this review, we summarize the latest trends in cardiac TF- or miRNA-based direct cardiac reprogramming and comprehensively discuses all molecular and cellular boosters and barriers affecting direct cardiac reprogramming. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Small Molecules Modulate Chromatin Accessibility to Promote NEUROG2-Mediated Fibroblast-to-Neuron Reprogramming

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    Derek K. Smith

    2016-11-01

    Full Text Available Pro-neural transcription factors and small molecules can induce the reprogramming of fibroblasts into functional neurons; however, the immediate-early molecular events that catalyze this conversion have not been well defined. We previously demonstrated that neurogenin 2 (NEUROG2, forskolin (F, and dorsomorphin (D can reprogram fibroblasts into functional neurons with high efficiency. Here, we used this model to define the genetic and epigenetic events that initiate an acquisition of neuronal identity. We demonstrate that NEUROG2 is a pioneer factor, FD enhances chromatin accessibility and H3K27 acetylation, and synergistic transcription activated by these factors is essential to successful reprogramming. CREB1 promotes neuron survival and acts with NEUROG2 to upregulate SOX4, which co-activates NEUROD1 and NEUROD4. In addition, SOX4 targets SWI/SNF subunits and SOX4 knockdown results in extensive loss of open chromatin and abolishes reprogramming. Applying these insights, adult human glioblastoma cell and skin fibroblast reprogramming can be improved using SOX4 or chromatin-modifying chemicals.

  19. Current Concept and Update of the Macrophage Plasticity Concept: Intracellular Mechanisms of Reprogramming and M3 Macrophage “Switch” Phenotype

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    Malyshev, Igor; Malyshev, Yuri

    2015-01-01

    Macrophages play a key role in immunity. In this review, we consider the traditional notion of macrophage plasticity, data that do not fit into existing concepts, and a hypothesis for existence of a new switch macrophage phenotype. Depending on the microenvironment, macrophages can reprogram their phenotype toward the proinflammatory M1 phenotype or toward the anti-inflammatory M2 phenotype. Macrophage reprogramming involves well-coordinated changes in activities of signalling and posttranslational mechanisms. Macrophage reprogramming is provided by JNK-, PI3K/Akt-, Notch-, JAK/STAT-, TGF-β-, TLR/NF-κB-, and hypoxia-dependent pathways. Posttranscriptional regulation is based on micro-mRNA. We have hypothesized that, in addition to the M1 and M2 phenotypes, an M3 switch phenotype exists. This switch phenotype responds to proinflammatory stimuli with reprogramming towards the anti-inflammatory M2 phenotype or, contrarily, it responds to anti-inflammatory stimuli with reprogramming towards the proinflammatory M1 phenotype. We have found signs of such a switch phenotype in lung diseases. Understanding the mechanisms of macrophage reprogramming will assist in the selection of new therapeutic targets for correction of impaired immunity. PMID:26366410

  20. Elements of successful communication skills of people with visually impaired

    OpenAIRE

    Zlodej, Marsela

    2013-01-01

    The main topic of the thesis are the answers to the questions, what are the factors of successful communication of persons with visual impairment, and what are the beliefs about communicating that affect on successful communication. The systematic review of the literature identifies communication as a concept and the specification of communication of people with visual impairment. Irrational beliefs are defined; what they are and why they arise. Some ways of learning communication of persons ...

  1. Understanding the molecular mechanisms of reprogramming

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    Krause, Marie N. [Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla 92037, CA (United States); University Hospital of Würzburg, Department of Pediatrics, 2 Josef-Schneiderstrasse, 97080 Würzburg (Germany); Sancho-Martinez, Ignacio [Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla 92037, CA (United States); Centre for Stem Cells and Regenerative Medicine, King' s College London, 28th Floor, Tower Wing, Guy' s Hospital, Great Maze Pond, London (United Kingdom); Izpisua Belmonte, Juan Carlos, E-mail: belmonte@salk.edu [Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla 92037, CA (United States)

    2016-05-06

    Despite the profound and rapid advancements in reprogramming technologies since the generation of the first induced pluripotent stem cells (iPSCs) in 2006[1], the molecular basics of the process and its implications are still not fully understood. Recent work has suggested that a subset of TFs, so called “Pioneer TFs”, play an important role during the stochastic phase of iPSC reprogramming [2–6]. Pioneer TFs activities differ from conventional transcription factors in their mechanism of action. They bind directly to condensed chromatin and elicit a series of chromatin remodeling events that lead to opening of the chromatin. Chromatin decondensation by pioneer factors progressively occurs during cell division and in turn exposes specific gene promoters in the DNA to which TFs can now directly bind to promoters that are readily accessible[2, 6]. Here, we will summarize recent advancements on our understanding of the molecular mechanisms underlying reprogramming to iPSC as well as the implications that pioneer Transcription Factor activities might play during different lineage conversion processes. - Highlights: • Pioneer transcription factor activity underlies the initial steps of iPSC generation. • Reprogramming can occur by cis- and/or trans- reprogramming events. • Cis-reprogramming implies remodeling of the chromatin for enabling TF accessibility. • Trans-reprogramming encompasses direct binding of Tfs to their target gene promoters.

  2. Epigenetic reprogramming in the porcine germ line

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    Matzen, Sara Maj Hyldig; Croxall, Nicola; Contreras, David A.

    2011-01-01

    BACKGROUND: Epigenetic reprogramming is critical for genome regulation during germ line development. Genome-wide demethylation in mouse primordial germ cells (PGC) is a unique reprogramming event essential for erasing epigenetic memory and preventing the transmission of epimutations to the next...... an increased proportion of cells in G2. CONCLUSIONS: Our study demonstrates that epigenetic reprogramming occurs in pig migratory and gonadal PGC, and establishes the window of time for the occurrence of these events. Reprogramming of histone H3K9me2 and H3K27me3 detected between E15-E21 precedes the dynamic...... DNA demethylation at imprinted loci and DNA repeats between E22-E42. Our findings demonstrate that major epigenetic reprogramming in the pig germ line follows the overall dynamics shown in mice, suggesting that epigenetic reprogramming of germ cells is conserved in mammals. A better understanding...

  3. Totipotency, Pluripotency and Nuclear Reprogramming

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    Mitalipov, Shoukhrat; Wolf, Don

    Mammalian development commences with the totipotent zygote which is capable of developing into all the specialized cells that make up the adult animal. As development unfolds, cells of the early embryo proliferate and differentiate into the first two lineages, the pluripotent inner cell mass and the trophectoderm. Pluripotent cells can be isolated, adapted and propagated indefinitely in vitro in an undifferentiated state as embryonic stem cells (ESCs). ESCs retain their ability to differentiate into cells representing the three major germ layers: endoderm, mesoderm or ectoderm or any of the 200+ cell types present in the adult body. Since many human diseases result from defects in a single cell type, pluripotent human ESCs represent an unlimited source of any cell or tissue type for replacement therapy thus providing a possible cure for many devastating conditions. Pluripotent cells resembling ESCs can also be derived experimentally by the nuclear reprogramming of somatic cells. Reprogrammed somatic cells may have an even more important role in cell replacement therapies since the patient's own somatic cells can be used for reprogramming thereby eliminating immune based rejection of transplanted cells. In this review, we summarize two major approaches to reprogramming: (1) somatic cell nuclear transfer and (2) direct reprogramming using genetic manipulations.

  4. Asymmetric Reprogramming Capacity of Parental Pronuclei in Mouse Zygotes

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    Wenqiang Liu

    2014-03-01

    Full Text Available It has been demonstrated that reprogramming factors are sequestered in the pronuclei of zygotes after fertilization, because zygotes enucleated at the M phase instead of interphase of the first mitosis can support the development of cloned embryos. However, the contribution of the parental pronucleus derived from either the sperm or the oocyte in reprogramming remains elusive. Here, we demonstrate that the parental pronuclei have asymmetric reprogramming capacities and that the reprogramming factors reside predominantly in the male pronucleus. As a result, only female pronucleus-depleted (FPD mouse zygotes can reprogram somatic cells to a pluripotent state and support the full-term development of cloned embryos; male pronucleus-depleted (MPD zygotes fail to support somatic cell reprogramming. We further demonstrate that fusion of an additional male pronucleus into a zygote greatly enhances reprogramming efficiency. Our data provide a clue to further identify critical reprogramming factors in the male pronucleus.

  5. Re: Epigenetics of Cellular Reprogramming

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    Fehmi Narter

    2016-12-01

    Full Text Available EDITORIAL COMMENT Cells have some specific molecular and physiological properties that act their functional process. However, many cells have an ability of efficient transition from one type to another. This ability is named plasticity. This process occurs due to epigenetic reprogramming that involves changes in transcription and chromatin structure. Some changes during reprogramming that have been identified in recent years as genomic demethylation (both histone and DNA, histone acetylation and loss of heterochromatin during the development of many diseases such as infertility and cancer progression. In this review, the authors focused on the latest work addressing the mechanisms surrounding the epigenetic regulation of various types of reprogramming, including somatic cell nuclear transfer, cell fusion and transcription factor- and microRNA-induced pluripotency. There are many responsible factors such as genes, cytokines, proteins, co-factors (i.e. vitamin C in this local area network. The exact mechanisms by which these changes are achieved and the detailed interplay between the players responsible, however, remain relatively unclear. In the treatment of diseases, such as infertility, urooncology, reconstructive urology, etc., epigenetic changes and cellular reprogramming will be crucial in the near future. Central to achieving that goal is a more thorough understanding of the epigenetic state of fully reprogrammed cells. By the progress of researches on this topic, new treatment modalities will be identified for these diseases.

  6. Rat embryonic fibroblasts improve reprogramming of human keratinocytes into induced pluripotent stem cells.

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    Linta, Leonhard; Stockmann, Marianne; Kleinhans, Karin N; Böckers, Anja; Storch, Alexander; Zaehres, Holm; Lin, Qiong; Barbi, Gotthold; Böckers, Tobias M; Kleger, Alexander; Liebau, Stefan

    2012-04-10

    Patient-specific human induced pluripotent stem (hiPS) cells not only provide a promising tool for cellular disease models in general, but also open up the opportunity to establish cell-type-specific systems for personalized medicine. One of the crucial prerequisites for these strategies, however, is a fast and efficient reprogramming strategy from easy accessible somatic cell populations. Keratinocytes from plucked human hair had been introduced as a superior cell source for reprogramming purposes compared with the widely used skin fibroblasts. The starting cell population is, however, limited and thereby further optimization in terms of time, efficiency, and quality is inevitable. Here we show that rat embryonic fibroblasts (REFs) should replace mouse embryonic fibroblasts as feeder cells in the reprogramming process. REFs enable a significantly more efficient reprogramming procedure as shown by colony number and total amount of SSEA4-positive cells. We successfully produced keratinocyte-derived hiPS (k-hiPS) cells from various donors. The arising k-hiPS cells display the hallmarks of pluripotency such as expression of stem cell markers and differentiation into all 3 germ layers. The increased reprogramming efficiency using REFs as a feeder layer occurred independent of the proliferation rate in the parental keratinocytes and acts, at least in part, in a non-cell autonomous way by secreting factors known to facilitate pluripotency such as Tgfb1, Inhba and Grem1. Hence, we provide an easy to use and highly efficient reprogramming system that could be very useful for a broad application to generate human iPS cells. © Mary Ann Liebert, Inc.

  7. Metabolome Profiling of Partial and Fully Reprogrammed Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Park, Soon-Jung; Lee, Sang A; Prasain, Nutan; Bae, Daekyeong; Kang, Hyunsu; Ha, Taewon; Kim, Jong Soo; Hong, Ki-Sung; Mantel, Charlie; Moon, Sung-Hwan; Broxmeyer, Hal E; Lee, Man Ryul

    2017-05-15

    Acquisition of proper metabolomic fate is required to convert somatic cells toward fully reprogrammed pluripotent stem cells. The majority of induced pluripotent stem cells (iPSCs) are partially reprogrammed and have a transcriptome different from that of the pluripotent stem cells. The metabolomic profile and mitochondrial metabolic functions required to achieve full reprogramming of somatic cells to iPSC status have not yet been elucidated. Clarification of the metabolites underlying reprogramming mechanisms should enable further optimization to enhance the efficiency of obtaining fully reprogrammed iPSCs. In this study, we characterized the metabolites of human fully reprogrammed iPSCs, partially reprogrammed iPSCs, and embryonic stem cells (ESCs). Using capillary electrophoresis time-of-flight mass spectrometry-based metabolomics, we found that 89% of analyzed metabolites were similarly expressed in fully reprogrammed iPSCs and human ESCs (hESCs), whereas partially reprogrammed iPSCs shared only 74% similarly expressed metabolites with hESCs. Metabolomic profiling analysis suggested that converting mitochondrial respiration to glycolytic flux is critical for reprogramming of somatic cells into fully reprogrammed iPSCs. This characterization of metabolic reprogramming in iPSCs may enable the development of new reprogramming parameters for enhancing the generation of fully reprogrammed human iPSCs.

  8. Fluctuating levels of reprogramming factor expression in cultured ...

    African Journals Online (AJOL)

    Although human undifferentiated keratinocytes (HUKs) can be reprogrammed to become induced pluripotent stem cells (iPSCs) with high efficiency and rapid kinetics by transducing reprogramming factors (RFs), the endogenous expression of reprogramming factors in cultured HUKs is not clear at different stages. In this ...

  9. The Epithelial-Mesenchymal Transition Factor SNAIL Paradoxically Enhances Reprogramming

    Directory of Open Access Journals (Sweden)

    Juli J. Unternaehrer

    2014-11-01

    Full Text Available Reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs entails a mesenchymal to epithelial transition (MET. While attempting to dissect the mechanism of MET during reprogramming, we observed that knockdown (KD of the epithelial-to-mesenchymal transition (EMT factor SNAI1 (SNAIL paradoxically reduced, while overexpression enhanced, reprogramming efficiency in human cells and in mouse cells, depending on strain. We observed nuclear localization of SNAI1 at an early stage of fibroblast reprogramming and using mouse fibroblasts expressing a knockin SNAI1-YFP reporter found cells expressing SNAI1 reprogrammed at higher efficiency. We further demonstrated that SNAI1 binds the let-7 promoter, which may play a role in reduced expression of let-7 microRNAs, enforced expression of which, early in the reprogramming process, compromises efficiency. Our data reveal an unexpected role for the EMT factor SNAI1 in reprogramming somatic cells to pluripotency.

  10. Heterozygous loss of TSC2 alters p53 signaling and human stem cell reprogramming.

    Science.gov (United States)

    Armstrong, Laura C; Westlake, Grant; Snow, John P; Cawthon, Bryan; Armour, Eric; Bowman, Aaron B; Ess, Kevin C

    2017-12-01

    Tuberous sclerosis complex (TSC) is a pediatric disorder of dysregulated growth and differentiation caused by loss of function mutations in either the TSC1 or TSC2 genes, which regulate mTOR kinase activity. To study aberrations of early development in TSC, we generated induced pluripotent stem cells using dermal fibroblasts obtained from patients with TSC. During validation, we found that stem cells generated from TSC patients had a very high rate of integration of the reprogramming plasmid containing a shRNA against TP53. We also found that loss of one allele of TSC2 in human fibroblasts is sufficient to increase p53 levels and impair stem cell reprogramming. Increased p53 was also observed in TSC2 heterozygous and homozygous mutant human stem cells, suggesting that the interactions between TSC2 and p53 are consistent across cell types and gene dosage. These results support important contributions of TSC2 heterozygous and homozygous mutant cells to the pathogenesis of TSC and the important role of p53 during reprogramming. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  11. Ataxia-telangiectasia mutated (ATM) deficiency decreases reprogramming efficiency and leads to genomic instability in iPS cells

    Energy Technology Data Exchange (ETDEWEB)

    Kinoshita, Taisuke [Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo 160-8582 (Japan); Nagamatsu, Go, E-mail: gonag@sc.itc.keio.ac.jp [Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo 160-8582 (Japan); Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012 (Japan); Kosaka, Takeo [Department of Urology, School of Medicine, Keio University, Tokyo 160-8582 (Japan); Takubo, Keiyo [Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo 160-8582 (Japan); Hotta, Akitsu [Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012 (Japan); Department of Reprogramming Science, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto (Japan); Ellis, James [Ontario Human iPS Cell Facility, Molecular Genetics, University of Toronto, Developmental and Stem Cell Biology, SickKids, Toronto, Canada MG1L7 (Canada); Suda, Toshio, E-mail: sudato@sc.itc.keio.ac.jp [Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo 160-8582 (Japan)

    2011-04-08

    Highlights: {yields} iPS cells were induced with a fluorescence monitoring system. {yields} ATM-deficient tail-tip fibroblasts exhibited quite a low reprogramming efficiency. {yields} iPS cells obtained from ATM-deficient cells had pluripotent cell characteristics. {yields} ATM-deficient iPS cells had abnormal chromosomes, which were accumulated in culture. -- Abstract: During cell division, one of the major features of somatic cell reprogramming by defined factors, cells are potentially exposed to DNA damage. Inactivation of the tumor suppressor gene p53 raised reprogramming efficiency but resulted in an increased number of abnormal chromosomes in established iPS cells. Ataxia-telangiectasia mutated (ATM), which is critical in the cellular response to DNA double-strand breaks, may also play an important role during reprogramming. To clarify the function of ATM in somatic cell reprogramming, we investigated reprogramming in ATM-deficient (ATM-KO) tail-tip fibroblasts (TTFs). Although reprogramming efficiency was greatly reduced in ATM-KO TTFs, ATM-KO iPS cells were successfully generated and showed the same proliferation activity as WT iPS cells. ATM-KO iPS cells had a gene expression profile similar to ES cells and WT iPS cells, and had the capacity to differentiate into all three germ layers. On the other hand, ATM-KO iPS cells accumulated abnormal genome structures upon continuous passages. Even with the abnormal karyotype, ATM-KO iPS cells retained pluripotent cell characteristics for at least 20 passages. These data indicate that ATM does participate in the reprogramming process, although its role is not essential.

  12. Ataxia-telangiectasia mutated (ATM) deficiency decreases reprogramming efficiency and leads to genomic instability in iPS cells

    International Nuclear Information System (INIS)

    Kinoshita, Taisuke; Nagamatsu, Go; Kosaka, Takeo; Takubo, Keiyo; Hotta, Akitsu; Ellis, James; Suda, Toshio

    2011-01-01

    Highlights: → iPS cells were induced with a fluorescence monitoring system. → ATM-deficient tail-tip fibroblasts exhibited quite a low reprogramming efficiency. → iPS cells obtained from ATM-deficient cells had pluripotent cell characteristics. → ATM-deficient iPS cells had abnormal chromosomes, which were accumulated in culture. -- Abstract: During cell division, one of the major features of somatic cell reprogramming by defined factors, cells are potentially exposed to DNA damage. Inactivation of the tumor suppressor gene p53 raised reprogramming efficiency but resulted in an increased number of abnormal chromosomes in established iPS cells. Ataxia-telangiectasia mutated (ATM), which is critical in the cellular response to DNA double-strand breaks, may also play an important role during reprogramming. To clarify the function of ATM in somatic cell reprogramming, we investigated reprogramming in ATM-deficient (ATM-KO) tail-tip fibroblasts (TTFs). Although reprogramming efficiency was greatly reduced in ATM-KO TTFs, ATM-KO iPS cells were successfully generated and showed the same proliferation activity as WT iPS cells. ATM-KO iPS cells had a gene expression profile similar to ES cells and WT iPS cells, and had the capacity to differentiate into all three germ layers. On the other hand, ATM-KO iPS cells accumulated abnormal genome structures upon continuous passages. Even with the abnormal karyotype, ATM-KO iPS cells retained pluripotent cell characteristics for at least 20 passages. These data indicate that ATM does participate in the reprogramming process, although its role is not essential.

  13. Fluorescent tagged episomals for stoichiometric induced pluripotent stem cell reprogramming.

    Science.gov (United States)

    Schmitt, Christopher E; Morales, Blanca M; Schmitz, Ellen M H; Hawkins, John S; Lizama, Carlos O; Zape, Joan P; Hsiao, Edward C; Zovein, Ann C

    2017-06-05

    Non-integrating episomal vectors have become an important tool for induced pluripotent stem cell reprogramming. The episomal vectors carrying the "Yamanaka reprogramming factors" (Oct4, Klf, Sox2, and L-Myc + Lin28) are critical tools for non-integrating reprogramming of cells to a pluripotent state. However, the reprogramming process remains highly stochastic, and is hampered by an inability to easily identify clones that carry the episomal vectors. We modified the original set of vectors to express spectrally separable fluorescent proteins to allow for enrichment of transfected cells. The vectors were then tested against the standard original vectors for reprogramming efficiency and for the ability to enrich for stoichiometric ratios of factors. The reengineered vectors allow for cell sorting based on reprogramming factor expression. We show that these vectors can assist in tracking episomal expression in individual cells and can select the reprogramming factor dosage. Together, these modified vectors are a useful tool for understanding the reprogramming process and improving induced pluripotent stem cell isolation efficiency.

  14. Marine Corps Budgetary Reprogramming Effectiveness

    Science.gov (United States)

    2015-03-01

    infrastructure (Appropriations Act of Congress, 2008). The environmental restoration is a transfer account controlled by the DOD. Usually in the case of...at an average just over 11 percent and the Marine Corps encircle the backend of the DOD portion of reprogramming with the Marine Corps reprogramming...blue force tracker (BFT), radio systems, high mobility multipurpose wheeled vehicle (HMMWV), medium tactical vehicle replacement (MTVR), and

  15. Concise review: reprogramming strategies for cardiovascular regenerative medicine: from induced pluripotent stem cells to direct reprogramming.

    Science.gov (United States)

    Budniatzky, Inbar; Gepstein, Lior

    2014-04-01

    Myocardial cell-replacement therapies are emerging as novel therapeutic paradigms for myocardial repair but are hampered by the lack of sources of autologous human cardiomyocytes. The recent advances in stem cell biology and in transcription factor-based reprogramming strategies may provide exciting solutions to this problem. In the current review, we describe the different reprogramming strategies that can give rise to cardiomyocytes for regenerative medicine purposes. Initially, we describe induced pluripotent stem cell technology, a method by which adult somatic cells can be reprogrammed to yield pluripotent stem cells that could later be coaxed ex vivo to differentiate into cardiomyocytes. The generated induced pluripotent stem cell-derived cardiomyocytes could then be used for myocardial cell transplantation and tissue engineering strategies. We also describe the more recent direct reprogramming approaches that aim to directly convert the phenotype of one mature cell type (fibroblast) to another (cardiomyocyte) without going through a pluripotent intermediate cell type. The advantages and shortcomings of each strategy for cardiac regeneration are discussed, along with the hurdles that need to be overcome on the road to clinical translation.

  16. Reprogramming Cells for Brain Repair

    Directory of Open Access Journals (Sweden)

    Randall D. McKinnon

    2013-08-01

    Full Text Available At present there are no clinical therapies that can repair traumatic brain injury, spinal cord injury or degenerative brain disease. While redundancy and rewiring of surviving circuits can recover some lost function, the brain and spinal column lack sufficient endogenous stem cells to replace lost neurons or their supporting glia. In contrast, pre-clinical studies have demonstrated that exogenous transplants can have remarkable efficacy for brain repair in animal models. Mesenchymal stromal cells (MSCs can provide paracrine factors that repair damage caused by ischemic injury, and oligodendrocyte progenitor cell (OPC grafts give dramatic functional recovery from spinal cord injury. These studies have progressed to clinical trials, including human embryonic stem cell (hESC-derived OPCs for spinal cord repair. However, ESC-derived allografts are less than optimal, and we need to identify a more appropriate donor graft population. The cell reprogramming field has developed the ability to trans-differentiate somatic cells into distinct cell types, a technology that has the potential to generate autologous neurons and glia which address the histocompatibility concerns of allografts and the tumorigenicity concerns of ESC-derived grafts. Further clarifying how cell reprogramming works may lead to more efficient direct reprogram approaches, and possibly in vivo reprogramming, in order to promote brain and spinal cord repair.

  17. Simultaneous Reprogramming and Gene Correction of Patient Fibroblasts

    Directory of Open Access Journals (Sweden)

    Sara E. Howden

    2015-12-01

    Full Text Available The derivation of genetically modified induced pluripotent stem (iPS cells typically involves multiple steps, requiring lengthy cell culture periods, drug selection, and several clonal events. We report the generation of gene-targeted iPS cell lines following a single electroporation of patient-specific fibroblasts using episomal-based reprogramming vectors and the Cas9/CRISPR system. Simultaneous reprogramming and gene targeting was tested and achieved in two independent fibroblast lines with targeting efficiencies of up to 8% of the total iPS cell population. We have successfully targeted the DNMT3B and OCT4 genes with a fluorescent reporter and corrected the disease-causing mutation in both patient fibroblast lines: one derived from an adult with retinitis pigmentosa, the other from an infant with severe combined immunodeficiency. This procedure allows the generation of gene-targeted iPS cell lines with only a single clonal event in as little as 2 weeks and without the need for drug selection, thereby facilitating “seamless” single base-pair changes.

  18. Heterogeneity of osteosarcoma cell lines led to variable responses in reprogramming.

    Science.gov (United States)

    Choong, Pei Feng; Teh, Hui Xin; Teoh, Hoon Koon; Ong, Han Kiat; Choo, Kong Bung; Sugii, Shigeki; Cheong, Soon Keng; Kamarul, Tunku

    2014-01-01

    Four osteosarcoma cell lines, Saos-2, MG-63, G-292 and U-2 OS, were reprogrammed to pluripotent state using Yamanaka factors retroviral transduction method. Embryonic stem cell (ESC)-like clusters started to appear between 15 to 20 days post transduction. Morphology of the colonies resembled that of ESC colonies with defined border and tightly-packed cells. The reprogrammed sarcomas expressed alkaline phosphatase and pluripotency markers, OCT4, SSEA4, TRA-1-60 and TRA-1-81, as in ESC up to Passage 15. All reprogrammed sarcomas could form embryoid body-like spheres when cultured in suspension in a low attachment dish for up to 10 days. Further testing on the directed differentiation capacity of the reprogrammed sarcomas showed all four reprogrammed sarcoma lines could differentiate into adipocytes while reprogrammed Saos-2-REP, MG-63-REP and G-292-REP could differentiate into osteocytes. Among the 4 osteosarcoma cell lines, U-2 OS reported the highest transduction efficiency but recorded the lowest reprogramming stability under long term culture. Thus, there may be intrinsic differences governing the variable responses of osteosarcoma cell lines towards reprogramming and long term culture effect of the reprogrammed cells. This is a first report to associate intrinsic factors in different osteosarcoma cell lines with variable reprogramming responses and effects on the reprogrammed cells after prolonged culture.

  19. Heterogeneity of Osteosarcoma Cell Lines Led to Variable Responses in Reprogramming

    Science.gov (United States)

    Choong, Pei Feng; Teh, Hui Xin; Teoh, Hoon Koon; Ong, Han Kiat; Choo, Kong Bung; Sugii, Shigeki; Cheong, Soon Keng; Kamarul, Tunku

    2014-01-01

    Four osteosarcoma cell lines, Saos-2, MG-63, G-292 and U-2 OS, were reprogrammed to pluripotent state using Yamanaka factors retroviral transduction method. Embryonic stem cell (ESC)-like clusters started to appear between 15 to 20 days post transduction. Morphology of the colonies resembled that of ESC colonies with defined border and tightly-packed cells. The reprogrammed sarcomas expressed alkaline phosphatase and pluripotency markers, OCT4, SSEA4, TRA-1-60 and TRA-1-81, as in ESC up to Passage 15. All reprogrammed sarcomas could form embryoid body-like spheres when cultured in suspension in a low attachment dish for up to 10 days. Further testing on the directed differentiation capacity of the reprogrammed sarcomas showed all four reprogrammed sarcoma lines could differentiate into adipocytes while reprogrammed Saos-2-REP, MG-63-REP and G-292-REP could differentiate into osteocytes. Among the 4 osteosarcoma cell lines, U-2 OS reported the highest transduction efficiency but recorded the lowest reprogramming stability under long term culture. Thus, there may be intrinsic differences governing the variable responses of osteosarcoma cell lines towards reprogramming and long term culture effect of the reprogrammed cells. This is a first report to associate intrinsic factors in different osteosarcoma cell lines with variable reprogramming responses and effects on the reprogrammed cells after prolonged culture. PMID:25170299

  20. Cellular reprogramming dynamics follow a simple 1D reaction coordinate

    Science.gov (United States)

    Teja Pusuluri, Sai; Lang, Alex H.; Mehta, Pankaj; Castillo, Horacio E.

    2018-01-01

    Cellular reprogramming, the conversion of one cell type to another, induces global changes in gene expression involving thousands of genes, and understanding how cells globally alter their gene expression profile during reprogramming is an ongoing problem. Here we reanalyze time-course data on cellular reprogramming from differentiated cell types to induced pluripotent stem cells (iPSCs) and show that gene expression dynamics during reprogramming follow a simple 1D reaction coordinate. This reaction coordinate is independent of both the time it takes to reach the iPSC state as well as the details of the experimental protocol used. Using Monte-Carlo simulations, we show that such a reaction coordinate emerges from epigenetic landscape models where cellular reprogramming is viewed as a ‘barrier-crossing’ process between cell fates. Overall, our analysis and model suggest that gene expression dynamics during reprogramming follow a canonical trajectory consistent with the idea of an ‘optimal path’ in gene expression space for reprogramming.

  1. Linking incomplete reprogramming to the improved pluripotency of murine embryonal carcinoma cell-derived pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Gang Chang

    Full Text Available Somatic cell nuclear transfer (SCNT has been proved capable of reprogramming various differentiated somatic cells into pluripotent stem cells. Recently, induced pluripotent stem cells (iPS have been successfully derived from mouse and human somatic cells by the over-expression of a combination of transcription factors. However, the molecular mechanisms underlying the reprogramming mediated by either the SCNT or iPS approach are poorly understood. Increasing evidence indicates that many tumor pathways play roles in the derivation of iPS cells. Embryonal carcinoma (EC cells have the characteristics of both stem cells and cancer cells and thus they might be the better candidates for elucidating the details of the reprogramming process. Although previous studies indicate that EC cells cannot be reprogrammed into real pluripotent stem cells, the reasons for this remain unclear. Here, nuclei from mouse EC cells (P19 were transplanted into enucleated oocytes and pluripotent stem cells (P19 NTES cells were subsequently established. Interestingly, P19 NTES cells prolonged the development of tetraploid aggregated embryos compared to EC cells alone. More importantly, we found that the expression recovery of the imprinted H19 gene was dependent on the methylation state in the differential methylation region (DMR. The induction of Nanog expression, however, was independent of the promoter region DNA methylation state in P19 NTES cells. A whole-genome transcriptome analysis further demonstrated that P19 NTES cells were indeed the intermediates between P19 cells and ES cells and many interesting genes were uncovered that may be responsible for the failed reprogramming of P19 cells. To our knowledge, for the first time, we linked incomplete reprogramming to the improved pluripotency of EC cell-derived pluripotent stem cells. The candidate genes we discovered may be useful not only for understanding the mechanisms of reprogramming, but also for deciphering the

  2. Artificial acceleration of mammalian cell reprogramming by bacterial proteins.

    Science.gov (United States)

    Ikeda, Takashi; Uchiyama, Ikuo; Iwasaki, Mio; Sasaki, Tetsuhiko; Nakagawa, Masato; Okita, Keisuke; Masui, Shinji

    2017-10-01

    The molecular mechanisms of cell reprogramming and differentiation involve various signaling factors. Small molecule compounds have been identified to artificially influence these factors through interacting cellular proteins. Although such small molecule compounds are useful to enhance reprogramming and differentiation and to show the mechanisms that underlie these events, the screening usually requires a large number of compounds to identify only a very small number of hits (e.g., one hit among several tens of thousands of compounds). Here, we show a proof of concept that xenospecific gene products can affect the efficiency of cell reprogramming to pluripotency. Thirty genes specific for the bacterium Wolbachia pipientis were forcibly expressed individually along with reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) that can generate induced pluripotent stem cells in mammalian cells, and eight were found to affect the reprogramming efficiency either positively or negatively (hit rate 26.7%). Mechanistic analysis suggested one of these proteins interacted with cytoskeleton to promote reprogramming. Our results raise the possibility that xenospecific gene products provide an alternative way to study the regulatory mechanism of cell identity. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  3. Pluripotent Conversion of Muscle Stem Cells Without Reprogramming Factors or Small Molecules.

    Science.gov (United States)

    Bose, Bipasha; Shenoy P, Sudheer

    2016-02-01

    Muscle derived stem cells (MDSCs) are multipotent stem cells that can differentiate into several lineages including skeletal muscle precursor cells. Here, we show that MDSCs from myostatin null mice (Mstn (-/-) ) can be readily induced into pluripotent stem cells without using reprogramming factors. Microarray studies revealed a strong upregulation of markers like Leukemia Inhibitory factor (LIF) and Leukemia Inhibitory factor receptor (LIFR) in Mstn (-/-) MDSCs as compared to wild type MDSCs (WT-MDSCs). Furthermore when cultured in mouse embryonic stem cell media with LIF for 95 days, Mstn (-/-) MDSCs formed embryonic stem cell (ES) like colonies. We termed such ES like cells as the culture-induced pluripotent stem cells (CiPSC). CiPSCs from Mstn (-/-) MDSCs were phenotypically similar to ESCs, expressed high levels of Oct4, Nanog, Sox2 and SSEA-1, maintained a normal karyotype. Furthermore, CiPSCs formed embryoid bodies and teratomas when injected into immunocompromised mice. In addition, CiPSCs differentiated into somatic cells of all three lineages. We further show that culturing in ES cell media, resulted in hypermethylation and downregulation of BMP2 in Mstn(-/-) MDSCs. Western blot further confirmed a down regulation of BMP2 signaling in Mstn (-/-) MDSCs in supportive of pluripotent reprogramming. Given that down regulation of BMP2 has been shown to induce pluripotency in cells, we propose that lack of myostatin epigenetically reprograms the MDSCs to become pluripotent stem cells. Thus, here we report the successful establishment of ES-like cells from adult stem cells of the non-germline origin under culture-induced conditions without introducing reprogramming genes.

  4. Genome-wide transcriptional reprogramming under drought stress

    KAUST Repository

    Chen, Hao

    2012-01-01

    Soil water deficit is one of the major factors limiting plant productivity. Plants cope with this adverse environmental condition by coordinating the up- or downregulation of an array of stress responsive genes. Reprogramming the expression of these genes leads to rebalanced development and growth that are in concert with the reduced water availability and that ultimately confer enhanced stress tolerance. Currently, several techniques have been employed to monitor genome-wide transcriptional reprogramming under drought stress. The results from these high throughput studies indicate that drought stress-induced transcriptional reprogramming is dynamic, has temporal and spatial specificity, and is coupled with the circadian clock and phytohormone signaling pathways. © 2012 Springer-Verlag Berlin Heidelberg. All rights are reserved.

  5. Advances in reprogramming somatic cells to induced pluripotent stem cells.

    Science.gov (United States)

    Patel, Minal; Yang, Shuying

    2010-09-01

    Traditionally, nuclear reprogramming of cells has been performed by transferring somatic cell nuclei into oocytes, by combining somatic and pluripotent cells together through cell fusion and through genetic integration of factors through somatic cell chromatin. All of these techniques changes gene expression which further leads to a change in cell fate. Here we discuss recent advances in generating induced pluripotent stem cells, different reprogramming methods and clinical applications of iPS cells. Viral vectors have been used to transfer transcription factors (Oct4, Sox2, c-myc, Klf4, and nanog) to induce reprogramming of mouse fibroblasts, neural stem cells, neural progenitor cells, keratinocytes, B lymphocytes and meningeal membrane cells towards pluripotency. Human fibroblasts, neural cells, blood and keratinocytes have also been reprogrammed towards pluripotency. In this review we have discussed the use of viral vectors for reprogramming both animal and human stem cells. Currently, many studies are also involved in finding alternatives to using viral vectors carrying transcription factors for reprogramming cells. These include using plasmid transfection, piggyback transposon system and piggyback transposon system combined with a non viral vector system. Applications of these techniques have been discussed in detail including its advantages and disadvantages. Finally, current clinical applications of induced pluripotent stem cells and its limitations have also been reviewed. Thus, this review is a summary of current research advances in reprogramming cells into induced pluripotent stem cells.

  6. Epigenetic reprogramming of breast cancer cells with oocyte extracts

    Directory of Open Access Journals (Sweden)

    Kumari Rajendra

    2011-01-01

    Full Text Available Abstract Background Breast cancer is a disease characterised by both genetic and epigenetic alterations. Epigenetic silencing of tumour suppressor genes is an early event in breast carcinogenesis and reversion of gene silencing by epigenetic reprogramming can provide clues to the mechanisms responsible for tumour initiation and progression. In this study we apply the reprogramming capacity of oocytes to cancer cells in order to study breast oncogenesis. Results We show that breast cancer cells can be directly reprogrammed by amphibian oocyte extracts. The reprogramming effect, after six hours of treatment, in the absence of DNA replication, includes DNA demethylation and removal of repressive histone marks at the promoters of tumour suppressor genes; also, expression of the silenced genes is re-activated in response to treatment. This activity is specific to oocytes as it is not elicited by extracts from ovulated eggs, and is present at very limited levels in extracts from mouse embryonic stem cells. Epigenetic reprogramming in oocyte extracts results in reduction of cancer cell growth under anchorage independent conditions and a reduction in tumour growth in mouse xenografts. Conclusions This study presents a new method to investigate tumour reversion by epigenetic reprogramming. After testing extracts from different sources, we found that axolotl oocyte extracts possess superior reprogramming ability, which reverses epigenetic silencing of tumour suppressor genes and tumorigenicity of breast cancer cells in a mouse xenograft model. Therefore this system can be extremely valuable for dissecting the mechanisms involved in tumour suppressor gene silencing and identifying molecular activities capable of arresting tumour growth. These applications can ultimately shed light on the contribution of epigenetic alterations in breast cancer and advance the development of epigenetic therapies.

  7. Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors

    Energy Technology Data Exchange (ETDEWEB)

    Yakubov, Eduard [Department of Molecular Cell Biology, Weizmann Institute of Science, 76100 Rehovot (Israel); Rechavi, Gidi [Cancer Research Center, Chaim Sheba Medical Center, Tel-Hashomer and Sackler School of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Rozenblatt, Shmuel [Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Tel-Aviv (Israel); Givol, David, E-mail: david.givol@weizmann.ac.il [Department of Molecular Cell Biology, Weizmann Institute of Science, 76100 Rehovot (Israel)

    2010-03-26

    Reprogramming of differentiated cells into induced pluripotent cells (iPS) was accomplished in 2006 by expressing four, or less, embryonic stem cell (ESC)-specific transcription factors. Due to the possible danger of DNA damage and the potential tumorigenicity associated with such DNA damage, attempts were made to minimize DNA integration by the vectors involved in this process without complete success. Here we present a method of using RNA transfection as a tool for reprogramming human fibroblasts to iPS. We used RNA synthesized in vitro from cDNA of the same reprogramming four transcription factors. After transfection of the RNA, we show intracellular expression and nuclear localization of the respective proteins in at least 70% of the cells. We used five consecutive transfections to support continuous protein expression resulting in the formation of iPS colonies that express alkaline phosphatase and several ESC markers and that can be expanded. This method completely avoids DNA integration and may be developed to replace the use of DNA vectors in the formation of iPS.

  8. DNA replication is an integral part of the mouse oocyte's reprogramming machinery.

    Directory of Open Access Journals (Sweden)

    Bingyuan Wang

    Full Text Available Many of the structural and mechanistic requirements of oocyte-mediated nuclear reprogramming remain elusive. Previous accounts that transcriptional reprogramming of somatic nuclei in mouse zygotes may be complete in 24-36 hours, far more rapidly than in other reprogramming systems, raise the question of whether the mere exposure to the activated mouse ooplasm is sufficient to enact reprogramming in a nucleus. We therefore prevented DNA replication and cytokinesis, which ensue after nuclear transfer, in order to assess their requirement for transcriptional reprogramming of the key pluripotency genes Oct4 (Pou5f1 and Nanog in cloned mouse embryos. Using transcriptome and allele-specific analysis, we observed that hundreds of mRNAs, but not Oct4 and Nanog, became elevated in nucleus-transplanted oocytes without DNA replication. Progression through the first round of DNA replication was essential but not sufficient for transcriptional reprogramming of Oct4 and Nanog, whereas cytokinesis and thereby cell-cell interactions were dispensable for transcriptional reprogramming. Responses similar to clones also were observed in embryos produced by fertilization in vitro. Our results link the occurrence of reprogramming to a previously unappreciated requirement of oocyte-mediated nuclear reprogramming, namely DNA replication. Nuclear transfer alone affords no immediate transition from a somatic to a pluripotent gene expression pattern unless DNA replication is also in place. This study is therefore a resource to appreciate that the quest for always faster reprogramming methods may collide with a limit that is dictated by the cell cycle.

  9. Glis family proteins are differentially implicated in the cellular reprogramming of human somatic cells.

    Science.gov (United States)

    Lee, Seo-Young; Noh, Hye Bin; Kim, Hyeong-Taek; Lee, Kang-In; Hwang, Dong-Youn

    2017-09-29

    The ground-breaking discovery of the reprogramming of somatic cells into pluripotent cells, termed induced pluripotent stem cells (iPSCs), was accomplished by delivering 4 transcription factors, Oct4, Sox2, Klf4, and c-Myc, into fibroblasts. Since then, several efforts have attempted to unveil other factors that are directly implicated in or might enhance reprogramming. Importantly, a number of transcription factors are reported to retain reprogramming activity. A previous study suggested Gli-similar 1 (Glis1) as a factor that enhances the reprogramming of fibroblasts during iPSC generation. However, the implication of other Glis members, including Glis2 and Glis3 (variants 1 and 2), in cellular reprogramming remains unknown. In this study, we investigated the potential involvement of human Glis family proteins, including hGlis1-3, in cellular reprogramming. Our results demonstrate that hGlis1, which is reported to reprogram human fibroblasts, promotes the reprogramming of human adipose-derived stromal cells (hADSCs), indicating that the reprogramming activity of Glis1 is not cell type-specific. Strikingly, hGlis3 promoted the reprogramming of hADSCs as efficiently as hGlis1. On the contrary, hGlis2 showed a strong negative effect on reprogramming. Together, our results reveal clear differences in the cellular reprogramming activity among Glis family members and provide valuable insight into the development of a new reprogramming strategy using Glis family proteins.

  10. Changes in Parthenogenetic Imprinting Patterns during Reprogramming by Cell Fusion.

    Directory of Open Access Journals (Sweden)

    Hyun Sik Jang

    Full Text Available Differentiated somatic cells can be reprogrammed into the pluripotent state by cell-cell fusion. In the pluripotent state, reprogrammed cells may then self-renew and differentiate into all three germ layers. Fusion-induced reprogramming also epigenetically modifies the somatic cell genome through DNA demethylation, X chromosome reactivation, and histone modification. In this study, we investigated whether fusion with embryonic stem cells (ESCs also reprograms genomic imprinting patterns in somatic cells. In particular, we examined imprinting changes in parthenogenetic neural stem cells fused with biparental ESCs, as well as in biparental neural stem cells fused with parthenogenetic ESCs. The resulting hybrid cells expressed the pluripotency markers Oct4 and Nanog. In addition, methylation of several imprinted genes except Peg3 was comparable between hybrid cells and ESCs. This finding indicates that reprogramming by cell fusion does not necessarily reverse the status of all imprinted genes to the state of pluripotent fusion partner.

  11. Local epigenetic reprogramming induced by G-quadruplex ligands

    Science.gov (United States)

    Guilbaud, Guillaume; Murat, Pierre; Recolin, Bénédicte; Campbell, Beth C.; Maiter, Ahmed; Sale, Julian E.; Balasubramanian, Shankar

    2017-11-01

    DNA and histone modifications regulate transcriptional activity and thus represent valuable targets to reprogram the activity of genes. Current epigenetic therapies target the machinery that regulates these modifications, leading to global transcriptional reprogramming with the potential for extensive undesired effects. Epigenetic information can also be modified as a consequence of disrupting processive DNA replication. Here, we demonstrate that impeding replication by small-molecule-mediated stabilization of G-quadruplex nucleic acid secondary structures triggers local epigenetic plasticity. We report the use of the BU-1 locus of chicken DT40 cells to screen for small molecules able to induce G-quadruplex-dependent transcriptional reprogramming. Further characterization of the top hit compound revealed its ability to induce a dose-dependent inactivation of BU-1 expression in two steps: the loss of H3K4me3 and then subsequent DNA cytosine methylation, changes that were heritable across cell divisions even after the compound was removed. Targeting DNA secondary structures thus represents a potentially new approach for locus-specific epigenetic reprogramming.

  12. Overcoming reprogramming resistance of Fanconi anemia cells

    Science.gov (United States)

    Müller, Lars U. W.; Milsom, Michael D.; Harris, Chad E.; Vyas, Rutesh; Brumme, Kristina M.; Parmar, Kalindi; Moreau, Lisa A.; Schambach, Axel; Park, In-Hyun; London, Wendy B.; Strait, Kelly; Schlaeger, Thorsten; DeVine, Alexander L.; Grassman, Elke; D'Andrea, Alan; Daley, George Q.

    2012-01-01

    Fanconi anemia (FA) is a recessive syndrome characterized by progressive fatal BM failure and chromosomal instability. FA cells have inactivating mutations in a signaling pathway that is critical for maintaining genomic integrity and protecting cells from the DNA damage caused by cross-linking agents. Transgenic expression of the implicated genes corrects the phenotype of hematopoietic cells, but previous attempts at gene therapy have failed largely because of inadequate numbers of hematopoietic stem cells available for gene correction. Induced pluripotent stem cells (iPSCs) constitute an alternate source of autologous cells that are amenable to ex vivo expansion, genetic correction, and molecular characterization. In the present study, we demonstrate that reprogramming leads to activation of the FA pathway, increased DNA double-strand breaks, and senescence. We also demonstrate that defects in the FA DNA-repair pathway decrease the reprogramming efficiency of murine and human primary cells. FA pathway complementation reduces senescence and restores the reprogramming efficiency of somatic FA cells to normal levels. Disease-specific iPSCs derived in this fashion maintain a normal karyotype and are capable of hematopoietic differentiation. These data define the role of the FA pathway in reprogramming and provide a strategy for future translational applications of patient-specific FA iPSCs. PMID:22371882

  13. Brief Report: Human Acute Myeloid Leukemia Reprogramming to Pluripotency Is a Rare Event and Selects for Patient Hematopoietic Cells Devoid of Leukemic Mutations.

    Science.gov (United States)

    Lee, Jong-Hee; Salci, Kyle R; Reid, Jennifer C; Orlando, Luca; Tanasijevic, Borko; Shapovalova, Zoya; Bhatia, Mickie

    2017-09-01

    Induced pluripotent stem cell reprogramming has provided critical insights into disease processes by modeling the genetics and related clinical pathophysiology. Human cancer represents highly diverse genetics, as well as inter- and intra-patient heterogeneity, where cellular model systems capable of capturing this disease complexity would be invaluable. Acute myeloid leukemia (AML) represents one of most heterogeneous cancers and has been divided into genetic subtypes correlated with unique risk stratification over the decades. Here, we report our efforts to induce pluripotency from the heterogeneous population of human patients that represents this disease in the clinic. Using robust optimized reprogramming methods, we demonstrate that reprogramming of AML cells harboring leukemic genomic aberrations is a rare event with the exception of those with de novo mixed-lineage leukemia (MLL) mutations that can be reprogrammed and model drug responses in vitro. Our findings indicate that unlike hematopoietic cells devoid of genomic aberrations, AML cells harboring driver mutations are refractory to reprogramming. Expression of MLL fusion proteins in AML cells did not contribute to induced reprogramming success, which continued to select for patient derived cells devoid of AML patient-specific aberrations. Our study reveals that unanticipated blockades to achieving pluripotency reside within the majority of transformed AML patient cells. Stem Cells 2017;35:2095-2102. © 2017 AlphaMed Press.

  14. Evaluating the potential of poly(beta-amino ester) nanoparticles for reprogramming human fibroblasts to become induced pluripotent stem cells.

    Science.gov (United States)

    Bhise, Nupura S; Wahlin, Karl J; Zack, Donald J; Green, Jordan J

    2013-01-01

    study shows that certain nonviral reprogramming methods may not necessarily be safer than viral approaches and that maximizing exogenous gene expression of reprogramming factors is not sufficient to ensure successful reprogramming.

  15. Excessive Cellular Proliferation Negatively Impacts Reprogramming Efficiency of Human Fibroblasts.

    Science.gov (United States)

    Gupta, Manoj K; Teo, Adrian Kee Keong; Rao, Tata Nageswara; Bhatt, Shweta; Kleinridders, Andre; Shirakawa, Jun; Takatani, Tomozumi; Hu, Jiang; De Jesus, Dario F; Windmueller, Rebecca; Wagers, Amy J; Kulkarni, Rohit N

    2015-10-01

    The impact of somatic cell proliferation rate on induction of pluripotent stem cells remains controversial. Herein, we report that rapid proliferation of human somatic fibroblasts is detrimental to reprogramming efficiency when reprogrammed using a lentiviral vector expressing OCT4, SOX2, KLF4, and cMYC in insulin-rich defined medium. Human fibroblasts grown in this medium showed higher proliferation, enhanced expression of insulin signaling and cell cycle genes, and a switch from glycolytic to oxidative phosphorylation metabolism, but they displayed poor reprogramming efficiency compared with cells grown in normal medium. Thus, in contrast to previous studies, our work reveals an inverse correlation between the proliferation rate of somatic cells and reprogramming efficiency, and also suggests that upregulation of proteins in the growth factor signaling pathway limits the ability to induce pluripotency in human somatic fibroblasts. The efficiency with which human cells can be reprogrammed is of interest to stem cell biology. In this study, human fibroblasts cultured in media containing different concentrations of growth factors such as insulin and insulin-like growth factor-1 exhibited variable abilities to proliferate, with consequences on pluripotency. This occurred in part because of changes in the expression of proteins involved in the growth factor signaling pathway, glycolysis, and oxidative phosphorylation. These findings have implications for efficient reprogramming of human cells. ©AlphaMed Press.

  16. A Blueprint for a Synthetic Genetic Feedback Controller to Reprogram Cell Fate.

    Science.gov (United States)

    Del Vecchio, Domitilla; Abdallah, Hussein; Qian, Yili; Collins, James J

    2017-01-25

    To artificially reprogram cell fate, experimentalists manipulate the gene regulatory networks (GRNs) that maintain a cell's phenotype. In practice, reprogramming is often performed by constant overexpression of specific transcription factors (TFs). This process can be unreliable and inefficient. Here, we address this problem by introducing a new approach to reprogramming based on mathematical analysis. We demonstrate that reprogramming GRNs using constant overexpression may not succeed in general. Instead, we propose an alternative reprogramming strategy: a synthetic genetic feedback controller that dynamically steers the concentration of a GRN's key TFs to any desired value. The controller works by adjusting TF expression based on the discrepancy between desired and actual TF concentrations. Theory predicts that this reprogramming strategy is guaranteed to succeed, and its performance is independent of the GRN's structure and parameters, provided that feedback gain is sufficiently high. As a case study, we apply the controller to a model of induced pluripotency in stem cells. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  17. Optical reprogramming with ultrashort femtosecond laser pulses

    Science.gov (United States)

    Uchugonova, Aisada; Breunig, Hans G.; Batista, Ana; König, Karsten

    2015-03-01

    The use of sub-15 femtosecond laser pulses in stem cell research is explored with particular emphasis on the optical reprogramming of somatic cells. The reprogramming of somatic cells into induced pluripotent stem (iPS) cells can be evoked through the ectopic expression of defined transcription factors. Conventional approaches utilize retro/lenti-viruses to deliver genes/transcription factors as well as to facilitate the integration of transcription factors into that of the host genome. However, the use of viruses may result in insertional mutations caused by the random integration of genes and as a result, this may limit the use within clinical applications due to the risk of the formation of cancer. In this study, a new approach is demonstrated in realizing non-viral reprogramming through the use of ultrashort laser pulses, to introduce transcription factors into the cell so as to generate iPS cells.

  18. A comparison of non-integrating reprogramming methods

    Science.gov (United States)

    Schlaeger, Thorsten M; Daheron, Laurence; Brickler, Thomas R; Entwisle, Samuel; Chan, Karrie; Cianci, Amelia; DeVine, Alexander; Ettenger, Andrew; Fitzgerald, Kelly; Godfrey, Michelle; Gupta, Dipti; McPherson, Jade; Malwadkar, Prerana; Gupta, Manav; Bell, Blair; Doi, Akiko; Jung, Namyoung; Li, Xin; Lynes, Maureen S; Brookes, Emily; Cherry, Anne B C; Demirbas, Didem; Tsankov, Alexander M; Zon, Leonard I; Rubin, Lee L; Feinberg, Andrew P; Meissner, Alexander; Cowan, Chad A; Daley, George Q

    2015-01-01

    Human induced pluripotent stem cells (hiPSCs1–3) are useful in disease modeling and drug discovery, and they promise to provide a new generation of cell-based therapeutics. To date there has been no systematic evaluation of the most widely used techniques for generating integration-free hiPSCs. Here we compare Sendai-viral (SeV)4, episomal (Epi)5 and mRNA transfection mRNA6 methods using a number of criteria. All methods generated high-quality hiPSCs, but significant differences existed in aneuploidy rates, reprogramming efficiency, reliability and workload. We discuss the advantages and shortcomings of each approach, and present and review the results of a survey of a large number of human reprogramming laboratories on their independent experiences and preferences. Our analysis provides a valuable resource to inform the use of specific reprogramming methods for different laboratories and different applications, including clinical translation. PMID:25437882

  19. Specific Cell (Re-)Programming: Approaches and Perspectives.

    Science.gov (United States)

    Hausburg, Frauke; Jung, Julia Jeannine; David, Robert

    2018-01-01

    Many disorders are manifested by dysfunction of key cell types or their disturbed integration in complex organs. Thereby, adult organ systems often bear restricted self-renewal potential and are incapable of achieving functional regeneration. This underlies the need for novel strategies in the field of cell (re-)programming-based regenerative medicine as well as for drug development in vitro. The regenerative field has been hampered by restricted availability of adult stem cells and the potentially hazardous features of pluripotent embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Moreover, ethical concerns and legal restrictions regarding the generation and use of ESCs still exist. The establishment of direct reprogramming protocols for various therapeutically valuable somatic cell types has overcome some of these limitations. Meanwhile, new perspectives for safe and efficient generation of different specified somatic cell types have emerged from numerous approaches relying on exogenous expression of lineage-specific transcription factors, coding and noncoding RNAs, and chemical compounds.It should be of highest priority to develop protocols for the production of mature and physiologically functional cells with properties ideally matching those of their endogenous counterparts. Their availability can bring together basic research, drug screening, safety testing, and ultimately clinical trials. Here, we highlight the remarkable successes in cellular (re-)programming, which have greatly advanced the field of regenerative medicine in recent years. In particular, we review recent progress on the generation of cardiomyocyte subtypes, with a focus on cardiac pacemaker cells. Graphical Abstract.

  20. Algorithm for cellular reprogramming.

    Science.gov (United States)

    Ronquist, Scott; Patterson, Geoff; Muir, Lindsey A; Lindsly, Stephen; Chen, Haiming; Brown, Markus; Wicha, Max S; Bloch, Anthony; Brockett, Roger; Rajapakse, Indika

    2017-11-07

    The day we understand the time evolution of subcellular events at a level of detail comparable to physical systems governed by Newton's laws of motion seems far away. Even so, quantitative approaches to cellular dynamics add to our understanding of cell biology. With data-guided frameworks we can develop better predictions about, and methods for, control over specific biological processes and system-wide cell behavior. Here we describe an approach for optimizing the use of transcription factors (TFs) in cellular reprogramming, based on a device commonly used in optimal control. We construct an approximate model for the natural evolution of a cell-cycle-synchronized population of human fibroblasts, based on data obtained by sampling the expression of 22,083 genes at several time points during the cell cycle. To arrive at a model of moderate complexity, we cluster gene expression based on division of the genome into topologically associating domains (TADs) and then model the dynamics of TAD expression levels. Based on this dynamical model and additional data, such as known TF binding sites and activity, we develop a methodology for identifying the top TF candidates for a specific cellular reprogramming task. Our data-guided methodology identifies a number of TFs previously validated for reprogramming and/or natural differentiation and predicts some potentially useful combinations of TFs. Our findings highlight the immense potential of dynamical models, mathematics, and data-guided methodologies for improving strategies for control over biological processes. Copyright © 2017 the Author(s). Published by PNAS.

  1. Developmental Programming of Adult Disease: Reprogramming by Melatonin?

    Science.gov (United States)

    Tain, You-Lin; Huang, Li-Tung; Hsu, Chien-Ning

    2017-02-16

    Adult-onset chronic non-communicable diseases (NCDs) can originate from early life through so-called the "developmental origins of health and disease" (DOHaD) or "developmental programming". The DOHaD concept offers the "reprogramming" strategy to shift the treatment from adulthood to early life, before clinical disease is apparent. Melatonin, an endogenous indoleamine produced by the pineal gland, has pleiotropic bioactivities those are beneficial in a variety of human diseases. Emerging evidence support that melatonin is closely inter-related to other proposed mechanisms contributing to the developmental programming of a variety of chronic NCDs. Recent animal studies have begun to unravel the multifunctional roles of melatonin in many experimental models of developmental programming. Even though some progress has been made in research on melatonin as a reprogramming strategy to prevent DOHaD-related NCDs, future human studies should aim at filling the translational gap between animal models and clinical trials. Here, we review several key themes on the reprogramming effects of melatonin in DOHaD research. We have particularly focused on the following areas: mechanisms of developmental programming; the interrelationship between melatonin and mechanisms underlying developmental programming; pathophysiological roles of melatonin in pregnancy and fetal development; and insight provided by animal models to support melatonin as a reprogramming therapy. Rates of NCDs are increasing faster than anticipated all over the world. Hence, there is an urgent need to understand reprogramming mechanisms of melatonin and to translate experimental research into clinical practice for halting a growing list of DOHaD-related NCDs.

  2. Reprogramming cancer cells: a novel approach for cancer therapy or a tool for disease-modeling?

    Science.gov (United States)

    Yilmazer, Açelya; de Lázaro, Irene; Taheri, Hadiseh

    2015-12-01

    Chromatin dynamics have been the major focus of many physiological and pathological processes over the past 20 years. Epigenetic mechanisms have been shown to be reshaped during both cellular reprogramming and tumorigenesis. For this reason, cancer cell reprogramming can provide a powerful tool to better understand both regenerative and cancer-fate processes, with a potential to develop novel therapeutic approaches. Recent studies showed that cancer cells can be reprogrammed to a pluripotent state by the overexpression of reprogramming transcription factors. Activation of transcription factors and modification of chromatin regulators may result in the remodeling of epigenetic status and refueling of tumorigenicity in these reprogrammed cancer cells. However, studies focusing on cancer cell reprogramming are contradictory; some studies reported increased tumor progression whereas others showed that cellular reprogramming has a treatment potential for cancer. In this review, first, the current knowledge on the epigenetic mechanisms involved during cancer development and cellular reprogramming will be presented. Later, different reports and key factors about pluripotency-based reprogramming of cancer cells will be reviewed in detail. New insights will be provided on cancer biology and therapy in the light of cellular reprogramming. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  3. Susceptibility of Human Oral Squamous Cell Carcinoma (OSCC H103 and H376 cell lines to Retroviral OSKM mediated reprogramming

    Directory of Open Access Journals (Sweden)

    Nalini Devi Verusingam

    2017-04-01

    Full Text Available Although numbers of cancer cell lines have been shown to be successfully reprogrammed into induced pluripotent stem cells (iPSCs, reprogramming Oral Squamous Cell Carcinoma (OSCC to pluripotency in relation to its cancer cell type and the expression pattern of pluripotent genes under later passage remain unexplored. In our study, we reprogrammed and characterised H103 and H376 oral squamous carcinoma cells using retroviral OSKM mediated method. Reprogrammed cells were characterized for their embryonic stem cells (ESCs like morphology, pluripotent gene expression via quantitative real-time polymerase chain reaction (RT-qPCR, immunofluorescence staining, embryoid bodies (EB formation and directed differentiation capacity. Reprogrammed H103 (Rep-H103 exhibited similar ESCs morphologies with flatten cells and clear borders on feeder layer. Reprogrammed H376 (Rep-H376 did not show ESCs morphologies but grow with a disorganized morphology. Critical pluripotency genes Oct4, Sox2 and Nanog were expressed higher in Rep-H103 against the parental counterpart from passage 5 to passage 10. As for Rep-H376, Nanog expression against its parental counterpart showed a significant decrease at passage 5 and although increased in passage 10, the level of expression was similar to the parental cells. Rep-H103 exhibited pluripotent signals (Oct4, Sox2, Nanog and Tra-1-60 and could form EB with the presence of three germ layers markers. Rep-H103 displayed differentiation capacity into adipocytes and osteocytes. The OSCC cell line H103 which was able to be reprogrammed into an iPSC like state showed high expression of Oct4, Sox2 and Nanog at late passage and may provide a potential iPSC model to study multi-stage oncogenesis in OSCC.

  4. RNAi Reveals Phase-Specific Global Regulators of Human Somatic Cell Reprogramming

    Directory of Open Access Journals (Sweden)

    Cheng-Xu Delon Toh

    2016-06-01

    Full Text Available Incomplete knowledge of the mechanisms at work continues to hamper efforts to maximize reprogramming efficiency. Here, we present a systematic genome-wide RNAi screen to determine the global regulators during the early stages of human reprogramming. Our screen identifies functional repressors and effectors that act to impede or promote the reprogramming process. Repressors and effectors form close interacting networks in pathways, including RNA processing, G protein signaling, protein ubiquitination, and chromatin modification. Combinatorial knockdown of five repressors (SMAD3, ZMYM2, SFRS11, SAE1, and ESET synergistically resulted in ∼85% TRA-1-60-positive cells. Removal of the novel splicing factor SFRS11 during reprogramming is accompanied by rapid acquisition of pluripotency-specific spliced forms. Mechanistically, SFRS11 regulates exon skipping and mutually exclusive splicing of transcripts in genes involved in cell differentiation, mRNA splicing, and chromatin modification. Our study provides insights into the reprogramming process, which comprises comprehensive and multi-layered transcriptional, splicing, and epigenetic machineries.

  5. Epigenetic reprogramming in mammalian species after SCNT-based cloning.

    Science.gov (United States)

    Niemann, Heiner

    2016-07-01

    The birth of "Dolly," the first mammal cloned from an adult mammary epithelial cell, abolished the decades-old scientific dogma implying that a terminally differentiated cell cannot be reprogrammed into a pluripotent embryonic state. The most dramatic epigenetic reprogramming occurs in SCNT when the expression profile of a differentiated cell is abolished and a new embryo-specific expression profile, involving 10,000 to 12,000 genes, and thus, most genes of the entire genome is established, which drives embryonic and fetal development. The initial release from somatic cell epigenetic constraints is followed by establishment of post-zygotic expression patterns, X-chromosome inactivation, and adjustment of telomere length. Somatic cell nuclear transfer may be associated with a variety of pathologic changes of the fetal and placental phenotype in a proportion of cloned offspring, specifically in ruminants, that are thought to be caused by aberrant epigenetic reprogramming. Improvements in our understanding of this dramatic epigenetic reprogramming event will be instrumental in realizing the great potential of SCNT for basic research and for important agricultural and biomedical applications. Here, current knowledge on epigenetic reprogramming after use of SCNT in livestock is reviewed, with emphasis on gene-specific and global DNA methylation, imprinting, X-chromosome inactivation, and telomere length restoration in early development. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. In vivo myomaker-mediated heterologous fusion and nuclear reprogramming.

    Science.gov (United States)

    Mitani, Yasuyuki; Vagnozzi, Ronald J; Millay, Douglas P

    2017-01-01

    Knowledge regarding cellular fusion and nuclear reprogramming may aid in cell therapy strategies for skeletal muscle diseases. An issue with cell therapy approaches to restore dystrophin expression in muscular dystrophy is obtaining a sufficient quantity of cells that normally fuse with muscle. Here we conferred fusogenic activity without transdifferentiation to multiple non-muscle cell types and tested dystrophin restoration in mouse models of muscular dystrophy. We previously demonstrated that myomaker, a skeletal muscle-specific transmembrane protein necessary for myoblast fusion, is sufficient to fuse 10T 1/2 fibroblasts to myoblasts in vitro. Whether myomaker-mediated heterologous fusion is functional in vivo and whether the newly introduced nonmuscle nuclei undergoes nuclear reprogramming has not been investigated. We showed that mesenchymal stromal cells, cortical bone stem cells, and tail-tip fibroblasts fuse to skeletal muscle when they express myomaker. These cells restored dystrophin expression in a fraction of dystrophin-deficient myotubes after fusion in vitro. However, dystrophin restoration was not detected in vivo although nuclear reprogramming of the muscle-specific myosin light chain promoter did occur. Despite the lack of detectable dystrophin reprogramming by immunostaining, this study indicated that myomaker could be used in nonmuscle cells to induce fusion with muscle in vivo, thereby providing a platform to deliver therapeutic material.-Mitani, Y., Vagnozzi, R. J., Millay, D. P. In vivo myomaker-mediated heterologous fusion and nuclear reprogramming. © FASEB.

  7. Cell reprogramming modelled as transitions in a hierarchy of cell cycles

    International Nuclear Information System (INIS)

    Hannam, Ryan; Annibale, Alessia; Kühn, Reimer

    2017-01-01

    We construct a model of cell reprogramming (the conversion of fully differentiated cells to a state of pluripotency, known as induced pluripotent stem cells, or iPSCs) which builds on key elements of cell biology viz. cell cycles and cell lineages. Although reprogramming has been demonstrated experimentally, much of the underlying processes governing cell fate decisions remain unknown. This work aims to bridge this gap by modelling cell types as a set of hierarchically related dynamical attractors representing cell cycles. Stages of the cell cycle are characterised by the configuration of gene expression levels, and reprogramming corresponds to triggering transitions between such configurations. Two mechanisms were found for reprogramming in a two level hierarchy: cycle specific perturbations and a noise induced switching. The former corresponds to a directed perturbation that induces a transition into a cycle-state of a different cell type in the potency hierarchy (mainly a stem cell) whilst the latter is a priori undirected and could be induced, e.g. by a (stochastic) change in the cellular environment. These reprogramming protocols were found to be effective in large regimes of the parameter space and make specific predictions concerning reprogramming dynamics which are broadly in line with experimental findings. (paper)

  8. Ionizing Radiation Potentiates High Fat Diet-Induced Insulin Resistance and Reprograms Skeletal Muscle and Adipose Progenitor Cells

    DEFF Research Database (Denmark)

    Nylander, Vibe; Ingerslev, Lars R; Andersen, Emil

    2016-01-01

    Exposure to ionizing radiation increases the risk of chronic metabolic disorders such as insulin resistance and type 2 diabetes later in life. We hypothesized that irradiation reprograms the epigenome of metabolic progenitor cells, which could account for impaired metabolism after cancer treatment...... mice. Mice subjected to total body irradiation showed alterations in glucose metabolism and, when challenged with HFD, marked hyperinsulinemia. Insulin signaling was chronically disrupted in skeletal muscle and adipose progenitor cells collected from irradiated mice and differentiated in culture...

  9. Cellular Reprogramming Employing Recombinant Sox2 Protein

    Directory of Open Access Journals (Sweden)

    Marc Thier

    2012-01-01

    Full Text Available Induced pluripotent stem (iPS cells represent an attractive option for the derivation of patient-specific pluripotent cells for cell replacement therapies as well as disease modeling. To become clinically meaningful, safe iPS cells need to be generated exhibiting no permanent genetic modifications that are caused by viral integrations of the reprogramming transgenes. Recently, various experimental strategies have been applied to accomplish transgene-free derivation of iPS cells, including the use of nonintegrating viruses, episomal expression, or excision of transgenes after reprogramming by site-specific recombinases or transposases. A straightforward approach to induce reprogramming factors is the direct delivery of either synthetic mRNA or biologically active proteins. We previously reported the generation of cell-permeant versions of Oct4 (Oct4-TAT and Sox2 (Sox2-TAT proteins and showed that Oct4-TAT is reprogramming-competent, that is, it can substitute for Oct4-encoding virus. Here, we explore conditions for enhanced Sox2-TAT protein stabilization and functional delivery into somatic cells. We show that cell-permeant Sox2 protein can be stabilized by lipid-rich albumin supplements in serum replacement or low-serum-supplemented media. Employing optimized conditions for protein delivery, we demonstrate that Sox2-TAT protein is able to substitute for viral Sox2. Sox2-piPS cells express pluripotency-associated markers and differentiate into all three germ layers.

  10. Plasticity of adult human pancreatic duct cells by neurogenin3-mediated reprogramming.

    Directory of Open Access Journals (Sweden)

    Nathalie Swales

    Full Text Available AIMS/HYPOTHESIS: Duct cells isolated from adult human pancreas can be reprogrammed to express islet beta cell genes by adenoviral transduction of the developmental transcription factor neurogenin3 (Ngn3. In this study we aimed to fully characterize the extent of this reprogramming and intended to improve it. METHODS: The extent of the Ngn3-mediated duct-to-endocrine cell reprogramming was measured employing genome wide mRNA profiling. By modulation of the Delta-Notch signaling or addition of pancreatic endocrine transcription factors Myt1, MafA and Pdx1 we intended to improve the reprogramming. RESULTS: Ngn3 stimulates duct cells to express a focused set of genes that are characteristic for islet endocrine cells and/or neural tissues. This neuro-endocrine shift however, is incomplete with less than 10% of full duct-to-endocrine reprogramming achieved. Transduction of exogenous Ngn3 activates endogenous Ngn3 suggesting auto-activation of this gene. Furthermore, pancreatic endocrine reprogramming of human duct cells can be moderately enhanced by inhibition of Delta-Notch signaling as well as by co-expressing the transcription factor Myt1, but not MafA and Pdx1. CONCLUSIONS/INTERPRETATION: The results provide further insight into the plasticity of adult human duct cells and suggest measurable routes to enhance Ngn3-mediated in vitro reprogramming protocols for regenerative beta cell therapy in diabetes.

  11. S-phase Synchronization Facilitates the Early Progression of Induced-Cardiomyocyte Reprogramming through Enhanced Cell-Cycle Exit.

    Science.gov (United States)

    Bektik, Emre; Dennis, Adrienne; Pawlowski, Gary; Zhou, Chen; Maleski, Danielle; Takahashi, Satoru; Laurita, Kenneth R; Deschênes, Isabelle; Fu, Ji-Dong

    2018-05-04

    Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) holds a great promise for regenerative medicine and has been studied in several major directions. However, cell-cycle regulation, a fundamental biological process, has not been investigated during iCM-reprogramming. Here, our time-lapse imaging on iCMs, reprogrammed by Gata4, Mef2c, and Tbx5 (GMT) monocistronic retroviruses, revealed that iCM-reprogramming was majorly initiated at late-G1- or S-phase and nearly half of GMT-reprogrammed iCMs divided soon after reprogramming. iCMs exited cell cycle along the process of reprogramming with decreased percentage of 5-ethynyl-20-deoxyuridine (EdU)⁺/α-myosin heavy chain (αMHC)-GFP⁺ cells. S-phase synchronization post-GMT-infection could enhance cell-cycle exit of reprogrammed iCMs and yield more GFP high iCMs, which achieved an advanced reprogramming with more expression of cardiac genes than GFP low cells. However, S-phase synchronization did not enhance the reprogramming with a polycistronic-viral vector, in which cell-cycle exit had been accelerated. In conclusion, post-infection synchronization of S-phase facilitated the early progression of GMT-reprogramming through a mechanism of enhanced cell-cycle exit.

  12. LIF-activated Jak signaling determines Esrrb expression during late-stage reprogramming

    Directory of Open Access Journals (Sweden)

    Delun Huang

    2018-01-01

    Full Text Available The regulatory process of naïve-state induced pluripotent stem cell (iPSC generation is not well understood. Leukemia inhibitory factor (LIF-activated Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3 is the master regulator for naïve-state pluripotency achievement and maintenance. The estrogen-related receptor beta (Esrrb serves as a naïve-state marker gene regulating self-renewal of embryonic stem cells (ESCs. However, the interconnection between Esrrb and LIF signaling for pluripotency establishment in reprogramming is unclear. We screened the marker genes critical for complete reprogramming during mouse iPSC generation, and identified genes including Esrrb that are responsive to LIF/Jak pathway signaling. Overexpression of Esrrb resumes the reprogramming halted by inhibition of Jak activity in partially reprogrammed cells (pre-iPSCs, and leads to the generation of pluripotent iPSCs. We further show that neither overexpression of Nanog nor stimulation of Wnt signaling, two upstream regulators of Esrrb in ESCs, stimulates the expression of Esrrb in reprogramming when LIF or Jak activity is blocked. Our study demonstrates that Esrrb is a specific reprogramming factor regulated downstream of the LIF/Jak signaling pathway. These results shed new light on the regulatory role of LIF pathway on complete pluripotency establishment during iPSC generation.

  13. Identifying Candidate Reprogramming Genes in Mouse Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Gao, Fang; Li, Jingyu; Zhang, Heng; Yang, Xu; An, Tiezhu

    2017-08-01

    Factor-based induced reprogramming approaches have tremendous potential for human regenerative medicine, but the efficiencies of these approaches are still low. In this study, we analyzed the global transcriptional profiles of mouse induced pluripotent stem cells (miPSCs) and mouse embryonic stem cells (mESCs) from seven different labs and present here the first successful clustering according to cell type, not by lab of origin. We identified 2131 different expression genes (DEs) as candidate pluripotency-associated genes by comparing mESCs/miPSCs with somatic cells and 720 DEs between miPSCs and mESCs. Interestingly, there was a significant overlap between the two DE sets. Therefore, we defined the overlap DEs as "consensus DEs" including 313 miPSC-specific genes expressed at a higher level in miPSCs versus mESCs and 184 mESC-specific genes in total and reasoned that these may contribute to the differences in pluripotency between mESCs and miPSCs. A classification of "consensus DEs" according to their different expression levels between somatic cells and mESCs/miPSCs shows that 86% of the miPSC-specific genes are more highly expressed in somatic cells, while 73% of mESC-specific genes are highly expressed in mESCs/miPSCs, indicating that the miPSCs have not efficiently silenced the expression pattern of the somatic cells from which they are derived and failed to completely induce the genes with high expression levels in mESCs. We further revealed a strong correlation between oocyte-enriched factors and insufficiently induced mESC-specific genes and identified 11 hub genes via network analysis. In light of these findings, we postulated that these key hub genes might not only drive somatic cell nuclear transfer (SCNT) reprogramming but also augment the efficiency and quality of miPSC reprogramming.

  14. Mitochondrial Spare Respiratory Capacity Is Negatively Correlated with Nuclear Reprogramming Efficiency

    DEFF Research Database (Denmark)

    Yan, Zhou; Al-Saaidi, Rasha Abdelkadhem; Fernandez Guerra, Paula

    2017-01-01

    Nuclear reprogramming efficiency has been shown to be highly variable among different types of somatic cells and different individuals, yet the underlying mechanism remains largely unknown. Several studies have shown that reprogramming of fibroblasts into induced pluripotent stem cells (i......, opposed to fibroblasts with the highest mitochondrial SRC, which showed lowest reprogramming efficiency. Furthermore, we found that targeted fluorescent tagging of endogenous genes (MYH6 and COL2A1) by CRISPR/Cas9-mediated homologous recombination was accompanied by an increase in the SRC level...

  15. Mitochondrial Spare Respiratory Capacity Is Negatively Correlated With Nuclear Reprogramming Efficiency

    DEFF Research Database (Denmark)

    Zhou, Yan; Al-Saaidi, Rasha Abdelkadhem; Guerra, Paula Fernandez

    2017-01-01

    Nuclear reprogramming efficiency has been shown to be highly variable among different types of somatic cells and different individuals, yet the underlying mechanism remains largely unknown. Several studies have shown that reprogramming of fibroblasts into induced pluripotent stem cells (i......, opposed to fibroblasts with the highest mitochondrial SRC, which showed lowest reprogramming efficiency. Furthermore, we found that targeted fluorescent tagging of endogenous genes (MYH6 and COL2A1) by CRISPR/Cas9-mediated homologous recombination was accompanied by an increase in the SRC level...

  16. Nuclear Reprogramming in Mouse Primordial Germ Cells: Epigenetic Contribution

    Directory of Open Access Journals (Sweden)

    Massimo De Felici

    2011-01-01

    Full Text Available The unique capability of germ cells to give rise to a new organism, allowing the transmission of primary genetic information from generation to generation, depends on their epigenetic reprogramming ability and underlying genomic totipotency. Recent studies have shown that genome-wide epigenetic modifications, referred to as “epigenetic reprogramming”, occur during the development of the gamete precursors termed primordial germ cells (PGCs in the embryo. This reprogramming is likely to be critical for the germ line development itself and necessary to erase the parental imprinting and setting the base for totipotency intrinsic to this cell lineage. The status of genome acquired during reprogramming and the associated expression of key pluripotency genes render PGCs susceptible to transform into pluripotent stem cells. This may occur in vivo under still undefined condition, and it is likely at the origin of the formation of germ cell tumors. The phenomenon appears to be reproduced under partly defined in vitro culture conditions, when PGCs are transformed into embryonic germ (EG cells. In the present paper, I will try to summarize the contribution that epigenetic modifications give to nuclear reprogramming in mouse PGCs.

  17. Genomic stability during cellular reprogramming: Mission impossible?

    Energy Technology Data Exchange (ETDEWEB)

    Joest, Mathieu von; Búa Aguín, Sabela; Li, Han, E-mail: han.li@pasteur.fr

    2016-06-15

    The generation of induced pluripotent stem cells (iPSCs) from adult somatic cells is one of the most exciting discoveries in recent biomedical research. It holds tremendous potential in drug discovery and regenerative medicine. However, a series of reports highlighting genomic instability in iPSCs raises concerns about their clinical application. Although the mechanisms cause genomic instability during cellular reprogramming are largely unknown, several potential sources have been suggested. This review summarizes current knowledge on this active research field and discusses the latest efforts to alleviate the genomic insults during cellular reprogramming to generate iPSCs with enhanced quality and safety.

  18. Reprogramming of round spermatids by the germinal vesicle cytoplasm in mice.

    Directory of Open Access Journals (Sweden)

    Peng-Cheng Kong

    Full Text Available The birthrate following round spermatid injection (ROSI remains low in current and evidence suggests that factors in the germinal vesicle (GV cytoplasm and certain substances in the GV such as the nucleolus might be responsible for genomic reprogramming and embryonic development. However, little is known whether the reprogramming factors in GV oocyte cytoplasm and/or nucleolus in GV are beneficial to the reprogramming of round spermatids and development of ROSI embryos. Here, round spermatids were treated with GV cytolysates and injected this round spermatid alone or co-injected with GV oocyte nucleolus into mature metaphase II oocytes. Subsequent embryonic development was assessed morphologically and by Oct4 expression in blastocysts. There was no significant difference between experimental groups at the zygote to four-cell development stages. Blastocysts derived from oocytes which were injected with cytolysate treated-round spermatid alone or co-injected with nucleoli injection yielded 63.6% and 70.3% high quality embryos, respectively; comparable to blastocysts derived by intracytoplasmic sperm injection (ICSI, but higher than these oocytes which were co-injected with lysis buffer-treated round spermatids and nucleoli or injected with the lysis buffer-treated round spermatids alone. Furthermore, the proportion of live offspring resulting from oocytes which were co-injected with cytolysate treated-round spermatids and nucleoli or injected with cytolysate treated-round spermatids alone was higher than those were injected with lysis buffer treated-round spermaids, but comparable with the ICSI group. Our results demonstrate that factors from the GV cytoplasm improve round spermatid reprogramming, and while injection of the extra nucleolus does not obviously improve reprogramming its potential contribution, although which cannot be definitively excluded. Thus, some reprogramming factors are evidently present in GV oocyte cytoplasm and could

  19. Reprogramming to pluripotency can conceal somatic cell chromosomal instability.

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    Masakazu Hamada

    Full Text Available The discovery that somatic cells are reprogrammable to pluripotency by ectopic expression of a small subset of transcription factors has created great potential for the development of broadly applicable stem-cell-based therapies. One of the concerns regarding the safe use of induced pluripotent stem cells (iPSCs in therapeutic applications is loss of genomic integrity, a hallmark of various human conditions and diseases, including cancer. Structural chromosome defects such as short telomeres and double-strand breaks are known to limit reprogramming of somatic cells into iPSCs, but whether defects that cause whole-chromosome instability (W-CIN preclude reprogramming is unknown. Here we demonstrate, using aneuploidy-prone mouse embryonic fibroblasts (MEFs in which chromosome missegregation is driven by BubR1 or RanBP2 insufficiency, that W-CIN is not a barrier to reprogramming. Unexpectedly, the two W-CIN defects had contrasting effects on iPSC genomic integrity, with BubR1 hypomorphic MEFs almost exclusively yielding aneuploid iPSC clones and RanBP2 hypomorphic MEFs karyotypically normal iPSC clones. Moreover, BubR1-insufficient iPSC clones were karyotypically unstable, whereas RanBP2-insufficient iPSC clones were rather stable. These findings suggest that aneuploid cells can be selected for or against during reprogramming depending on the W-CIN gene defect and present the novel concept that somatic cell W-CIN can be concealed in the pluripotent state. Thus, karyotypic analysis of somatic cells of origin in addition to iPSC lines is necessary for safe application of reprogramming technology.

  20. Cocktail of chemical compounds robustly promoting cell reprogramming protects liver against acute injury

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    Yuewen Tang

    2017-02-01

    Full Text Available Abstract Tissue damage induces cells into reprogramming-like cellular state, which contributes to tissue regeneration. However, whether factors promoting the cell reprogramming favor tissue regeneration remains elusive. Here we identified combination of small chemical compounds including drug cocktails robustly promoting in vitro cell reprogramming. We then administrated the drug cocktails to mice with acute liver injuries induced by partial hepatectomy or toxic treatment. Our results demonstrated that the drug cocktails which promoted cell reprogramming in vitro improved liver regeneration and hepatic function in vivo after acute injuries. The underlying mechanism could be that expression of pluripotent genes activated after injury is further upregulated by drug cocktails. Thus our study offers proof-of-concept evidence that cocktail of clinical compounds improving cell reprogramming favors tissue recovery after acute damages, which is an attractive strategy for regenerative purpose.

  1. Looking into the Black Box: Insights into the Mechanisms of Somatic Cell Reprogramming

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    Jeffrey L. Wrana

    2011-01-01

    Full Text Available The dramatic discovery that somatic cells could be reprogrammed to induced pluripotent stem cells (iPSCs, by the expression of just four factors, has opened new opportunities for regenerative medicine and novel ways of modeling human diseases. Extensive research over the short time since the first iPSCs were generated has yielded the ability to reprogram various cell types using a diverse range of methods. However the duration, efficiency, and safety of induced reprogramming have remained a persistent limitation to achieving a robust experimental and therapeutic system. The field has worked to resolve these issues through technological advances using non-integrative approaches, factor replacement or complementation with microRNA, shRNA and drugs. Despite these advances, the molecular mechanisms underlying the reprogramming process remain poorly understood. Recently, through the use of inducible secondary reprogramming systems, researchers have now accessed more rigorous mechanistic experiments to decipher this complex process. In this review we will discuss some of the major recent findings in reprogramming, pertaining to proliferation and cellular senescence, epigenetic and chromatin remodeling, and other complex cellular processes such as morphological changes and mesenchymal-to-epithelial transition. We will focus on the implications of this work in the construction of a mechanistic understanding of reprogramming and discuss unexplored areas in this rapidly expanding field.

  2. Reprogramming stem cells is a microenvironmental task

    Energy Technology Data Exchange (ETDEWEB)

    Bissell, Mina J; Inman, Jamie

    2008-10-14

    That tumor cells for all practical purposes are unstable and plastic could be expected. However, the astonishing ability of the nuclei from cells of normal adult tissues to be reprogrammed - given the right embryonic context - found its final truth even for mammals in the experiments that allowed engineering Dolly (1). The landmark experiments showed that nuclei originating from cells of frozen mammary tissues were capable of being reprogrammed by the embryonic cytoplasm and its microenvironment to produce a normal sheep. The rest is history. However, whether microenvironments other than those of the embryos can also reprogram adult cells of different tissue origins still containing their cytoplasm is of obvious interest. In this issue of PNAS, the laboratory of Gilbert Smith (2) reports on how the mammary gland microenvironment can reprogram both embryonic and adult stem neuronal cells. The work is a follow-up to their previous report on testis stem cells that were reprogrammed by the mammary microenvironment (3). They demonstrated that cells isolated from the seminiferous tubules of the mature testis, mixed with normal mammary epithelial cells, contributed a sizable number of epithelial progeny to normal mammary outgrowths in transplanted mammary fat pads. However, in those experiments they were unable to distinguish which subpopulation of the testis cells contributed progeny to the mammary epithelial tree. The current work adds new, compelling, and provocative information to our understanding of stem cell plasticity. Booth et al. (2) use neuronal stem cells (NSCs) isolated from WAP-cre/R26R mice combined with unlabeled mammary epithelial cells that subsequently are implanted in cleared mammary fat pads. In this new microenvironment, the NSCs that are incorporated into the branching mammary tree make chimeric glands (Fig. 1) that remarkably can also express the milk protein {beta}-casein, progesterone receptor, and estrogen receptor {alpha}. Remarkably, the

  3. Evaluating the potential of poly(beta-amino ester nanoparticles for reprogramming human fibroblasts to become induced pluripotent stem cells

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    Bhise NS

    2013-12-01

    reagents, including Lipofectamine® 2000, FuGENE® HD, and 25 kDa branched polyethylenimine, for nonviral gene transfer. B4S4E7 nanoparticles showed effective gene delivery to IMR-90 human primary fibroblasts and to dermal fibroblasts derived from a patient with retinitis pigmentosa, and enabled coexpression of exogenously delivered genes, as is needed for reprogramming. The karyotypically normal hiPSC-like cells generated by conventional electroporation, but not by poly(beta-amino ester reprogramming, could be differentiated toward the neuronal lineage, specifically pseudostratified optic cups.Conclusion: This study shows that certain nonviral reprogramming methods may not necessarily be safer than viral approaches and that maximizing exogenous gene expression of reprogramming factors is not sufficient to ensure successful reprogramming.Keywords: poly(beta-amino ester nanoparticles, reprogramming, human fibroblasts, induced pluripotent stem cells

  4. DMPD: Cellular reprogramming by gram-positive bacterial components: a review. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 16885502 Cellular reprogramming by gram-positive bacterial components: a review. Bu...(.csml) Show Cellular reprogramming by gram-positive bacterial components: a review. PubmedID 16885502 Title... Cellular reprogramming by gram-positive bacterial components: a review. Authors

  5. Naked Mole Rat Cells Have a Stable Epigenome that Resists iPSC Reprogramming

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    Li Tan

    2017-11-01

    Full Text Available Naked mole rat (NMR is a valuable model for aging and cancer research due to its exceptional longevity and cancer resistance. We observed that the reprogramming efficiency of NMR fibroblasts in response to OSKM was drastically lower than that of mouse fibroblasts. Expression of SV40 LargeT antigen (LT dramatically improved reprogramming of NMR fibroblasts. Inactivation of Rb alone, but not p53, was sufficient to improve reprogramming efficiency, suggesting that NMR chromatin may be refractory to reprogramming. Analysis of the global histone landscape revealed that NMR had higher levels of repressive H3K27 methylation marks and lower levels of activating H3K27 acetylation marks than mouse. ATAC-seq revealed that in NMR, promoters of reprogramming genes were more closed than mouse promoters, while expression of LT led to massive opening of the NMR promoters. These results suggest that NMR displays a more stable epigenome that resists de-differentiation, contributing to the cancer resistance and longevity of this species.

  6. Histone H3 Methylated at Arginine 17 Is Essential for Reprogramming the Paternal Genome in Zygotes

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    Yuki Hatanaka

    2017-09-01

    Full Text Available At fertilization, the paternal genome undergoes extensive reprogramming through protamine-histone exchange and active DNA demethylation, but only a few maternal factors have been defined in these processes. We identified maternal Mettl23 as a protein arginine methyltransferase (PRMT, which most likely catalyzes the asymmetric dimethylation of histone H3R17 (H3R17me2a, as indicated by in vitro assays and treatment with TBBD, an H3R17 PRMT inhibitor. Maternal histone H3.3, which is essential for paternal nucleosomal assembly, is unable to be incorporated into the male pronucleus when it lacks R17me2a. Mettl23 interacts with Tet3, a 5mC-oxidizing enzyme responsible for active DNA demethylation, by binding to another maternal factor, GSE (gonad-specific expression. Depletion of Mettl23 from oocytes resulted in impaired accumulation of GSE, Tet3, and 5hmC in the male pronucleus, suggesting that Mettl23 may recruit GSE-Tet3 to chromatin. Our findings establish H3R17me2a and its catalyzing enzyme Mettl23 as key regulators of paternal genome reprogramming.

  7. A unique Oct4 interface is crucial for reprogramming to pluripotency

    NARCIS (Netherlands)

    Esch, Daniel; Vahokoski, Juha; Groves, Matthew R; Pogenberg, Vivian; Cojocaru, Vlad; Vom Bruch, Hermann; Han, Dong; Drexler, Hannes C A; Araúzo-Bravo, Marcos J; Ng, Calista K L; Jauch, Ralf; Wilmanns, Matthias; Schöler, Hans R

    Terminally differentiated cells can be reprogrammed to pluripotency by the forced expression of Oct4, Sox2, Klf4 and c-Myc. However, it remains unknown how this leads to the multitude of epigenetic changes observed during the reprogramming process. Interestingly, Oct4 is the only factor that cannot

  8. Direct lineage reprogramming of mouse fibroblasts to functional midbrain dopaminergic neuronal progenitors

    Directory of Open Access Journals (Sweden)

    Han-Seop Kim

    2014-01-01

    Full Text Available The direct lineage reprogramming of somatic cells to other lineages by defined factors has led to innovative cell-fate-change approaches for providing patient-specific cells. Recent reports have demonstrated that four pluripotency factors (Oct4, Sox2, Klf4, and c-Myc are sufficient to directly reprogram fibroblasts to other specific cells, including induced neural stem cells (iNSCs. Here, we show that mouse fibroblasts can be directly reprogrammed into midbrain dopaminergic neuronal progenitors (DPs by temporal expression of the pluripotency factors and environment containing sonic hedgehog and fibroblast growth factor 8. Within thirteen days, self-renewing and functional induced DPs (iDPs were generated. Interestingly, the inhibition of both Jak and Gsk3β notably enhanced the iDP reprogramming efficiency. We confirmed the functionality of the iDPs by showing that the dopaminergic neurons generated from iDPs express midbrain markers, release dopamine, and show typical electrophysiological profiles. Our results demonstrate that the pluripotency factors-mediated direct reprogramming is an invaluable strategy for supplying functional and proliferating iDPs and may be useful for other neural progenitors required for disease modeling and cell therapies for neurodegenerative disorders.

  9. Homologous Recombination DNA Repair Genes Play a Critical Role in Reprogramming to a Pluripotent State

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    Federico González

    2013-03-01

    Full Text Available Induced pluripotent stem cells (iPSCs hold great promise for personalized regenerative medicine. However, recent studies show that iPSC lines carry genetic abnormalities, suggesting that reprogramming may be mutagenic. Here, we show that the ectopic expression of reprogramming factors increases the level of phosphorylated histone H2AX, one of the earliest cellular responses to DNA double-strand breaks (DSBs. Additional mechanistic studies uncover a direct role of the homologous recombination (HR pathway, a pathway essential for error-free repair of DNA DSBs, in reprogramming. This role is independent of the use of integrative or nonintegrative methods in introducing reprogramming factors, despite the latter being considered a safer approach that circumvents genetic modifications. Finally, deletion of the tumor suppressor p53 rescues the reprogramming phenotype in HR-deficient cells primarily through the restoration of reprogramming-dependent defects in cell proliferation and apoptosis. These mechanistic insights have important implications for the design of safer approaches to creating iPSCs.

  10. Developmental Programming of Adult Disease: Reprogramming by Melatonin?

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    You-Lin Tain

    2017-02-01

    Full Text Available Adult-onset chronic non-communicable diseases (NCDs can originate from early life through so-called the “developmental origins of health and disease” (DOHaD or “developmental programming”. The DOHaD concept offers the “reprogramming” strategy to shift the treatment from adulthood to early life, before clinical disease is apparent. Melatonin, an endogenous indoleamine produced by the pineal gland, has pleiotropic bioactivities those are beneficial in a variety of human diseases. Emerging evidence support that melatonin is closely inter-related to other proposed mechanisms contributing to the developmental programming of a variety of chronic NCDs. Recent animal studies have begun to unravel the multifunctional roles of melatonin in many experimental models of developmental programming. Even though some progress has been made in research on melatonin as a reprogramming strategy to prevent DOHaD-related NCDs, future human studies should aim at filling the translational gap between animal models and clinical trials. Here, we review several key themes on the reprogramming effects of melatonin in DOHaD research. We have particularly focused on the following areas: mechanisms of developmental programming; the interrelationship between melatonin and mechanisms underlying developmental programming; pathophysiological roles of melatonin in pregnancy and fetal development; and insight provided by animal models to support melatonin as a reprogramming therapy. Rates of NCDs are increasing faster than anticipated all over the world. Hence, there is an urgent need to understand reprogramming mechanisms of melatonin and to translate experimental research into clinical practice for halting a growing list of DOHaD-related NCDs.

  11. Grandpaternal-induced transgenerational dietary reprogramming of the unfolded protein response in skeletal muscle

    DEFF Research Database (Denmark)

    Alm, Petter S; de Castro Barbosa, Thais; Barrès, Romain

    2017-01-01

    OBJECTIVE: Parental nutrition and lifestyle impact the metabolic phenotype of the offspring. We have reported that grandpaternal chronic high-fat diet (HFD) transgenerationally impairs glucose metabolism in subsequent generations. Here we determined whether grandpaternal diet transgenerationally....... Gene set enrichment analysis (GSEA) was performed to determine pathways reprogrammed by grandpaternal diet. RESULTS: GSEA revealed an enrichment of the unfolded protein response pathway in skeletal muscle of grand-offspring from HFD-fed grandfathers compared to grand-offspring of chow-fed males....... Activation of the stress sensor (ATF6α), may be a pivotal point whereby this pathway is activated. Interestingly, skeletal muscle from F1-offspring was not affected in a similar manner. No major changes were observed in the skeletal muscle lipidome profile due to grandpaternal diet. CONCLUSIONS...

  12. The Epigenetic Reprogramming Roadmap in Generation of iPSCs from Somatic Cells

    DEFF Research Database (Denmark)

    Brix, Jacob; Zhou, Yan; Luo, Yonglun

    2015-01-01

    Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is a comprehensive epigenetic process involving genome-wide modifications of histones and DNA methylation. This process is often incomplete, which subsequently affects iPSC reprograming, pluripotency, and differentiation cap...

  13. The Effect of Substrate Topography on Direct Reprogramming of Fibroblasts to Induced Neurons

    Science.gov (United States)

    Kulangara, Karina; Adler, Andrew F.; Wang, Hong; Chellappan, Malathi; Hammett, Ellen; Yasuda, Ryohei; Leong, Kam W.

    2014-01-01

    Cellular reprogramming holds tremendous potential for cell therapy and regenerative medicine. Recently, fibroblasts have been directly converted into induced neurons (iNs) by overexpression of the neuronal transcription factors Ascl1, Brn2 and Myt1L. Hypothesizing that cell-topography interactions could influence the fibroblast-to-neuron reprogramming process, we investigated the effects of various topographies on iNs produced by direct reprogramming. Final iN purity and conversion efficiency were increased on micrograting substrates. Neurite branching was increased on microposts and decreased on microgratings, with a simplified dendritic arbor characterized by the reduction of MAP2+ neurites. Neurite outgrowth increased significantly on various topographies. DNA microarray analysis detected 20 differentially expressed genes in iNs reprogrammed on smooth versus microgratings, and quantitative PCR (qPCR) confirmed the upregulation of Vip and downregulation of Thy1 and Bmp5 on microgratings. Electrophysiology and calcium imaging verified the functionality of these iNs. This study demonstrates the potential of applying topographical cues to optimize cellular reprogramming. PMID:24709523

  14. The Current State of Nanoparticle-Induced Macrophage Polarization and Reprogramming Research

    Directory of Open Access Journals (Sweden)

    Xiaoyuan Miao

    2017-02-01

    Full Text Available Macrophages are vital regulators of the host defense in organisms. In response to different local microenvironments, resting macrophages (M0 can be polarized into different phenotypes, pro-inflammatory (M1 or anti-inflammatory (M2, and perform different roles in different physiological or pathological conditions. Polarized macrophages can also be further reprogrammed by reversing their phenotype according to the changed milieu. Macrophage polarization and reprogramming play essential roles in maintaining the steady state of the immune system and are involved in the processes of many diseases. As foreign substances, nanoparticles (NPs mainly target macrophages after entering the body. NPs can perturb the polarization and reprogramming of macrophages, affect their immunological function and, therefore, affect the pathological process of disease. Optimally-designed NPs for the modulation of macrophage polarization and reprogramming might provide new solutions for treating diseases. Systematically investigating how NPs affect macrophage polarization is crucial for understanding the regulatory effects of NPs on immune cells in vivo. In this review, macrophage polarization by NPs is summarized and discussed.

  15. Generation of Patient-Specific induced Pluripotent Stem Cell from Peripheral Blood Mononuclear Cells by Sendai Reprogramming Vectors.

    Science.gov (United States)

    Quintana-Bustamante, Oscar; Segovia, Jose C

    2016-01-01

    Induced pluripotent stem cells (iPSC) technology has changed preclinical research since their generation was described by Shinya Yamanaka in 2006. iPSCs are derived from somatic cells after being reprogrammed back to an embryonic state by specific combination of reprogramming factors. These reprogrammed cells resemble all the characteristic of embryonic stem cells (ESC). The reprogramming technology is even more valuable to research diseases biology and treatment by opening gene and cell therapies in own patient's iPSC. Patient-specific iPSC can be generated from a large variety of patient cells by any of the myriad of reprogramming platforms described. Here, we describe the generation of patient-specific iPSC from patient peripheral blood mononuclear cells by Sendai Reprogramming vectors.

  16. In vivo reprogramming for heart regeneration: A glance at efficiency, environmental impacts, challenges and future directions.

    Science.gov (United States)

    Ebrahimi, Behnam

    2017-07-01

    Replacing dying or diseased cells of a tissue with new ones that are converted from patient's own cells is an attractive strategy in regenerative medicine. In vivo reprogramming is a novel strategy that can circumvent the hurdles of autologous/allogeneic cell injection therapies. Interestingly, studies have demonstrated that direct injection of cardiac transcription factors or specific miRNAs into the infarct border zone of murine hearts following myocardial infarction converts resident cardiac fibroblasts into functional cardiomyocytes. Moreover, in vivo cardiac reprogramming not only drives cardiac tissue regeneration, but also improves cardiac function and survival rate after myocardial infarction. Thanks to the influence of cardiac microenvironment and the same developmental origin, cardiac fibroblasts seem to be more amenable to reprogramming toward cardiomyocyte fate than other cell sources (e.g. skin fibroblasts). Thus, reprogramming of cardiac fibroblasts to functional induced cardiomyocytes in the cardiac environment holds great promises for induced regeneration and potential clinical purposes. Application of small molecules in future studies may represent a major advancement in this arena and pharmacological reprogramming would convey reprogramming technology to the translational medicine paradigm. This study reviews accomplishments in the field of in vitro and in vivo mouse cardiac reprogramming and then deals with strategies for the enhancement of the efficiency and quality of the process. Furthermore, it discusses challenges ahead and provides suggestions for future research. Human cardiac reprogramming is also addressed as a foundation for possible application of in vivo cardiac reprogramming for human heart regeneration in the future. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Electromagnetic fields mediate efficient cell reprogramming into a pluripotent state.

    Science.gov (United States)

    Baek, Soonbong; Quan, Xiaoyuan; Kim, Soochan; Lengner, Christopher; Park, Jung-Keug; Kim, Jongpil

    2014-10-28

    Life on Earth is constantly exposed to natural electromagnetic fields (EMFs), and it is generally accepted that EMFs may exert a variety of effects on biological systems. Particularly, extremely low-frequency electromagnetic fields (EL-EMFs) affect biological processes such as cell development and differentiation; however, the fundamental mechanisms by which EMFs influence these processes remain unclear. Here we show that EMF exposure induces epigenetic changes that promote efficient somatic cell reprogramming to pluripotency. These epigenetic changes resulted from EMF-induced activation of the histone lysine methyltransferase Mll2. Remarkably, an EMF-free system that eliminates Earth's naturally occurring magnetic field abrogates these epigenetic changes, resulting in a failure to undergo reprogramming. Therefore, our results reveal that EMF directly regulates dynamic epigenetic changes through Mll2, providing an efficient tool for epigenetic reprogramming including the acquisition of pluripotency.

  18. Gender Differences in Global but Not Targeted Demethylation in iPSC Reprogramming

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    Inês Milagre

    2017-01-01

    Full Text Available Global DNA demethylation is an integral part of reprogramming processes in vivo and in vitro, but whether it occurs in the derivation of induced pluripotent stem cells (iPSCs is not known. Here, we show that iPSC reprogramming involves both global and targeted demethylation, which are separable mechanistically and by their biological outcomes. Cells at intermediate-late stages of reprogramming undergo transient genome-wide demethylation, which is more pronounced in female cells. Global demethylation requires activation-induced cytidine deaminase (AID-mediated downregulation of UHRF1 protein, and abolishing demethylation leaves thousands of hypermethylated regions in the iPSC genome. Independently of AID and global demethylation, regulatory regions, particularly ESC enhancers and super-enhancers, are specifically targeted for hypomethylation in association with transcription of the pluripotency network. Our results show that global and targeted DNA demethylation are conserved and distinct reprogramming processes, presumably because of their respective roles in epigenetic memory erasure and in the establishment of cell identity.

  19. Current reprogramming systems in regenerative medicine: from somatic cells to induced pluripotent stem cells.

    Science.gov (United States)

    Hu, Chenxia; Li, Lanjuan

    2016-01-01

    Induced pluripotent stem cells (iPSCs) paved the way for research fields including cell therapy, drug screening, disease modeling and the mechanism of embryonic development. Although iPSC technology has been improved by various delivery systems, direct transduction and small molecule regulation, low reprogramming efficiency and genomic modification steps still inhibit its clinical use. Improvements in current vectors and the exploration of novel vectors are required to balance efficiency and genomic modification for reprogramming. Herein, we set out a comprehensive analysis of current reprogramming systems for the generation of iPSCs from somatic cells. By clarifying advantages and disadvantages of the current reprogramming systems, we are striding toward an effective route to generate clinical grade iPSCs.

  20. Molecular Imaging Of Metabolic Reprogramming In Mutant IDH Cells

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    Pavithra eViswanath

    2016-03-01

    Full Text Available Mutations in the metabolic enzyme isocitrate dehydrogenase (IDH have recently been identified as drivers in the development of several tumor types. Most notably, cytosolic IDH1 is mutated in 70-90% of low-grade gliomas and upgraded glioblastomas, and mitochondrial IDH2 is mutated in ~20% of acute myeloid leukemia cases. Wild-type IDH catalyzes the interconversion of isocitrate to α-ketoglutarate (α-KG. Mutations in the enzyme lead to loss of wild-type enzymatic activity and a neomorphic activity that converts α-KG to 2-hydroxyglutarate (2-HG. In turn, 2-HG, which has been termed an oncometabolite, inhibits key α-KG- dependent enzymes, resulting in alterations of the cellular epigenetic profile and, subsequently, inhibition of differentiation and initiation of tumorigenesis. In addition, it is now clear that the IDH mutation also induces a broad metabolic reprogramming that extends beyond 2-HG production, and this reprogramming often differs from what has been previously reported in other cancer types. In this review we will discuss in detail what is known to date about the metabolic reprogramming of mutant IDH cells and how this reprogramming has been investigated using molecular metabolic imaging. We will describe how metabolic imaging has helped shed light on the basic biology of mutant IDH cells and how this information can be leveraged to identify new therapeutic targets and to develop new clinically translatable imaging methods to detect and monitor mutant IDH tumors in vivo.

  1. Generating pluripotent stem cells: Differential epigenetic changes during cellular reprogramming

    OpenAIRE

    Tobin, Stacey C.; Kim, Kitai

    2012-01-01

    Pluripotent stem cells hold enomous potential for therapuetic applications in tissue replacement therapy. Reprogramming somatic cells from a patient donor to generate pluripotent stem cells involves both ethical concerns inherent in the use of embryonic and oocyte-derived stem cells, as well as issues of histocompatibility. Among the various pluripotent stem cells, induced pluripotent stem cells (iPSC)—derived by ectopic expression of four reprogramming factors in donor somatic cells—are supe...

  2. Robust Differentiation of mRNA-Reprogrammed Human Induced Pluripotent Stem Cells Toward a Retinal Lineage.

    Science.gov (United States)

    Sridhar, Akshayalakshmi; Ohlemacher, Sarah K; Langer, Kirstin B; Meyer, Jason S

    2016-04-01

    The derivation of human induced pluripotent stem cells (hiPSCs) from patient-specific sources has allowed for the development of novel approaches to studies of human development and disease. However, traditional methods of generating hiPSCs involve the risks of genomic integration and potential constitutive expression of pluripotency factors and often exhibit low reprogramming efficiencies. The recent description of cellular reprogramming using synthetic mRNA molecules might eliminate these shortcomings; however, the ability of mRNA-reprogrammed hiPSCs to effectively give rise to retinal cell lineages has yet to be demonstrated. Thus, efforts were undertaken to test the ability and efficiency of mRNA-reprogrammed hiPSCs to yield retinal cell types in a directed, stepwise manner. hiPSCs were generated from human fibroblasts via mRNA reprogramming, with parallel cultures of isogenic human fibroblasts reprogrammed via retroviral delivery of reprogramming factors. New lines of mRNA-reprogrammed hiPSCs were established and were subsequently differentiated into a retinal fate using established protocols in a directed, stepwise fashion. The efficiency of retinal differentiation from these lines was compared with retroviral-derived cell lines at various stages of development. On differentiation, mRNA-reprogrammed hiPSCs were capable of robust differentiation to a retinal fate, including the derivation of photoreceptors and retinal ganglion cells, at efficiencies often equal to or greater than their retroviral-derived hiPSC counterparts. Thus, given that hiPSCs derived through mRNA-based reprogramming strategies offer numerous advantages owing to the lack of genomic integration or constitutive expression of pluripotency genes, such methods likely represent a promising new approach for retinal stem cell research, in particular, those for translational applications. In the current report, the ability to derive mRNA-reprogrammed human induced pluripotent stem cells (hi

  3. Analysis of nucleolar morphology and protein localization as an indicator of nuclear reprogramming

    DEFF Research Database (Denmark)

    Østrup, Olga; Pedersen, Hanne Skovsgaard; Holm, Hanne M.

    2015-01-01

    When a cell is reprogrammed to a new phenotype, the nucleolus undergoes more or less dramatic modulations, which can be used as a marker for the occurrence of the reprogramming. This phenomenon is most pronounced when differentiated cells are reprogrammed to totipotency when they are submitted...... of the nucleolus are summarized in this developmental context, but also as they occur in assisted reproductive technologies such as in vitro fertilization and somatic cell nuclear transfer. Moreover, detailed protocols for monitoring the nucleolar changes by transmission electron microscopy and immunocytochemistry...

  4. Deletion of the Mitochondrial Chaperone TRAP-1 Uncovers Global Reprogramming of Metabolic Networks

    Directory of Open Access Journals (Sweden)

    Sofia Lisanti

    2014-08-01

    Full Text Available Reprogramming of metabolic pathways contributes to human disease, especially cancer, but the regulators of this process are unknown. Here, we have generated a mouse knockout for the mitochondrial chaperone TRAP-1, a regulator of bioenergetics in tumors. TRAP-1−/− mice are viable and showed reduced incidence of age-associated pathologies, including obesity, inflammatory tissue degeneration, dysplasia, and spontaneous tumor formation. This was accompanied by global upregulation of oxidative phosphorylation and glycolysis transcriptomes, causing deregulated mitochondrial respiration, oxidative stress, impaired cell proliferation, and a switch to glycolytic metabolism in vivo. These data identify TRAP-1 as a central regulator of mitochondrial bioenergetics, and this pathway could contribute to metabolic rewiring in tumors.

  5. Cell-of-Origin-Specific 3D Genome Structure Acquired during Somatic Cell Reprogramming

    NARCIS (Netherlands)

    Krijger, Peter Hugo Lodewijk; Di Stefano, Bruno; de Wit, Elzo; Limone, Francesco; van Oevelen, Chris; de Laat, Wouter; Graf, Thomas

    2016-01-01

    Forced expression of reprogramming factors can convert somatic cells into induced pluripotent stem cells (iPSCs). Here we studied genome topology dynamics during reprogramming of different somatic cell types with highly distinct genome conformations. We find large-scale topologically associated

  6. Genome-wide transcriptional reprogramming under drought stress

    KAUST Repository

    Chen, Hao; Xiong, Liming

    2012-01-01

    Soil water deficit is one of the major factors limiting plant productivity. Plants cope with this adverse environmental condition by coordinating the up- or downregulation of an array of stress responsive genes. Reprogramming the expression

  7. Effects of mechanical stimulation on the reprogramming of somatic cells into human-induced pluripotent stem cells.

    Science.gov (United States)

    Kim, Young Mi; Kang, Yun Gyeong; Park, So Hee; Han, Myung-Kwan; Kim, Jae Ho; Shin, Ji Won; Shin, Jung-Woog

    2017-06-08

    Mechanical stimuli play important roles in the proliferation and differentiation of adult stem cells. However, few studies on their effects on induced pluripotent stem cells (iPSCs) have been published. Human dermal fibroblasts were seeded onto flexible membrane-bottom plates, and infected with retrovirus expressing the four reprogramming factors OCT4, SOX2, KLF, and c-MYC (OSKM). The cells were subjected to equiaxial stretching (3% or 8% for 2, 4, or 7 days) and seeded on feeder cells (STO). The reprogramming into iPSCs was evaluated by the expression of pluripotent markers, in vitro differentiation into three germ layers, and teratoma formation. Equiaxial stretching enhanced reprogramming efficiency without affecting the viral transduction rate. iPSCs induced by transduction of four reprogramming factors and application of equiaxial stretching had characteristics typical of iPSCs in terms of pluripotency and differentiation potentials. This is the first study to show that mechanical stimuli can increase reprogramming efficiency. However, it did not enhance the infection rate, indicating that mechanical stimuli, defined as stretching in this study, have positive effects on reprogramming rather than on infection. Additional studies should evaluate the mechanism underlying the modulation of reprogramming of somatic cells into iPSCs.

  8. mTOR-regulated senescence and autophagy during reprogramming of somatic cells to pluripotency: a roadmap from energy metabolism to stem cell renewal and aging.

    Science.gov (United States)

    Menendez, Javier A; Vellon, Luciano; Oliveras-Ferraros, Cristina; Cufí, Sílvia; Vazquez-Martin, Alejandro

    2011-11-01

    Molecular controllers of the number and function of tissue stem cells may share common regulatory pathways for the nuclear reprogramming of somatic cells to become induced Pluripotent Stem Cells (iPSCs). If this hypothesis is true, testing the ability of longevity-promoting chemicals to improve reprogramming efficiency may provide a proof-of-concept validation tool for pivotal housekeeping pathways that limit the numerical and/or functional decline of adult stem cells. Reprogramming is a slow, stochastic process due to the complex and apparently unrelated cellular processes that are involved. First, forced expression of the Yamanaka cocktail of stemness factors, OSKM, is a stressful process that activates apoptosis and cellular senescence, which are the two primary barriers to cancer development and somatic reprogramming. Second, the a priori energetic infrastructure of somatic cells appears to be a crucial stochastic feature for optimal successful routing to pluripotency. If longevity-promoting compounds can ablate the drivers and effectors of cellular senescence while concurrently enhancing a bioenergetic shift from somatic oxidative mitochondria toward an alternative ATP-generating glycolytic metabotype, they could maximize the efficiency of somatic reprogramming to pluripotency. Support for this hypothesis is evidenced by recent findings that well-characterized mTOR inhibitors and autophagy activators (e.g., PP242, rapamycin and resveratrol) notably improve the speed and efficiency of iPSC generation. This article reviews the existing research evidence that the most established mTOR inhibitors can notably decelerate the cellular senescence that is imposed by DNA damage-like responses, which are somewhat equivalent to the responses caused by reprogramming factors. These data suggest that fine-tuning mTOR signaling can impact mitochondrial dynamics to segregate mitochondria that are destined for clearance through autophagy, which results in the loss of

  9. Generation of Induced Progenitor-like Cells from Mature Epithelial Cells Using Interrupted Reprogramming

    Directory of Open Access Journals (Sweden)

    Li Guo

    2017-12-01

    Full Text Available Summary: A suitable source of progenitor cells is required to attenuate disease or affect cure. We present an “interrupted reprogramming” strategy to generate “induced progenitor-like (iPL cells” using carefully timed expression of induced pluripotent stem cell reprogramming factors (Oct4, Sox2, Klf4, and c-Myc; OSKM from non-proliferative Club cells. Interrupted reprogramming allowed controlled expansion yet preservation of lineage commitment. Under clonogenic conditions, iPL cells expanded and functioned as a bronchiolar progenitor-like population to generate mature Club cells, mucin-producing goblet cells, and cystic fibrosis transmembrane conductance regulator (CFTR-expressing ciliated epithelium. In vivo, iPL cells can repopulate CFTR-deficient epithelium. This interrupted reprogramming process could be metronomically applied to achieve controlled progenitor-like proliferation. By carefully controlling the duration of expression of OSKM, iPL cells do not become pluripotent, and they maintain their memory of origin and retain their ability to efficiently return to their original phenotype. A generic technique to produce highly specified populations may have significant implications for regenerative medicine. : In this article Waddell, Nagy, and colleagues present an “interrupted reprogramming” strategy to produce highly specified functional “induced progenitor-like cells” from mature quiescent cells. They propose that careful control of the duration of transient expression of iPSC reprogramming factors (OSKM allows controlled expansion yet preservation of parental lineage without traversing the pluripotent state. Keywords: generation of induced progenitor-like cells

  10. Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming.

    Directory of Open Access Journals (Sweden)

    Ming-Wai Poon

    Full Text Available A variety of pluripotency reprogramming frequencies from different somatic cells has been observed, indicating cell origin is a critical contributor for efficiency of pluripotency reprogramming. Identifying the cell sources for efficient induced pluripotent stem cells (iPSCs generation, and defining its advantages or disadvantages on reprogramming, is therefore important. Human ocular tissue-derived conjunctival epithelial cells (OECs exhibited endogenous expression of reprogramming factors OCT4A (the specific OCT 4 isoform on pluripotency reprogramming and SOX2. We therefore determined whether OECs could be used for high efficiency of iPSCs generation. We compared the endogenous expression levels of four pluripotency factors and the pluripotency reprograming efficiency of human OECs with that of ocular stromal cells (OSCs. Real-time PCR, microarray analysis, Western blotting and immunostaining assays were employed to compare OECiPSCs with OSCiPSCs on molecular bases of reprogramming efficiency and preferred lineage-differentiation potential. Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2 reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%. Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs. The findings in this study suggest that conjunctival-derived epithelial (OECs cells can be easier converted to iPSCs than conjunctival-derived stromal cells (OSCs. This cell type may also have advantages in retinal pigmented epithelial differentiation.

  11. Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming.

    Science.gov (United States)

    Poon, Ming-Wai; He, Jia; Fang, Xiaowei; Zhang, Zhao; Wang, Weixin; Wang, Junwen; Qiu, Fangfang; Tse, Hung-Fat; Li, Wei; Liu, Zuguo; Lian, Qizhou

    2015-01-01

    A variety of pluripotency reprogramming frequencies from different somatic cells has been observed, indicating cell origin is a critical contributor for efficiency of pluripotency reprogramming. Identifying the cell sources for efficient induced pluripotent stem cells (iPSCs) generation, and defining its advantages or disadvantages on reprogramming, is therefore important. Human ocular tissue-derived conjunctival epithelial cells (OECs) exhibited endogenous expression of reprogramming factors OCT4A (the specific OCT 4 isoform on pluripotency reprogramming) and SOX2. We therefore determined whether OECs could be used for high efficiency of iPSCs generation. We compared the endogenous expression levels of four pluripotency factors and the pluripotency reprograming efficiency of human OECs with that of ocular stromal cells (OSCs). Real-time PCR, microarray analysis, Western blotting and immunostaining assays were employed to compare OECiPSCs with OSCiPSCs on molecular bases of reprogramming efficiency and preferred lineage-differentiation potential. Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2) reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%). Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells) was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs. The findings in this study suggest that conjunctival-derived epithelial (OECs) cells can be easier converted to iPSCs than conjunctival-derived stromal cells (OSCs). This cell type may also have advantages in retinal pigmented epithelial differentiation.

  12. Efficient production of retroviruses using PLGA/bPEI-DNA nanoparticles and application for reprogramming somatic cells.

    Directory of Open Access Journals (Sweden)

    Eun Jin Seo

    Full Text Available Reprogramming of somatic cells to pluripotent cells requires the introduction of factors driving fate switches. Viral delivery has been the most efficient method for generation of induced pluripotent stem cells. Transfection, which precedes virus production, is a commonly-used process for delivery of nucleic acids into cells. The aim of this study is to evaluate the efficiency of PLGA/ bPEI nanoparticles in transfection and virus production. Using a modified method of producing PLGA nanoparticles, PLGA/bPEI-DNA nanoparticles were examined for transfection efficiency and virus production yield in comparison with PLGA-DNA, bPEI-DNA nanoparticles or liposome-DNA complexes. After testing various ratios of PLGA, bPEI, and DNA, the ratio of 6:3:1 (PLGA:bPEI:DNA, w/w/w was determined to be optimal, with acceptable cellular toxicity. PLGA/bPEI-DNA (6:3:1 nanoparticles showed superior transfection efficiency, especially in multiple gene transfection, and viral yield when compared with liposome-DNA complexes. The culture supernatants of HEK293FT cells transfected with PLGA/bPEI-DNA of viral constructs containing reprogramming factors (Oct4, Sox2, Klf4, or c-Myc successfully and more efficiently generated induced pluripotent stem cell colonies from mouse embryonic fibroblasts. These results strongly suggest that PLGA/bPEI-DNA nanoparticles can provide significant advantages in studying the effect of multiple factor delivery such as in reprogramming or direct conversion of cell fate.

  13. The HIST1 Locus Escapes Reprogramming in Cloned Bovine Embryos

    Directory of Open Access Journals (Sweden)

    Byungkuk Min

    2016-05-01

    Full Text Available Epigenetic reprogramming is necessary in somatic cell nuclear transfer (SCNT embryos in order to erase the differentiation-associated epigenetic marks of donor cells. However, such epigenetic memories often persist throughout the course of clonal development, thus decreasing cloning efficiency. Here, we explored reprogramming-refractory regions in bovine SCNT blastocyst transcriptomes. We observed that histone genes residing in the 1.5 Mb spanning the cow HIST1 cluster were coordinately downregulated in SCNT blastocysts. In contrast, both the nonhistone genes of this cluster, and histone genes elsewhere remained unaffected. This indicated that the downregulation was specific to HIST1 histone genes. We found that, after trichostatin A treatment, HIST1 histone genes were derepressed, and DNA methylation at their promoters was decreased to the level of in vitro fertilization embryos. Therefore, our results indicate that the reduced expression of HIST1 histone genes is a consequence of poor epigenetic reprogramming in SCNT blastocysts.

  14. Chemically Induced Reprogramming of Somatic Cells to Pluripotent Stem Cells and Neural Cells.

    Science.gov (United States)

    Biswas, Dhruba; Jiang, Peng

    2016-02-06

    The ability to generate transplantable neural cells in a large quantity in the laboratory is a critical step in the field of developing stem cell regenerative medicine for neural repair. During the last few years, groundbreaking studies have shown that cell fate of adult somatic cells can be reprogrammed through lineage specific expression of transcription factors (TFs)-and defined culture conditions. This key concept has been used to identify a number of potent small molecules that could enhance the efficiency of reprogramming with TFs. Recently, a growing number of studies have shown that small molecules targeting specific epigenetic and signaling pathways can replace all of the reprogramming TFs. Here, we provide a detailed review of the studies reporting the generation of chemically induced pluripotent stem cells (ciPSCs), neural stem cells (ciNSCs), and neurons (ciN). We also discuss the main mechanisms of actions and the pathways that the small molecules regulate during chemical reprogramming.

  15. Factor-Reduced Human Induced Pluripotent Stem Cells Efficiently Differentiate into Neurons Independent of the Number of Reprogramming Factors

    Directory of Open Access Journals (Sweden)

    Andreas Hermann

    2016-01-01

    Full Text Available Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs by overexpression of the transcription factors OCT4, SOX2, KLF4, and c-Myc holds great promise for the development of personalized cell replacement therapies. In an attempt to minimize the risk of chromosomal disruption and to simplify reprogramming, several studies demonstrated that a reduced set of reprogramming factors is sufficient to generate iPSC. We recently showed that a reduction of reprogramming factors in murine cells not only reduces reprogramming efficiency but also may worsen subsequent differentiation. To prove whether this is also true for human cells, we compared the efficiency of neuronal differentiation of iPSC generated from fetal human neural stem cells with either one (OCT4; hiPSC1F-NSC or two (OCT4, KLF4; hiPSC2F-NSC reprogramming factors with iPSC produced from human fibroblasts using three (hiPSC3F-FIB or four reprogramming factors (hiPSC4F-FIB. After four weeks of coculture with PA6 stromal cells, neuronal differentiation of hiPSC1F-NSC and hiPSC2F-NSC was as efficient as iPSC3F-FIB or iPSC4F-FIB. We conclude that a reduction of reprogramming factors in human cells does reduce reprogramming efficiency but does not alter subsequent differentiation into neural lineages. This is of importance for the development of future application of iPSC in cell replacement therapies.

  16. precise delta extraction scheme for reprogramming of wireless

    African Journals Online (AJOL)

    eobe

    Keywords- reprogramming; operating system, wireless sensor network, Delta. 1. INTRODUCTION ... It entails the transmission of only modified modules that are then ... higher power consumption and slow system execution are drawbacks ...

  17. Epigenetics of cell fate reprogramming and its implications for neurological disorders modelling.

    Science.gov (United States)

    Grzybek, Maciej; Golonko, Aleksandra; Walczak, Marta; Lisowski, Pawel

    2017-03-01

    The reprogramming of human induced pluripotent stem cells (hiPSCs) proceeds in a stepwise manner with reprogramming factors binding and epigenetic composition changes during transition to maintain the epigenetic landscape, important for pluripotency. There arises a question as to whether the aberrant epigenetic state after reprogramming leads to epigenetic defects in induced stem cells causing unpredictable long term effects in differentiated cells. In this review, we present a comprehensive view of epigenetic alterations accompanying reprogramming, cell maintenance and differentiation as factors that influence applications of hiPSCs in stem cell based technologies. We conclude that sample heterogeneity masks DNA methylation signatures in subpopulations of cells and thus believe that beside a genetic evaluation, extensive epigenomic screening should become a standard procedure to ensure hiPSCs state before they are used for genome editing and differentiation into neurons of interest. In particular, we suggest that exploitation of the single-cell composition of the epigenome will provide important insights into heterogeneity within hiPSCs subpopulations to fast forward development of reliable hiPSC-based analytical platforms in neurological disorders modelling and before completed hiPSC technology will be implemented in clinical approaches. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Induced pluripotent stem cells reprogramming: Epigenetics and applications in the regenerative medicine

    Directory of Open Access Journals (Sweden)

    Kátia Maria Sampaio Gomes

    Full Text Available Summary Induced pluripotent stem cells (iPSCs are somatic cells reprogrammed into an embryonic-like pluripotent state by the expression of specific transcription factors. iPSC technology is expected to revolutionize regenerative medicine in the near future. Despite the fact that these cells have the capacity to self-renew, they present low efficiency of reprogramming. Recent studies have demonstrated that the previous somatic epigenetic signature is a limiting factor in iPSC performance. Indeed, the process of effective reprogramming involves a complete remodeling of the existing somatic epigenetic memory, followed by the establishment of a "new epigenetic signature" that complies with the new type of cell to be differentiated. Therefore, further investigations of epigenetic modifications associated with iPSC reprogramming are required in an attempt to improve their self-renew capacity and potency, as well as their application in regenerative medicine, with a new strategy to reduce the damage in degenerative diseases. Our review aimed to summarize the most recent findings on epigenetics and iPSC, focusing on DNA methylation, histone modifications and microRNAs, highlighting their potential in translating cell therapy into clinics.

  19. (Re-)programming of subtype specific cardiomyocytes.

    Science.gov (United States)

    Hausburg, Frauke; Jung, Julia Jeannine; Hoch, Matti; Wolfien, Markus; Yavari, Arash; Rimmbach, Christian; David, Robert

    2017-10-01

    Adult cardiomyocytes (CMs) possess a highly restricted intrinsic regenerative potential - a major barrier to the effective treatment of a range of chronic degenerative cardiac disorders characterized by cellular loss and/or irreversible dysfunction and which underlies the majority of deaths in developed countries. Both stem cell programming and direct cell reprogramming hold promise as novel, potentially curative approaches to address this therapeutic challenge. The advent of induced pluripotent stem cells (iPSCs) has introduced a second pluripotent stem cell source besides embryonic stem cells (ESCs), enabling even autologous cardiomyocyte production. In addition, the recent achievement of directly reprogramming somatic cells into cardiomyocytes is likely to become of great importance. In either case, different clinical scenarios will require the generation of highly pure, specific cardiac cellular-subtypes. In this review, we discuss these themes as related to the cardiovascular stem cell and programming field, including a focus on the emergent topic of pacemaker cell generation for the development of biological pacemakers and in vitro drug testing. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Enhanced human somatic cell reprogramming efficiency by fusion of the MYC transactivation domain and OCT4

    Directory of Open Access Journals (Sweden)

    Ling Wang

    2017-12-01

    Full Text Available The development of human induced pluripotent stem cells (iPSCs holds great promise for regenerative medicine. However the iPSC induction efficiency is still very low and with lengthy reprogramming process. We utilized the highly potent transactivation domain (TAD of MYC protein to engineer the human OCT4 fusion proteins. Applying the MYC-TAD-OCT4 fusion proteins in mouse iPSC generation leads to shorter reprogramming dynamics, with earlier activation of pluripotent markers in reprogrammed cells than wild type OCT4 (wt-OCT4. Dramatic enhancement of iPSC colony induction efficiency and shortened reprogramming dynamics were observed when these MYC-TAD-OCT4 fusion proteins were used to reprogram primary human cells. The OCT4 fusion proteins induced human iPSCs are pluripotent. We further show that the MYC Box I (MBI is dispensable while both MBII and the linking region between MBI/II are essential for the enhanced reprogramming activity of MYC-TAD-OCT4 fusion protein. Consistent with an enhanced transcription activity, the engineered OCT4 significantly stimulated the expression of genes specifically targeted by OCT4-alone, OCT4/SOX2, and OCT4/SOX2/KLF4 during human iPSC induction, compared with the wt-OCT4. The MYC-TAD-OCT4 fusion proteins we generated will be valuable tools for studying the reprogramming mechanisms and for efficient iPSC generation for humans as well as for other species.

  1. SCL, LMO1 and Notch1 Reprogram Thymocytes into Self-Renewing Cells

    Science.gov (United States)

    Rojas-Sutterlin, Shanti; Herblot, Sabine; Hébert, Josée; Sauvageau, Guy; Lemieux, Sébastien; Lécuyer, Eric; Veiga, Diogo F. T.; Hoang, Trang

    2014-01-01

    The molecular determinants that render specific populations of normal cells susceptible to oncogenic reprogramming into self-renewing cancer stem cells are poorly understood. Here, we exploit T-cell acute lymphoblastic leukemia (T-ALL) as a model to define the critical initiating events in this disease. First, thymocytes that are reprogrammed by the SCL and LMO1 oncogenic transcription factors into self-renewing pre-leukemic stem cells (pre-LSCs) remain non-malignant, as evidenced by their capacities to generate functional T cells. Second, we provide strong genetic evidence that SCL directly interacts with LMO1 to activate the transcription of a self-renewal program coordinated by LYL1. Moreover, LYL1 can substitute for SCL to reprogram thymocytes in concert with LMO1. In contrast, inhibition of E2A was not sufficient to substitute for SCL, indicating that thymocyte reprogramming requires transcription activation by SCL-LMO1. Third, only a specific subset of normal thymic cells, known as DN3 thymocytes, is susceptible to reprogramming. This is because physiological NOTCH1 signals are highest in DN3 cells compared to other thymocyte subsets. Consistent with this, overexpression of a ligand-independent hyperactive NOTCH1 allele in all immature thymocytes is sufficient to sensitize them to SCL-LMO1, thereby increasing the pool of self-renewing cells. Surprisingly, hyperactive NOTCH1 cannot reprogram thymocytes on its own, despite the fact that NOTCH1 is activated by gain of function mutations in more than 55% of T-ALL cases. Rather, elevating NOTCH1 triggers a parallel pathway involving Hes1 and Myc that dramatically enhances the activity of SCL-LMO1 We conclude that the acquisition of self-renewal and the genesis of pre-LSCs from thymocytes with a finite lifespan represent a critical first event in T-ALL. Finally, LYL1 and LMO1 or LMO2 are co-expressed in most human T-ALL samples, except the cortical T subtype. We therefore anticipate that the self-renewal network

  2. Reprogramming suppresses premature senescence phenotypes of Werner syndrome cells and maintains chromosomal stability over long-term culture.

    Science.gov (United States)

    Shimamoto, Akira; Kagawa, Harunobu; Zensho, Kazumasa; Sera, Yukihiro; Kazuki, Yasuhiro; Osaki, Mitsuhiko; Oshimura, Mitsuo; Ishigaki, Yasuhito; Hamasaki, Kanya; Kodama, Yoshiaki; Yuasa, Shinsuke; Fukuda, Keiichi; Hirashima, Kyotaro; Seimiya, Hiroyuki; Koyama, Hirofumi; Shimizu, Takahiko; Takemoto, Minoru; Yokote, Koutaro; Goto, Makoto; Tahara, Hidetoshi

    2014-01-01

    Werner syndrome (WS) is a premature aging disorder characterized by chromosomal instability and cancer predisposition. Mutations in WRN are responsible for the disease and cause telomere dysfunction, resulting in accelerated aging. Recent studies have revealed that cells from WS patients can be successfully reprogrammed into induced pluripotent stem cells (iPSCs). In the present study, we describe the effects of long-term culture on WS iPSCs, which acquired and maintained infinite proliferative potential for self-renewal over 2 years. After long-term cultures, WS iPSCs exhibited stable undifferentiated states and differentiation capacity, and premature upregulation of senescence-associated genes in WS cells was completely suppressed in WS iPSCs despite WRN deficiency. WS iPSCs also showed recapitulation of the phenotypes during differentiation. Furthermore, karyotype analysis indicated that WS iPSCs were stable, and half of the descendant clones had chromosomal profiles that were similar to those of parental cells. These unexpected properties might be achieved by induced expression of endogenous telomerase gene during reprogramming, which trigger telomerase reactivation leading to suppression of both replicative senescence and telomere dysfunction in WS cells. These findings demonstrated that reprogramming suppressed premature senescence phenotypes in WS cells and WS iPSCs could lead to chromosomal stability over the long term. WS iPSCs will provide opportunities to identify affected lineages in WS and to develop a new strategy for the treatment of WS.

  3. Reprogramming suppresses premature senescence phenotypes of Werner syndrome cells and maintains chromosomal stability over long-term culture.

    Directory of Open Access Journals (Sweden)

    Akira Shimamoto

    Full Text Available Werner syndrome (WS is a premature aging disorder characterized by chromosomal instability and cancer predisposition. Mutations in WRN are responsible for the disease and cause telomere dysfunction, resulting in accelerated aging. Recent studies have revealed that cells from WS patients can be successfully reprogrammed into induced pluripotent stem cells (iPSCs. In the present study, we describe the effects of long-term culture on WS iPSCs, which acquired and maintained infinite proliferative potential for self-renewal over 2 years. After long-term cultures, WS iPSCs exhibited stable undifferentiated states and differentiation capacity, and premature upregulation of senescence-associated genes in WS cells was completely suppressed in WS iPSCs despite WRN deficiency. WS iPSCs also showed recapitulation of the phenotypes during differentiation. Furthermore, karyotype analysis indicated that WS iPSCs were stable, and half of the descendant clones had chromosomal profiles that were similar to those of parental cells. These unexpected properties might be achieved by induced expression of endogenous telomerase gene during reprogramming, which trigger telomerase reactivation leading to suppression of both replicative senescence and telomere dysfunction in WS cells. These findings demonstrated that reprogramming suppressed premature senescence phenotypes in WS cells and WS iPSCs could lead to chromosomal stability over the long term. WS iPSCs will provide opportunities to identify affected lineages in WS and to develop a new strategy for the treatment of WS.

  4. MicroRNAs Induce Epigenetic Reprogramming and Suppress Malignant Phenotypes of Human Colon Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Hisataka Ogawa

    Full Text Available Although cancer is a genetic disease, epigenetic alterations are involved in its initiation and progression. Previous studies have shown that reprogramming of colon cancer cells using Oct3/4, Sox2, Klf4, and cMyc reduces cancer malignancy. Therefore, cancer reprogramming may be a useful treatment for chemo- or radiotherapy-resistant cancer cells. It was also reported that the introduction of endogenous small-sized, non-coding ribonucleotides such as microRNA (miR 302s and miR-369-3p or -5p resulted in the induction of cellular reprogramming. miRs are smaller than the genes of transcription factors, making them possibly suitable for use in clinical strategies. Therefore, we reprogrammed colon cancer cells using miR-302s and miR-369-3p or -5p. This resulted in inhibition of cell proliferation and invasion and the stimulation of the mesenchymal-to-epithelial transition phenotype in colon cancer cells. Importantly, the introduction of the ribonucleotides resulted in epigenetic reprogramming of DNA demethylation and histone modification events. Furthermore, in vivo administration of the ribonucleotides in mice elicited the induction of cancer cell apoptosis, which involves the mitochondrial Bcl2 protein family. The present study shows that the introduction of miR-302s and miR-369s could induce cellular reprogramming and modulate malignant phenotypes of human colorectal cancer, suggesting that the appropriate delivery of functional small-sized ribonucleotides may open a new avenue for therapy against human malignant tumors.

  5. Mesenchymal to Epithelial Transition Mediated by CDH1 Promotes Spontaneous Reprogramming of Male Germline Stem Cells to Pluripotency

    Directory of Open Access Journals (Sweden)

    Junhui An

    2017-02-01

    Full Text Available Cultured spermatogonial stem cells (GSCs can spontaneously form pluripotent cells in certain culture conditions. However, GSC reprogramming is a rare event that is largely unexplained. We show GSCs have high expression of mesenchymal to epithelial transition (MET suppressors resulting in a developmental barrier inhibiting GSC reprogramming. Either increasing OCT4 or repressing transforming growth factor β (TGF-β signaling promotes GSC reprogramming by upregulating CDH1 and boosting MET. Reducing ZEB1 also enhances GSC reprogramming through its direct effect on CDH1. RNA sequencing shows that rare GSCs, identified as CDH1+ after trypsin digestion, are epithelial-like cells. CDH1+ GSCs exhibit enhanced reprogramming and become more prevalent during the course of reprogramming. Our results provide a mechanistic explanation for the spontaneous emergence of pluripotent cells from GSC cultures; namely, rare GSCs upregulate CDH1 and initiate MET, processes normally kept in check by ZEB1 and TGF-β signaling, thereby ensuring germ cells are protected from aberrant acquisition of pluripotency.

  6. Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation

    Science.gov (United States)

    Plate, Lars; Cooley, Christina B; Chen, John J; Paxman, Ryan J; Gallagher, Ciara M; Madoux, Franck; Genereux, Joseph C; Dobbs, Wesley; Garza, Dan; Spicer, Timothy P; Scampavia, Louis; Brown, Steven J; Rosen, Hugh; Powers, Evan T; Walter, Peter; Hodder, Peter; Wiseman, R Luke; Kelly, Jeffery W

    2016-01-01

    Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases. DOI: http://dx.doi.org/10.7554/eLife.15550.001 PMID:27435961

  7. Reprogramming Methods Do Not Affect Gene Expression Profile of Human Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Trevisan, Marta; Desole, Giovanna; Costanzi, Giulia; Lavezzo, Enrico; Palù, Giorgio; Barzon, Luisa

    2017-01-20

    Induced pluripotent stem cells (iPSCs) are pluripotent cells derived from adult somatic cells. After the pioneering work by Yamanaka, who first generated iPSCs by retroviral transduction of four reprogramming factors, several alternative methods to obtain iPSCs have been developed in order to increase the yield and safety of the process. However, the question remains open on whether the different reprogramming methods can influence the pluripotency features of the derived lines. In this study, three different strategies, based on retroviral vectors, episomal vectors, and Sendai virus vectors, were applied to derive iPSCs from human fibroblasts. The reprogramming efficiency of the methods based on episomal and Sendai virus vectors was higher than that of the retroviral vector-based approach. All human iPSC clones derived with the different methods showed the typical features of pluripotent stem cells, including the expression of alkaline phosphatase and stemness maker genes, and could give rise to the three germ layer derivatives upon embryoid bodies assay. Microarray analysis confirmed the presence of typical stem cell gene expression profiles in all iPSC clones and did not identify any significant difference among reprogramming methods. In conclusion, the use of different reprogramming methods is equivalent and does not affect gene expression profile of the derived human iPSCs.

  8. Hierarchical Oct4 Binding in Concert with Primed Epigenetic Rearrangements during Somatic Cell Reprogramming

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    Jun Chen

    2016-02-01

    Full Text Available The core pluripotency factor Oct4 plays key roles in somatic cell reprogramming through transcriptional control. Here, we profile Oct4 occupancy, epigenetic changes, and gene expression in reprogramming. We find that Oct4 binds in a hierarchical manner to target sites with primed epigenetic modifications. Oct4 binding is temporally continuous and seldom switches between bound and unbound. Oct4 occupancy in most of promoters is maintained throughout the entire reprogramming process. In contrast, somatic cell-specific enhancers are silenced in the early and intermediate stages, whereas stem cell-specific enhancers are activated in the late stage in parallel with cell fate transition. Both epigenetic remodeling and Oct4 binding contribute to the hyperdynamic enhancer signature transitions. The hierarchical Oct4 bindings are associated with distinct functional themes at different stages. Collectively, our results provide a comprehensive molecular roadmap of Oct4 binding in concert with epigenetic rearrangements and rich resources for future reprogramming studies.

  9. Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency

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    Cacchiarelli, Davide; Trapnell, Cole; Ziller, Michael J.; Soumillon, Magali; Cesana, Marcella; Karnik, Rahul; Donaghey, Julie; Smith, Zachary D.; Ratanasirintrawoot, Sutheera; Zhang, Xiaolan; Ho Sui, Shannan J.; Wu, Zhaoting; Akopian, Veronika; Gifford, Casey A.; Doench, John; Rinn, John L.; Daley, George Q.; Meissner, Alexander; Lander, Eric S.; Mikkelsen, Tarjei S.

    2015-01-01

    Summary Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered reactivation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. PMID:26186193

  10. Direct Cardiac Reprogramming: Advances in Cardiac Regeneration

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    Olivia Chen

    2015-01-01

    Full Text Available Heart disease is one of the lead causes of death worldwide. Many forms of heart disease, including myocardial infarction and pressure-loading cardiomyopathies, result in irreversible cardiomyocyte death. Activated fibroblasts respond to cardiac injury by forming scar tissue, but ultimately this response fails to restore cardiac function. Unfortunately, the human heart has little regenerative ability and long-term outcomes following acute coronary events often include chronic and end-stage heart failure. Building upon years of research aimed at restoring functional cardiomyocytes, recent advances have been made in the direct reprogramming of fibroblasts toward a cardiomyocyte cell fate both in vitro and in vivo. Several experiments show functional improvements in mouse models of myocardial infarction following in situ generation of cardiomyocyte-like cells from endogenous fibroblasts. Though many of these studies are in an early stage, this nascent technology holds promise for future applications in regenerative medicine. In this review, we discuss the history, progress, methods, challenges, and future directions of direct cardiac reprogramming.

  11. Efficient generation of fully reprogrammed human iPS cells via polycistronic retroviral vector and a new cocktail of chemical compounds.

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    Zhonghui Zhang

    Full Text Available Direct reprogramming of human somatic cells into induced pluripotent stem (iPS cells by defined transcription factors (TFs provides great potential for regenerative medicine and biomedical research. This procedure has many challenges, including low reprogramming efficiency, many partially reprogrammed colonies, somatic coding mutations in the genome, etc. Here, we describe a simple approach for generating fully reprogrammed human iPS cells by using a single polycistronic retroviral vector expressing four human TFs in a single open reading frame (ORF, combined with a cocktail containing three small molecules (Sodium butyrate, SB431542, and PD0325901. Our results demonstrate that human iPS cells generated by this approach express human ES cells markers and exhibit pluripotency demonstrated by their abilities to differentiate into the three germ layers in vitro and in vivo. Notably, this approach not only provides a much faster reprogramming process but also significantly diminishes partially reprogrammed iPS cell colonies, thus facilitating efficient isolation of desired fully reprogrammed iPS cell colonies.

  12. Fluctuating levels of reprogramming factor expression in cultured ...

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    pluripotent stem cells (iPSCs) with high efficiency and rapid kinetics by transducing reprogramming factors (RFs), the ... could serve as disease models and aid in the discovery of drugs and genes; furthermore, this approach to gene-.

  13. Improved methods for reprogramming human dermal fibroblasts using fluorescence activated cell sorting.

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    David J Kahler

    Full Text Available Current methods to derive induced pluripotent stem cell (iPSC lines from human dermal fibroblasts by viral infection rely on expensive and lengthy protocols. One major factor contributing to the time required to derive lines is the ability of researchers to identify fully reprogrammed unique candidate clones from a mixed cell population containing transformed or partially reprogrammed cells and fibroblasts at an early time point post infection. Failure to select high quality colonies early in the derivation process results in cell lines that require increased maintenance and unreliable experimental outcomes. Here, we describe an improved method for the derivation of iPSC lines using fluorescence activated cell sorting (FACS to isolate single cells expressing the cell surface marker signature CD13(NEGSSEA4(POSTra-1-60(POS on day 7-10 after infection. This technique prospectively isolates fully reprogrammed iPSCs, and depletes both parental and "contaminating" partially reprogrammed fibroblasts, thereby substantially reducing the time and reagents required to generate iPSC lines without the use of defined small molecule cocktails. FACS derived iPSC lines express common markers of pluripotency, and possess spontaneous differentiation potential in vitro and in vivo. To demonstrate the suitability of FACS for high-throughput iPSC generation, we derived 228 individual iPSC lines using either integrating (retroviral or non- integrating (Sendai virus reprogramming vectors and performed extensive characterization on a subset of those lines. The iPSC lines used in this study were derived from 76 unique samples from a variety of tissue sources, including fresh or frozen fibroblasts generated from biopsies harvested from healthy or disease patients.

  14. Nuclear reprogramming: kinetics of cell cycle and metabolic progression as determinants of success.

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    Sebastian Thomas Balbach

    Full Text Available Establishment of totipotency after somatic cell nuclear transfer (NT requires not only reprogramming of gene expression, but also conversion of the cell cycle from quiescence to the precisely timed sequence of embryonic cleavage. Inadequate adaptation of the somatic nucleus to the embryonic cell cycle regime may lay the foundation for NT embryo failure and their reported lower cell counts. We combined bright field and fluorescence imaging of histone H(2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This allowed us to quantitatively analyze cleavage kinetics of cloned embryos and revealed an extended and inconstant duration of the second and third cell cycles compared to fertilized controls generated by intracytoplasmic sperm injection (ICSI. Compared to fertilized embryos, slow and fast cleaving NT embryos presented similar rates of errors in M phase, but were considerably less tolerant to mitotic errors and underwent cleavage arrest. Although NT embryos vary substantially in their speed of cell cycle progression, transcriptome analysis did not detect systematic differences between fast and slow NT embryos. Profiling of amino acid turnover during pre-implantation development revealed that NT embryos consume lower amounts of amino acids, in particular arginine, than fertilized embryos until morula stage. An increased arginine supplementation enhanced development to blastocyst and increased embryo cell numbers. We conclude that a cell cycle delay, which is independent of pluripotency marker reactivation, and metabolic restraints reduce cell counts of NT embryos and impede their development.

  15. Cellular reprogramming through mitogen-activated protein kinases

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    Justin eLee

    2015-10-01

    Full Text Available Mitogen-activated protein kinase (MAPK cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554 in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression – including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding and degradation steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

  16. Reprogrammed Functional Brown Adipocytes Ameliorate Insulin Resistance and Dyslipidemia in Diet-Induced Obesity and Type 2 Diabetes

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    Tsunao Kishida

    2015-10-01

    Full Text Available Brown adipocytes (BAs play important roles in body temperature regulation, energy balance, and carbohydrate and lipid metabolism. Activities of BAs are remarkably diminished in obese and diabetic patients, providing possibilities of transplanting functional BAs resulting in therapeutic benefit. Here, we show generation of functional BAs by cellular reprogramming procedures. Transduction of the PRDM16 gene into iPSC-derived embryoid bodies induced BA phenotypes (iBAs. Moreover, normal human fibroblasts were directly converted into BAs (dBAs by C/EBP-β and C-MYC gene transduction. Approximately 90% of the fibroblasts were successfully converted within 12 days. The dBAs were highly active in mitochondrial biogenesis and oxidative metabolism. Mouse dBAs were induced by Prdm16, C/ebp-β, and L-myc genes, and after transplantation, they significantly reduced diet-induced obesity and insulin resistance in an UCP1-dependent manner. Thus, highly functional BAs can be generated by cellular reprogramming, suggesting a promising tailor-made cell therapy against metabolic disorders including type 2 diabetes mellitus.

  17. Reprogrammed Functional Brown Adipocytes Ameliorate Insulin Resistance and Dyslipidemia in Diet-Induced Obesity and Type 2 Diabetes.

    Science.gov (United States)

    Kishida, Tsunao; Ejima, Akika; Yamamoto, Kenta; Tanaka, Seiji; Yamamoto, Toshiro; Mazda, Osam

    2015-10-13

    Brown adipocytes (BAs) play important roles in body temperature regulation, energy balance, and carbohydrate and lipid metabolism. Activities of BAs are remarkably diminished in obese and diabetic patients, providing possibilities of transplanting functional BAs resulting in therapeutic benefit. Here, we show generation of functional BAs by cellular reprogramming procedures. Transduction of the PRDM16 gene into iPSC-derived embryoid bodies induced BA phenotypes (iBAs). Moreover, normal human fibroblasts were directly converted into BAs (dBAs) by C/EBP-β and C-MYC gene transduction. Approximately 90% of the fibroblasts were successfully converted within 12 days. The dBAs were highly active in mitochondrial biogenesis and oxidative metabolism. Mouse dBAs were induced by Prdm16, C/ebp-β, and L-myc genes, and after transplantation, they significantly reduced diet-induced obesity and insulin resistance in an UCP1-dependent manner. Thus, highly functional BAs can be generated by cellular reprogramming, suggesting a promising tailor-made cell therapy against metabolic disorders including type 2 diabetes mellitus. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Elixir of Life: Thwarting Aging With Regenerative Reprogramming.

    Science.gov (United States)

    Beyret, Ergin; Martinez Redondo, Paloma; Platero Luengo, Aida; Izpisua Belmonte, Juan Carlos

    2018-01-05

    All living beings undergo systemic physiological decline after ontogeny, characterized as aging. Modern medicine has increased the life expectancy, yet this has created an aged society that has more predisposition to degenerative disorders. Therefore, novel interventions that aim to extend the healthspan in parallel to the life span are needed. Regeneration ability of living beings maintains their biological integrity and thus is the major leverage against aging. However, mammalian regeneration capacity is low and further declines during aging. Therefore, modalities that reinforce regeneration can antagonize aging. Recent advances in the field of regenerative medicine have shown that aging is not an irreversible process. Conversion of somatic cells to embryonic-like pluripotent cells demonstrated that the differentiated state and age of a cell is not fixed. Identification of the pluripotency-inducing factors subsequently ignited the idea that cellular features can be reprogrammed by defined factors that specify the desired outcome. The last decade consequently has witnessed a plethora of studies that modify cellular features including the hallmarks of aging in addition to cellular function and identity in a variety of cell types in vitro. Recently, some of these reprogramming strategies have been directly used in animal models in pursuit of rejuvenation and cell replacement. Here, we review these in vivo reprogramming efforts and discuss their potential use to extend the longevity by complementing or augmenting the regenerative capacity. © 2017 American Heart Association, Inc.

  19. Rationale and Methodology of Reprogramming for Generation of Induced Pluripotent Stem Cells and Induced Neural Progenitor Cells

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    Zuojun Tian

    2016-04-01

    Full Text Available Great progress has been made regarding the capabilities to modify somatic cell fate ever since the technology for generation of induced pluripotent stem cells (iPSCs was discovered in 2006. Later, induced neural progenitor cells (iNPCs were generated from mouse and human cells, bypassing some of the concerns and risks of using iPSCs in neuroscience applications. To overcome the limitation of viral vector induced reprogramming, bioactive small molecules (SM have been explored to enhance the efficiency of reprogramming or even replace transcription factors (TFs, making the reprogrammed cells more amenable to clinical application. The chemical induced reprogramming process is a simple process from a technical perspective, but the choice of SM at each step is vital during the procedure. The mechanisms underlying cell transdifferentiation are still poorly understood, although, several experimental data and insights have indicated the rationale of cell reprogramming. The process begins with the forced expression of specific TFs or activation/inhibition of cell signaling pathways by bioactive chemicals in defined culture condition, which initiates the further reactivation of endogenous gene program and an optimal stoichiometric expression of the endogenous pluri- or multi-potency genes, and finally leads to the birth of reprogrammed cells such as iPSCs and iNPCs. In this review, we first outline the rationale and discuss the methodology of iPSCs and iNPCs in a stepwise manner; and then we also discuss the chemical-based reprogramming of iPSCs and iNPCs.

  20. Reserve stem cells: Reprogramming of differentiated cells fuels repair, metaplasia, and neoplasia in the adult gastrointestinal tract

    Science.gov (United States)

    Mills, Jason C.; Sansom, Owen J.

    2016-01-01

    It has long been known that differentiated cells can switch fates, especially in vitro, but only recently has there been a critical mass of publications describing the mechanisms adult, post-mitotic cells use in vivo to reverse their differentiation state. We propose that this sort of cellular reprogramming is a fundamental cellular process akin to apoptosis or mitosis. Because reprogramming can invoke regenerative cells from mature cells, it is critical to the longterm maintenance of tissues like the pancreas, which encounter large insults during adulthood but lack constitutively active adult stem cells to repair the damage. However, even in tissues with adult stem cells, like stomach and intestine, reprogramming may allow mature cells to serve as reserve (“quiescent”) stem cells when normal stem cells are compromised. We propose that the potential downside to reprogramming is that it increases risk for cancers that occur late in adulthood. Mature, long-lived cells may have years of exposure to mutagens. Mutations that affect the physiological function of differentiated, post-mitotic cells may lead to apoptosis, but mutations in genes that govern proliferation might not be selected against. Hence, reprogramming with reentry into the cell cycle might unmask those mutations, causing an irreversible progenitor-like, proliferative state. We review recent evidence showing that reprogramming fuels irreversible metaplastic and precancerous proliferations in stomach and pancreas. Finally, we illustrate how we think reprogrammed differentiated cells are likely candidates as cells of origin for cancers of the intestine. PMID:26175494

  1. Histone deacetylase inhibitor valproic acid promotes the induction of pluripotency in mouse fibroblasts by suppressing reprogramming-induced senescence stress

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    Zhai, Yingying; Chen, Xi; Yu, Dehai [Stem Cell and Cancer Center, First Affiliated Hospital, Jilin University, Changchun, Jilin 130061 (China); Stanford University Medical School, Palo Alto Veterans Institute for Research, Palo Alto, CA 94304 (United States); Li, Tao [Stanford University Medical School, Palo Alto Veterans Institute for Research, Palo Alto, CA 94304 (United States); Cui, Jiuwei; Wang, Guanjun [Stem Cell and Cancer Center, First Affiliated Hospital, Jilin University, Changchun, Jilin 130061 (China); Hu, Ji-Fan, E-mail: jifan@stanford.edu [Stem Cell and Cancer Center, First Affiliated Hospital, Jilin University, Changchun, Jilin 130061 (China); Stanford University Medical School, Palo Alto Veterans Institute for Research, Palo Alto, CA 94304 (United States); Li, Wei, E-mail: jdyylw@163.com [Stem Cell and Cancer Center, First Affiliated Hospital, Jilin University, Changchun, Jilin 130061 (China)

    2015-09-10

    Histone deacetylase inhibitor valproic acid (VPA) has been used to increase the reprogramming efficiency of induced pluripotent stem cell (iPSC) from somatic cells, yet the specific molecular mechanisms underlying this effect is unknown. Here, we demonstrate that reprogramming with lentiviruses carrying the iPSC-inducing factors (Oct4-Sox2-Klf4-cMyc, OSKM) caused senescence in mouse fibroblasts, establishing a stress barrier for cell reprogramming. Administration of VPA protected cells from reprogramming-induced senescent stress. Using an in vitro pre-mature senescence model, we found that VPA treatment increased cell proliferation and inhibited apoptosis through the suppression of the p16/p21 pathway. In addition, VPA also inhibited the G2/M phase blockage derived from the senescence stress. These findings highlight the role of VPA in breaking the cell senescence barrier required for the induction of pluripotency. - Highlights: • Histone deacetylase inhibitor valproic acid enhances iPSC induction. • Valproic acid suppresses reprogramming-induced senescence stress. • Valproic acid downregulates the p16/p21 pathway in reprogramming. • This study demonstrates a new mechanistic role of valproic acid in enhancing reprogramming.

  2. Reprogramming Methods Do Not Affect Gene Expression Profile of Human Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Marta Trevisan

    2017-01-01

    Full Text Available Induced pluripotent stem cells (iPSCs are pluripotent cells derived from adult somatic cells. After the pioneering work by Yamanaka, who first generated iPSCs by retroviral transduction of four reprogramming factors, several alternative methods to obtain iPSCs have been developed in order to increase the yield and safety of the process. However, the question remains open on whether the different reprogramming methods can influence the pluripotency features of the derived lines. In this study, three different strategies, based on retroviral vectors, episomal vectors, and Sendai virus vectors, were applied to derive iPSCs from human fibroblasts. The reprogramming efficiency of the methods based on episomal and Sendai virus vectors was higher than that of the retroviral vector-based approach. All human iPSC clones derived with the different methods showed the typical features of pluripotent stem cells, including the expression of alkaline phosphatase and stemness maker genes, and could give rise to the three germ layer derivatives upon embryoid bodies assay. Microarray analysis confirmed the presence of typical stem cell gene expression profiles in all iPSC clones and did not identify any significant difference among reprogramming methods. In conclusion, the use of different reprogramming methods is equivalent and does not affect gene expression profile of the derived human iPSCs.

  3. The Wnt/β-catenin signaling pathway tips the balance between apoptosis and reprograming of cell fusion hybrids.

    Science.gov (United States)

    Lluis, Frederic; Pedone, Elisa; Pepe, Stefano; Cosma, Maria Pia

    2010-11-01

    Cell-cell fusion contributes to cell differentiation and developmental processes. We have previously showed that activation of Wnt/β-catenin enhances somatic cell reprograming after polyethylene glycol (PEG)-mediated fusion. Here, we show that neural stem cells and ESCs can fuse spontaneously in cocultures, although with very low efficiency (about 2%), as the hybrids undergo apoptosis. In contrast, when Wnt/β-catenin signaling is activated in ESCs and leads to accumulation of low amounts of β-catenin in the nucleus, activated ESCs can reprogram somatic cells with very high efficiency after spontaneous fusion. Furthermore, we also show that different levels of β-catenin accumulation in the ESC nuclei can modulate cell proliferation, although in our experimental setting, cell proliferation does not modulate the reprograming efficiency per se. Overall, the present study provides evidence that spontaneous fusion occurs, while the survival of the reprogramed clones is strictly dependent on induction of a Wnt-mediated reprograming pathway. Copyright © 2010 AlphaMed Press.

  4. Generation of human β-thalassemia induced pluripotent cell lines by reprogramming of bone marrow-derived mesenchymal stromal cells using modified mRNA.

    Science.gov (United States)

    Varela, Ioanna; Karagiannidou, Angeliki; Oikonomakis, Vasilis; Tzetis, Maria; Tzanoudaki, Marianna; Siapati, Elena-Konstantina; Vassilopoulos, George; Graphakos, Stelios; Kanavakis, Emmanuel; Goussetis, Evgenios

    2014-12-01

    Synthetic modified mRNA molecules encoding pluripotency transcription factors have been used successfully in reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs). We have applied this method on bone marrow-derived mesenchymal stromal cells (BM-MSCs) obtained from a patient with β-thalassemia (β-thal) with the aim to generate trangene-free β-thal-iPSCs. Transfection of 10(4) BM-MSCs by lipofection with mRNA encoding the reprogramming factors Oct4, Klf4, Sox2, cMyc, and Lin28 resulted in formation of five iPSC colonies, from which three were picked up and expanded in β-thal-iPSC lines. After 10 serial passages in vitro, β-thal-iPSCs maintain genetic stability as shown by array comparative genomic hybridization (aCGH) and are capable of forming embryoid bodies in vitro and teratomas in vivo. Their gene expression profile compared to human embryonic stem cells (ESCs) and BM-MSCs seems to be similar to that of ESCs, whereas it differs from the profile of the parental BM-MSCs. Differentiation cultures toward a hematopoietic lineage showed the generation of CD34(+) progenitors up to 10%, but with a decreased hematopoietic colony-forming capability. In conclusion, we report herein the generation of transgene-free β-thal-iPSCs that could be widely used for disease modeling and gene therapy applications. Moreover, it was demonstrated that the mRNA-based reprogramming method, used mainly in fibroblasts, is also suitable for reprogramming of human BM-MSCs.

  5. CtIP-Specific Roles during Cell Reprogramming Have Long-Term Consequences in the Survival and Fitness of Induced Pluripotent Stem Cells

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    Daniel Gómez-Cabello

    2017-02-01

    Full Text Available Acquired genomic instability is one of the major concerns for the clinical use of induced pluripotent stem cells (iPSCs. All reprogramming methods are accompanied by the induction of DNA damage, of which double-strand breaks are the most cytotoxic and mutagenic. Consequently, DNA repair genes seem to be relevant for accurate reprogramming to minimize the impact of such DNA damage. Here, we reveal that reprogramming is associated with high levels of DNA end resection, a critical step in homologous recombination. Moreover, the resection factor CtIP is essential for cell reprogramming and establishment of iPSCs, probably to repair reprogramming-induced DNA damage. Our data reveal a new role for DNA end resection in maintaining genomic stability during cell reprogramming, allowing DNA repair fidelity to be retained in both human and mouse iPSCs. Moreover, we demonstrate that reprogramming in a resection-defective environment has long-term consequences on stem cell self-renewal and differentiation.

  6. Reserve stem cells: Differentiated cells reprogram to fuel repair, metaplasia, and neoplasia in the adult gastrointestinal tract.

    Science.gov (United States)

    Mills, Jason C; Sansom, Owen J

    2015-07-14

    It has long been known that differentiated cells can switch fates, especially in vitro, but only recently has there been a critical mass of publications describing the mechanisms adult, postmitotic cells use in vivo to reverse their differentiation state. We propose that this sort of cellular reprogramming is a fundamental cellular process akin to apoptosis or mitosis. Because reprogramming can invoke regenerative cells from mature cells, it is critical to the long-term maintenance of tissues like the pancreas, which encounter large insults during adulthood but lack constitutively active adult stem cells to repair the damage. However, even in tissues with adult stem cells, like the stomach and intestine, reprogramming may allow mature cells to serve as reserve ("quiescent") stem cells when normal stem cells are compromised. We propose that the potential downside to reprogramming is that it increases risk for cancers that occur late in adulthood. Mature, long-lived cells may have years of exposure to mutagens. Mutations that affect the physiological function of differentiated, postmitotic cells may lead to apoptosis, but mutations in genes that govern proliferation might not be selected against. Hence, reprogramming with reentry into the cell cycle might unmask those mutations, causing an irreversible progenitor-like, proliferative state. We review recent evidence showing that reprogramming fuels irreversible metaplastic and precancerous proliferation in the stomach and pancreas. Finally, we illustrate how we think reprogrammed differentiated cells are likely candidates as cells of origin for cancers of the intestine. Copyright © 2015, American Association for the Advancement of Science.

  7. In vitro reprogramming of rat bmMSCs into pancreatic endocrine-like cells.

    Science.gov (United States)

    Li, Hong-Tu; Jiang, Fang-Xu; Shi, Ping; Zhang, Tao; Liu, Xiao-Yu; Lin, Xue-Wen; San, Zhong-Yan; Pang, Xi-Ning

    2017-02-01

    Islet transplantation provides curative treatments to patients with type 1 diabetes, but donor shortage restricts the broad use of this therapy. Thus, generation of alternative transplantable cell sources is intensively investigated worldwide. We previously showed that bone marrow-derived mesenchymal stem cells (bmMSCs) can be reprogrammed to pancreatic-like cells through simultaneously forced suppression of Rest/Nrsf (repressor element-1 silencing transcription factor/neuronal restrictive silencing factor) and Shh (sonic hedgehog) and activation of Pdx1 (pancreas and duodenal transcription factor 1). We here aimed to reprogram bmMSCs further along the developmental pathway towards the islet lineages by improving our previous strategy and by overexpression of Ngn3 (neurogenin 3) and NeuroD1 (neurogenic differentiation 1), critical regulators of the development of endocrine pancreas. We showed that compared to the previous protocol, the overexpression of only Pdx1 and Ngn3 reprogrammed bmMSCs into cells with more characteristics of islet endocrine lineages verified with bioinformatic analyses of our RNA-Seq datasets. These analyses indicated 2325 differentially expressed genes including those involved in the pancreas and islet development. We validated with qRT-PCR analysis selective genes identified from the RNA-Seq datasets. Thus, we reprogrammed bmMSCs into islet endocrine-like cells and advanced the endeavor to generate surrogate functional insulin-secreting cells.

  8. Direct Reprogramming of Fibroblasts via a Chemically Induced XEN-like State.

    Science.gov (United States)

    Li, Xiang; Liu, Defang; Ma, Yantao; Du, Xiaomin; Jing, Junzhan; Wang, Lipeng; Xie, Bingqing; Sun, Da; Sun, Shaoqiang; Jin, Xueqin; Zhang, Xu; Zhao, Ting; Guan, Jingyang; Yi, Zexuan; Lai, Weifeng; Zheng, Ping; Huang, Zhuo; Chang, Yanzhong; Chai, Zhen; Xu, Jun; Deng, Hongkui

    2017-08-03

    Direct lineage reprogramming, including with small molecules, has emerged as a promising approach for generating desired cell types. We recently found that during chemical induction of induced pluripotent stem cells (iPSCs) from mouse fibroblasts, cells pass through an extra-embryonic endoderm (XEN)-like state. Here, we show that these chemically induced XEN-like cells can also be induced to directly reprogram into functional neurons, bypassing the pluripotent state. The induced neurons possess neuron-specific expression profiles, form functional synapses in culture, and further mature after transplantation into the adult mouse brain. Using similar principles, we were also able to induce hepatocyte-like cells from the XEN-like cells. Cells in the induced XEN-like state were readily expandable over at least 20 passages and retained genome stability and lineage specification potential. Our study therefore establishes a multifunctional route for chemical lineage reprogramming and may provide a platform for generating a diverse range of cell types via application of this expandable XEN-like state. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Chemical compound-based direct reprogramming for future clinical applications

    Science.gov (United States)

    Takeda, Yukimasa; Harada, Yoshinori; Yoshikawa, Toshikazu; Dai, Ping

    2018-01-01

    Recent studies have revealed that a combination of chemical compounds enables direct reprogramming from one somatic cell type into another without the use of transgenes by regulating cellular signaling pathways and epigenetic modifications. The generation of induced pluripotent stem (iPS) cells generally requires virus vector-mediated expression of multiple transcription factors, which might disrupt genomic integrity and proper cell functions. The direct reprogramming is a promising alternative to rapidly prepare different cell types by bypassing the pluripotent state. Because the strategy also depends on forced expression of exogenous lineage-specific transcription factors, the direct reprogramming in a chemical compound-based manner is an ideal approach to further reduce the risk for tumorigenesis. So far, a number of reported research efforts have revealed that combinations of chemical compounds and cell-type specific medium transdifferentiate somatic cells into desired cell types including neuronal cells, glial cells, neural stem cells, brown adipocytes, cardiomyocytes, somatic progenitor cells, and pluripotent stem cells. These desired cells rapidly converted from patient-derived autologous fibroblasts can be applied for their own transplantation therapy to avoid immune rejection. However, complete chemical compound-induced conversions remain challenging particularly in adult human-derived fibroblasts compared with mouse embryonic fibroblasts (MEFs). This review summarizes up-to-date progress in each specific cell type and discusses prospects for future clinical application toward cell transplantation therapy. PMID:29739872

  10. Mouse cloning and somatic cell reprogramming using electrofused blastomeres.

    Science.gov (United States)

    Riaz, Amjad; Zhao, Xiaoyang; Dai, Xiangpeng; Li, Wei; Liu, Lei; Wan, Haifeng; Yu, Yang; Wang, Liu; Zhou, Qi

    2011-05-01

    Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.

  11. Combined negative effect of donor age and time in culture on the reprogramming efficiency into induced pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Ras Trokovic

    2015-07-01

    Full Text Available Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSC by the forced expression of the transcription factors OCT4, SOX2, KLF4 and c-MYC. Pluripotent reprogramming appears as a slow and inefficient process because of genetic and epigenetic barriers of somatic cells. In this report, we have extended previous observations concerning donor age and passage number of human fibroblasts as critical determinants of the efficiency of iPSC induction. Human fibroblasts from 11 different donors of variable age were reprogrammed by ectopic expression of reprogramming factors. Although all fibroblasts gave rise to iPSC colonies, the reprogramming efficiency correlated negatively and declined rapidly with increasing donor age. In addition, the late passage fibroblasts gave less reprogrammed colonies than the early passage cell counterparts, a finding associated with the cellular senescence-induced upregulation of p21. Knockdown of p21 restored iPSC generation even in long-term passaged fibroblasts of an old donor, highlighting the central role of the p53/p21 pathway in cellular senescence induced by both donor age and culture time.

  12. The effects of musical training on movement pre-programming and re-programming abilities: an event-related potential investigation.

    Science.gov (United States)

    Anatürk, Melis; Jentzsch, Ines

    2015-03-01

    Two response precuing experiments were conducted to investigate effects of musical skill level on the ability to pre- and re-programme simple movements. Participants successfully used advance information to prepare forthcoming responses and showed response slowing when precue information was invalid rather than valid. This slowing was, however, only observed for partially invalid but not fully invalid precues. Musicians were generally faster than non-musicians, but no group differences in the efficiency of movement pre-programming or re-programming were observed. Interestingly, only musicians exhibited a significant foreperiod lateralized readiness potential (LRP) when response hand was pre-specified or full advance information was provided. These LRP findings suggest increased effector-specific motor preparation in musicians than non-musicians. However, here the levels of effector-specific preparation did not predict preparatory advantages observed in behaviour. In sum, combining the response precuing and ERP paradigms serves a valuable tool to examine influences of musical training on movement pre- or re-programming processes. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. SOX2 Reprograms Resident Astrocytes into Neural Progenitors in the Adult Brain

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    Wenze Niu

    2015-05-01

    Full Text Available Glial cells can be in vivo reprogrammed into functional neurons in the adult CNS; however, the process by which this reprogramming occurs is unclear. Here, we show that a distinct cellular sequence is involved in SOX2-driven in situ conversion of adult astrocytes to neurons. This includes ASCL1+ neural progenitors and DCX+ adult neuroblasts (iANBs as intermediates. Importantly, ASCL1 is required, but not sufficient, for the robust generation of iANBs in the adult striatum. These progenitor-derived iANBs predominantly give rise to calretinin+ interneurons when supplied with neurotrophic factors or the small-molecule valproic acid. Patch-clamp recordings from the induced neurons reveal subtype heterogeneity, though all are functionally mature, fire repetitive action potentials, and receive synaptic inputs. Together, these results show that SOX2-mediated in vivo reprogramming of astrocytes to neurons passes through proliferative intermediate progenitors, which may be exploited for regenerative medicine.

  14. NRF2 Orchestrates the Metabolic Shift during Induced Pluripotent Stem Cell Reprogramming

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    Kate E. Hawkins

    2016-03-01

    Full Text Available The potential of induced pluripotent stem cells (iPSCs in disease modeling and regenerative medicine is vast, but current methodologies remain inefficient. Understanding the cellular mechanisms underlying iPSC reprogramming, such as the metabolic shift from oxidative to glycolytic energy production, is key to improving its efficiency. We have developed a lentiviral reporter system to assay longitudinal changes in cell signaling and transcription factor activity in living cells throughout iPSC reprogramming of human dermal fibroblasts. We reveal early NF-κB, AP-1, and NRF2 transcription factor activation prior to a temporal peak in hypoxia inducible factor α (HIFα activity. Mechanistically, we show that an early burst in oxidative phosphorylation and elevated reactive oxygen species generation mediates increased NRF2 activity, which in turn initiates the HIFα-mediated glycolytic shift and may modulate glucose redistribution to the pentose phosphate pathway. Critically, inhibition of NRF2 by KEAP1 overexpression compromises metabolic reprogramming and results in reduced efficiency of iPSC colony formation.

  15. Combinatorial Modulation of Signaling Pathways Reveals Cell-Type-Specific Requirements for Highly Efficient and Synchronous iPSC Reprogramming

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    Simon E. Vidal

    2014-10-01

    Full Text Available The differentiated state of somatic cells provides barriers for the derivation of induced pluripotent stem cells (iPSCs. To address why some cell types reprogram more readily than others, we studied the effect of combined modulation of cellular signaling pathways. Surprisingly, inhibition of transforming growth factor β (TGF-β together with activation of Wnt signaling in the presence of ascorbic acid allows >80% of murine fibroblasts to acquire pluripotency after 1 week of reprogramming factor expression. In contrast, hepatic and blood progenitors predominantly required only TGF-β inhibition or canonical Wnt activation, respectively, to reprogram at efficiencies approaching 100%. Strikingly, blood progenitors reactivated endogenous pluripotency loci in a highly synchronous manner, and we demonstrate that expression of specific chromatin-modifying enzymes and reduced TGF-β/mitogen-activated protein (MAP kinase activity are intrinsic properties associated with the unique reprogramming response of these cells. Our observations define cell-type-specific requirements for the rapid and synchronous reprogramming of somatic cells.

  16. Reprogramming of various cell types to a beta-like state by Pdx1, Ngn3 and MafA.

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    Ersin Akinci

    Full Text Available The three transcription factors, PDX1, NGN3 and MAFA, are very important in pancreatic development. Overexpression of these three factors can reprogram both pancreatic exocrine cells and SOX9-positive cells of the liver into cells resembling pancreatic beta cells. In this study we investigate whether other cell types can be reprogrammed. Eight cell types are compared and the results are consistent with the idea that reprogramming occurs to a greater degree for developmentally related cells (pancreas, liver than for other types, such as fibroblasts. Using a line of mouse hepatocyte-derived cells we screened 13 compounds for the ability to increase the yield of reprogrammed cells. Three are active and when used in combination they can increase the yield of insulin-immunopositive cells by a factor of six. These results should contribute to the eventual ability to develop a new cure for diabetes based on the ability to reprogram other cells in the body to a beta cell phenotype.

  17. A stochastic model of epigenetic dynamics in somatic cell reprogramming

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    Max eFloettmann

    2012-06-01

    Full Text Available Somatic cell reprogramming has dramatically changed stem cell research inrecent years. The high pace of new findings in the field and an ever increasingamount of data from new high throughput techniques make it challengingto isolate core principles of the process. In order to analyze suchmechanisms, we developed an abstract mechanistic model of a subset of theknown regulatory processes during cell differentiation and production of inducedpluripotent stem cells. This probabilistic Boolean network describesthe interplay between gene expression, chromatin modifications and DNAmethylation. The model incorporates recent findings in epigenetics and reproducesexperimentally observed reprogramming efficiencies and changes inmethylation and chromatin remodeling. It enables us to investigate in detail,how the temporal progression of the process is regulated. It also explicitlyincludes the transduction of factors using viral vectors and their silencing inreprogrammed cells, since this is still a standard procedure in somatic cellreprogramming. Based on the model we calculate an epigenetic landscape.Simulation results show good reproduction of experimental observations duringreprogramming, despite the simple stucture of the model. An extensiveanalysis and introduced variations hint towards possible optimizations of theprocess, that could push the technique closer to clinical applications. Fasterchanges in DNA methylation increase the speed of reprogramming at theexpense of efficiency, while accelerated chromatin modifications moderatelyimprove efficiency.

  18. Human X chromosome inactivation and reactivation: implications for cell reprogramming and disease.

    Science.gov (United States)

    Cantone, Irene; Fisher, Amanda G

    2017-11-05

    X-chromosome inactivation (XCI) is an exemplar of epigenetic regulation that is set up as pluripotent cells differentiate. Once established, XCI is stably propagated, but can be reversed in vivo or by pluripotent reprogramming in vitro Although reprogramming provides a useful model for inactive X (Xi) reactivation in mouse, the relative instability and heterogeneity of human embryonic stem (ES) cells and induced pluripotent stem cells hampers comparable progress in human. Here we review studies aimed at reactivating the human Xi using different reprogramming strategies. We outline our recent results using mouse ES cells to reprogramme female human fibroblasts by cell-cell fusion. We show that pluripotent reprogramming induces widespread and rapid chromatin remodelling in which the human Xi loses XIST and H3K27m3 enrichment and selected Xi genes become reactivated, ahead of mitotic division. Using RNA sequencing to map the extent of human Xi reactivation, and chromatin-modifying drugs to potentiate reactivation, we outline how this approach could be used to better design strategies to re-express human X-linked loci. As cell fusion induces the expression of human pluripotency genes that represent both the 'primed' and 'naive' states, this approach may also offer a fresh opportunity to segregate human pluripotent states with distinct Xi expression profiles, using single-cell-based approaches.This article is part of the themed issue 'X-chromosome inactivation: a tribute to Mary Lyon'. © 2017 The Author(s).

  19. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA.

    Science.gov (United States)

    Warren, Luigi; Manos, Philip D; Ahfeldt, Tim; Loh, Yuin-Han; Li, Hu; Lau, Frank; Ebina, Wataru; Mandal, Pankaj K; Smith, Zachary D; Meissner, Alexander; Daley, George Q; Brack, Andrew S; Collins, James J; Cowan, Chad; Schlaeger, Thorsten M; Rossi, Derrick J

    2010-11-05

    Clinical application of induced pluripotent stem cells (iPSCs) is limited by the low efficiency of iPSC derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPSCs toward clinically useful cell types are lacking. Here we describe a simple, nonintegrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate antiviral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem cells (RiPSCs) into terminally differentiated myogenic cells. This technology represents a safe, efficient strategy for somatic cell reprogramming and directing cell fate that has broad applicability for basic research, disease modeling, and regenerative medicine. Copyright © 2010 Elsevier Inc. All rights reserved.

  20. Direct Reprogramming of Human Bone Marrow Stromal Cells into Functional Renal Cells Using Cell-free Extracts

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    Evangelia Papadimou

    2015-04-01

    Full Text Available The application of cell-based therapies in regenerative medicine is gaining recognition. Here, we show that human bone marrow stromal cells (BMSCs, also known as bone-marrow-derived mesenchymal cells, can be reprogrammed into renal proximal tubular-like epithelial cells using cell-free extracts. Streptolysin-O-permeabilized BMSCs exposed to HK2-cell extracts underwent morphological changes—formation of “domes” and tubule-like structures—and acquired epithelial functional properties such as transepithelial-resistance, albumin-binding, and uptake and specific markers E-cadherin and aquaporin-1. Transmission electron microscopy revealed the presence of brush border microvilli and tight intercellular contacts. RNA sequencing showed tubular epithelial transcript abundance and revealed the upregulation of components of the EGFR pathway. Reprogrammed BMSCs integrated into self-forming kidney tissue and formed tubular structures. Reprogrammed BMSCs infused in immunodeficient mice with cisplatin-induced acute kidney injury engrafted into proximal tubuli, reduced renal injury and improved function. Thus, reprogrammed BMSCs are a promising cell resource for future cell therapy.

  1. BIX-01294 increases pig cloning efficiency by improving epigenetic reprogramming of somatic cell nuclei.

    Science.gov (United States)

    Huang, Jiaojiao; Zhang, Hongyong; Yao, Jing; Qin, Guosong; Wang, Feng; Wang, Xianlong; Luo, Ailing; Zheng, Qiantao; Cao, Chunwei; Zhao, Jianguo

    2016-01-01

    Accumulating evidence suggests that faulty epigenetic reprogramming leads to the abnormal development of cloned embryos and results in the low success rates observed in all mammals produced through somatic cell nuclear transfer (SCNT). The aberrant methylation status of H3K9me and H3K9me2 has been reported in cloned mouse embryos. To explore the role of H3K9me2 and H3K9me in the porcine somatic cell nuclear reprogramming, BIX-01294, known as a specific inhibitor of G9A (histone-lysine methyltransferase of H3K9), was used to treat the nuclear-transferred (NT) oocytes for 14-16 h after activation. The results showed that the developmental competence of porcine SCNT embryos was significantly enhanced both in vitro (blastocyst rate 16.4% vs 23.2%, Pcloning rate 1.59% vs 2.96%) after 50 nm BIX-01294 treatment. BIX-01294 treatment significantly decreased the levels of H3K9me2 and H3K9me at the 2- and 4-cell stages, which are associated with embryo genetic activation, and increased the transcriptional expression of the pluripotency genes SOX2, NANOG and OCT4 in cloned blastocysts. Furthermore, the histone acetylation levels of H3K9, H4K8 and H4K12 in cloned embryos were decreased after BIX-01294 treatment. However, co-treatment of activated NT oocytes with BIX-01294 and Scriptaid rescued donor nuclear chromatin from decreased histone acetylation of H4K8 that resulted from exposure to BIX-01294 only and consequently improved the preimplantation development of SCNT embryos (blastocyst formation rates of 23.7% vs 21.5%). These results indicated that treatment with BIX-01294 enhanced the developmental competence of porcine SCNT embryos through improvements in epigenetic reprogramming and gene expression. © 2016 Society for Reproduction and Fertility.

  2. Diversity among POU transcription factors in chromatin recognition and cell fate reprogramming.

    Science.gov (United States)

    Malik, Vikas; Zimmer, Dennis; Jauch, Ralf

    2018-05-01

    The POU (Pit-Oct-Unc) protein family is an evolutionary ancient group of transcription factors (TFs) that bind specific DNA sequences to direct gene expression programs. The fundamental importance of POU TFs to orchestrate embryonic development and to direct cellular fate decisions is well established, but the molecular basis for this activity is insufficiently understood. POU TFs possess a bipartite 'two-in-one' DNA binding domain consisting of two independently folding structural units connected by a poorly conserved and flexible linker. Therefore, they represent a paradigmatic example to study the molecular basis for the functional versatility of TFs. Their modular architecture endows POU TFs with the capacity to accommodate alternative composite DNA sequences by adopting different quaternary structures. Moreover, associations with partner proteins crucially influence the selection of their DNA binding sites. The plentitude of DNA binding modes confers the ability to POU TFs to regulate distinct genes in the context of different cellular environments. Likewise, different binding modes of POU proteins to DNA could trigger alternative regulatory responses in the context of different genomic locations of the same cell. Prominent POU TFs such as Oct4, Brn2, Oct6 and Brn4 are not only essential regulators of development but have also been successfully employed to reprogram somatic cells to pluripotency and neural lineages. Here we review biochemical, structural, genomic and cellular reprogramming studies to examine how the ability of POU TFs to select regulatory DNA, alone or with partner factors, is tied to their capacity to epigenetically remodel chromatin and drive specific regulatory programs that give cells their identities.

  3. Lineage Reprogramming of Astroglial Cells from Different Origins into Distinct Neuronal Subtypes

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    Malek Chouchane

    2017-07-01

    Full Text Available Astroglial cells isolated from the rodent postnatal cerebral cortex are particularly susceptible to lineage reprogramming into neurons. However, it remains unknown whether other astroglial populations retain the same potential. Likewise, little is known about the fate of induced neurons (iNs in vivo. In this study we addressed these questions using two different astroglial populations isolated from the postnatal brain reprogrammed either with Neurogenin-2 (Neurog2 or Achaete scute homolog-1 (Ascl1. We show that cerebellum (CerebAstro and cerebral cortex astroglia (CtxAstro generates iNs with distinctive neurochemical and morphological properties. Both astroglial populations contribute iNs to the olfactory bulb following transplantation in the postnatal and adult mouse subventricular zone. However, only CtxAstro transfected with Neurog2 differentiate into pyramidal-like iNs after transplantation in the postnatal cerebral cortex. Altogether, our data indicate that the origin of the astroglial population and transcription factors used for reprogramming, as well as the region of integration, affect the fate of iNs.

  4. SOX2 and SOX2-MYC Reprogramming Process of Fibroblasts to the Neural Stem Cells Compromised by Senescence.

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    Marta Winiecka-Klimek

    Full Text Available Tumorigenic potential of induced pluripotent stem cells (iPSCs infiltrating population of induced neural stem cells (iNSCs generated from iPSCs may limit their medical applications. To overcome such a difficulty, direct reprogramming of adult somatic cells into iNSCs was proposed. The aim of this study was the systematic comparison of induced neural cells (iNc obtained with different methods-direct reprogramming of human adult fibroblasts with either SOX2 (SiNSc-like or SOX2 and c-MYC (SMiNSc-like and induced pluripotent stem cells differentiation to ebiNSc-in terms of gene expression profile, differentiation potential as well as proliferation properties. Immunocytochemistry and real-time PCR analyses were used to evaluate gene expression profile and differentiation potential of various iNc types. Bromodeoxyuridine (BrdU incorporation and senescence-associated beta-galactosidase (SA-β-gal assays were used to estimate proliferation potential. All three types of iNc were capable of neuronal differentiation; however, astrocytic differentiation was possible only in case of ebiNSc. Contrary to ebiNSc generation, the direct reprogramming was rarely a propitious process, despite 100% transduction efficiency. The potency of direct iNSCs-like cells generation was lower as compared to iNSCs obtained by iPSCs differentiation, and only slightly improved when c-MYC was added. Directly reprogrammed iNSCs-like cells were lacking the ability to differentiate into astrocytic cells and characterized by poor efficiency of neuronal cells formation. Such features indicated that these cells could not be fully reprogrammed, as confirmed mainly with senescence detection. Importantly, SiNSc-like and SMiNSc-like cells were unable to achieve the long-term survival and became senescent, which limits their possible therapeutic applicability. Our results suggest that iNSCs-like cells, generated in the direct reprogramming attempts, were either not fully reprogrammed or

  5. NANOG priming before full reprogramming may generate germ cell tumours

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    I Grad

    2011-11-01

    Full Text Available Reprogramming somatic cells into a pluripotent state brings patient-tailored, ethical controversy-free cellular therapy closer to reality. However, stem cells and cancer cells share many common characteristics; therefore, it is crucial to be able to discriminate between them. We generated two induced pluripotent stem cell (iPSC lines, with NANOG pre-transduction followed by OCT3/4, SOX2, and LIN28 overexpression. One of the cell lines, CHiPS W, showed normal pluripotent stem cell characteristics, while the other, CHiPS A, though expressing pluripotency markers, failed to differentiate and gave rise to germ cell-like tumours in vivo. Comparative genomic hybridisation analysis of the generated iPS lines revealed that they were genetically more stable than human embryonic stem cell counterparts. This analysis proved to be predictive for the differentiation potential of analysed cells. Moreover, the CHiPS A line expressed a lower ratio of p53/p21 when compared to CHiPS W. NANOG pre-induction followed by OCT3/4, SOX2, MYC, and KLF4 induction resulted in the same tumour-inducing phenotype. These results underline the importance of a re-examination of the role of NANOG during reprogramming. Moreover, this reprogramming method may provide insights into primordial cell tumour formation and cancer stem cell transformation.

  6. Reprogramming mediated radio-resistance of 3D-grown cancer cells

    International Nuclear Information System (INIS)

    Xue Gang; Ren Zhenxin; Chen Yaxiong; Zhu Jiayun; Du Yarong; Pan Dong; Li Xiaoman; Hu Burong; Grabham, Peter W.

    2015-01-01

    In vitro 3D growth of tumors is a new cell culture model that more closely mimics the features of the in vivo environment and is being used increasingly in the field of biological and medical research. It has been demonstrated that cancer cells cultured in 3D matrices are more radio-resistant compared with cells in monolayers. However, the mechanisms causing this difference remain unclear. Here we show that cancer cells cultured in a 3D microenvironment demonstrated an increase in cells with stem cell properties. This was confirmed by the finding that cells in 3D cultures upregulated the gene and protein expression of the stem cell reprogramming factors such as OCT4, SOX2, NANOG, LIN28 and miR-302a, compared with cells in monolayers. Moreover, the expression of β-catenin, a regulating molecule of reprogramming factors, also increased in 3D-grown cancer cells. These findings suggest that cancer cells were reprogrammed to become stem cell-like cancer cells in a 3D growth culture microenvironment. Since cancer stem cell-like cells demonstrate an increased radio-resistance and chemo-resistance, our results offer a new perspective as to why. Our findings shed new light on understanding the features of the 3D growth cell model and its application in basic research into clinical radiotherapy and medicine. (author)

  7. Transient acquisition of pluripotency during somatic cell transdifferentiation with iPSC reprogramming factors.

    Science.gov (United States)

    Maza, Itay; Caspi, Inbal; Zviran, Asaf; Chomsky, Elad; Rais, Yoach; Viukov, Sergey; Geula, Shay; Buenrostro, Jason D; Weinberger, Leehee; Krupalnik, Vladislav; Hanna, Suhair; Zerbib, Mirie; Dutton, James R; Greenleaf, William J; Massarwa, Rada; Novershtern, Noa; Hanna, Jacob H

    2015-07-01

    Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced 'transdifferentiation' pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by various methods.

  8. Fast-ball sports experts depend on an inhibitory strategy to reprogram their movement timing.

    Science.gov (United States)

    Nakamoto, Hiroki; Ikudome, Sachi; Yotani, Kengo; Maruyama, Atsuo; Mori, Shiro

    2013-07-01

    The purpose of our study was to clarify whether an inhibitory strategy is used for reprogramming of movement timing by experts in fast-ball sports when they correct their movement timing due to unexpected environmental changes. We evaluated the influence of disruption of inhibitory function of the right inferior frontal gyrus (rIFG) on reprogramming of movement timing of experts and non-experts in fast-ball sports. The task was to manually press a button to coincide with the arrival of a moving target. The target moved at a constant velocity, and its velocity was suddenly either increased or decreased in some trials. The task was performed either with or without transcranial magnetic stimulation (TMS), which was delivered to the region of the rIFG. Under velocity change conditions without TMS, the experts showed significantly smaller timing errors and a higher rate of reprogramming of movement timing than the non-experts. Moreover, TMS application during the task significantly diminished the expert group's performance, but not the control group, particularly in the condition where the target velocity decreases. These results suggest that experts use an inhibitory strategy for reprogramming of movement timing. In addition, the rIFG inhibitory function contributes to the superior movement correction of experts in fast-ball sports.

  9. Cell-Intrinsic Glycogen Metabolism Supports Early Glycolytic Reprogramming Required for Dendritic Cell Immune Responses.

    Science.gov (United States)

    Thwe, Phyu M; Pelgrom, Leonard; Cooper, Rachel; Beauchamp, Saritha; Reisz, Julie A; D'Alessandro, Angelo; Everts, Bart; Amiel, Eyal

    2017-09-05

    Dendritic cell (DC) activation by Toll-like receptor (TLR) agonists causes rapid glycolytic reprogramming that is required to meet the metabolic demands of their immune activation. Recent efforts in the field have identified an important role for extracellular glucose sourcing to support DC activation. However, the contributions of intracellular glucose stores to these processes have not been well characterized. We demonstrate that DCs possess intracellular glycogen stores and that cell-intrinsic glycogen metabolism supports the early effector functions of TLR-activated DCs. Inhibition of glycogenolysis significantly attenuates TLR-mediated DC maturation and impairs their ability to initiate lymphocyte activation. We further report that DCs exhibit functional compartmentalization of glucose- and glycogen-derived carbons, where these substrates preferentially contribute to distinct metabolic pathways. This work provides novel insights into nutrient homeostasis in DCs, demonstrating that differential utilization of glycogen and glucose metabolism regulates their optimal immune function. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Lentiviral Vector Design and Imaging Approaches to Visualize the Early Stages of Cellular Reprogramming

    OpenAIRE

    Warlich, Eva; Kuehle, Johannes; Cantz, Tobias; Brugman, Martijn H; Maetzig, Tobias; Galla, Melanie; Filipczyk, Adam A; Halle, Stephan; Klump, Hannes; Schöler, Hans R; Baum, Christopher; Schroeder, Timm; Schambach, Axel

    2011-01-01

    Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by gene transfer of reprogramming transcription factors. Expression levels of these factors strongly influence the overall efficacy to form iPSC colonies, but additional contribution of stochastic cell-intrinsic factors has been proposed. Here, we present engineered color-coded lentiviral vectors in which codon-optimized reprogramming factors are co-expressed by a strong retroviral promoter that is rapidly silenced in iP...

  11. Small Molecules Facilitate Single Factor-Mediated Hepatic Reprogramming

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    Kyung Tae Lim

    2016-04-01

    Full Text Available Recent studies have shown that defined factors could lead to the direct conversion of fibroblasts into induced hepatocyte-like cells (iHeps. However, reported conversion efficiencies are very low, and the underlying mechanism of the direct hepatic reprogramming is largely unknown. Here, we report that direct conversion into iHeps is a stepwise transition involving the erasure of somatic memory, mesenchymal-to-epithelial transition, and induction of hepatic cell fate in a sequential manner. Through screening for additional factors that could potentially enhance the conversion kinetics, we have found that c-Myc and Klf4 (CK dramatically accelerate conversion kinetics, resulting in remarkably improved iHep generation. Furthermore, we identified small molecules that could lead to the robust generation of iHeps without CK. Finally, we show that Hnf1α supported by small molecules is sufficient to efficiently induce direct hepatic reprogramming. This approach might help to fully elucidate the direct conversion process and also facilitate the translation of iHep into the clinic.

  12. Regenerating the human heart: direct reprogramming strategies and their current limitations.

    Science.gov (United States)

    Ghiroldi, Andrea; Piccoli, Marco; Ciconte, Giuseppe; Pappone, Carlo; Anastasia, Luigi

    2017-10-27

    Cardiovascular diseases are the leading cause of death in the Western world. Unfortunately, current therapies are often only palliative, consequently essentially making heart transplantation necessary for many patients. However, several novel therapeutic approaches in the past two decades have yielded quite encouraging results. The generation of induced pluripotent stem cells, through the forced expression of stem cell-specific transcription factors, has inspired the most promising strategies for heart regeneration by direct reprogramming of cardiac fibroblasts into functional cardiomyocytes. Initial attempts at this reprogramming were conducted using a similar approach to the one used with transcription factors, but during years, novel strategies have been tested, e.g., miRNAs, recombinant proteins and chemical molecules. Although preliminary results on animal models are promising, the low reprogramming efficiency, as well as the incomplete maturation of the cardiomyocytes, still represents important obstacles. This review covers direct transdifferentiation strategies that have been proposed and developed and illustrates the pros and cons of each approach. Indeed, as described in the manuscript, there are still many unanswered questions and drawbacks that require a better understanding of the basic signaling pathways and transcription factor networks before functional cells, suitable for cardiac regeneration and safe for the patients, can be generated and used for human therapies.

  13. Identification of potential nuclear reprogramming and differentiation factors by a novel selection method for cloning chromatin-binding proteins

    International Nuclear Information System (INIS)

    Wang Liu; Zheng Aihua; Yi Ling; Xu Chongren; Ding Mingxiao; Deng Hongkui

    2004-01-01

    Nuclear reprogramming is critical for animal cloning and stem cell creation through nuclear transfer, which requires extensive remodeling of chromosomal architecture involving dramatic changes in chromatin-binding proteins. To understand the mechanism of nuclear reprogramming, it is critical to identify chromatin-binding factors specify the reprogramming process. In this report, we have developed a high-throughput selection method, based on T7 phage display and chromatin immunoprecipitation, to isolate chromatin-binding factors expressed in mouse embryonic stem cells using primary mouse embryonic fibroblast chromatin. Seven chromatin-binding proteins have been isolated by this method. We have also isolated several chromatin-binding proteins involved in hepatocyte differentiation. Our method provides a powerful tool to rapidly and selectively identify chromatin-binding proteins. The method can be used to study epigenetic modification of chromatin during nuclear reprogramming, cell differentiation, and transdifferentiation

  14. Direct Reprogramming of Spiral Ganglion Non-neuronal Cells into Neurons: Toward Ameliorating Sensorineural Hearing Loss by Gene Therapy

    Directory of Open Access Journals (Sweden)

    Teppei Noda

    2018-02-01

    Full Text Available Primary auditory neurons (PANs play a critical role in hearing by transmitting sound information from the inner ear to the brain. Their progressive degeneration is associated with excessive noise, disease and aging. The loss of PANs leads to permanent hearing impairment since they are incapable of regenerating. Spiral ganglion non-neuronal cells (SGNNCs, comprised mainly of glia, are resident within the modiolus and continue to survive after PAN loss. These attributes make SGNNCs an excellent target for replacing damaged PANs through cellular reprogramming. We used the neurogenic pioneer transcription factor Ascl1 and the auditory neuron differentiation factor NeuroD1 to reprogram SGNNCs into induced neurons (iNs. The overexpression of both Ascl1 and NeuroD1 in vitro generated iNs at high efficiency. Transcriptome analyses revealed that iNs displayed a transcriptome profile resembling that of endogenous PANs, including expression of several key markers of neuronal identity: Tubb3, Map2, Prph, Snap25, and Prox1. Pathway analyses indicated that essential pathways in neuronal growth and maturation were activated in cells upon neuronal induction. Furthermore, iNs extended projections toward cochlear hair cells and cochlear nucleus neurons when cultured with each respective tissue. Taken together, our study demonstrates that PAN-like neurons can be generated from endogenous SGNNCs. This work suggests that gene therapy can be a viable strategy to treat sensorineural hearing loss caused by degeneration of PANs.

  15. Genetic reprogramming of host cells by bacterial pathogens.

    Science.gov (United States)

    Tran Van Nhieu, Guy; Arbibe, Laurence

    2009-10-29

    During the course of infection, pathogens often induce changes in gene expression in host cells and these changes can be long lasting and global or transient and of limited amplitude. Defining how, when, and why bacterial pathogens reprogram host cells represents an exciting challenge that opens up the opportunity to grasp the essence of pathogenesis and its molecular details.

  16. High School Teachers' Perspectives on Supporting Students with Visual Impairments toward Higher Education: Access, Barriers, and Success

    Science.gov (United States)

    Reed, Maureen; Curtis, Kathryn

    2011-01-01

    The objective of the study presented here was to understand the experiences of teachers in assisting students with visual impairments in making the transition to higher education. The teachers reported barriers in high school that affect students' access to and success in higher education. Furthermore, institutions of higher education provided…

  17. Recombinase-mediated reprogramming and dystrophin gene addition in mdx mouse induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Chunli Zhao

    Full Text Available A cell therapy strategy utilizing genetically-corrected induced pluripotent stem cells (iPSC may be an attractive approach for genetic disorders such as muscular dystrophies. Methods for genetic engineering of iPSC that emphasize precision and minimize random integration would be beneficial. We demonstrate here an approach in the mdx mouse model of Duchenne muscular dystrophy that focuses on the use of site-specific recombinases to achieve genetic engineering. We employed non-viral, plasmid-mediated methods to reprogram mdx fibroblasts, using phiC31 integrase to insert a single copy of the reprogramming genes at a safe location in the genome. We next used Bxb1 integrase to add the therapeutic full-length dystrophin cDNA to the iPSC in a site-specific manner. Unwanted DNA sequences, including the reprogramming genes, were then precisely deleted with Cre resolvase. Pluripotency of the iPSC was analyzed before and after gene addition, and ability of the genetically corrected iPSC to differentiate into myogenic precursors was evaluated by morphology, immunohistochemistry, qRT-PCR, FACS analysis, and intramuscular engraftment. These data demonstrate a non-viral, reprogramming-plus-gene addition genetic engineering strategy utilizing site-specific recombinases that can be applied easily to mouse cells. This work introduces a significant level of precision in the genetic engineering of iPSC that can be built upon in future studies.

  18. Zfp296 is a novel, pluripotent-specific reprogramming factor.

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    Gerrit Fischedick

    Full Text Available Expression of the four transcription factors Oct4, Sox2, Klf4, and c-Myc (OSKM is sufficient to reprogram somatic cells into induced pluripotent stem (iPSCs. However, this process is slow and inefficient compared with the fusion of somatic cells with embryonic stem cells (ESCs, indicating that ESCs express additional factors that can enhance the efficiency of reprogramming. We had previously developed a method to detect and isolate early neural induction intermediates during the differentiation of mouse ESCs. Using the gene expression profiles of these intermediates, we identified 23 ESC-specific transcripts and tested each for the ability to enhance iPSC formation. Of the tested factors, zinc finger protein 296 (Zfp296 led to the largest increase in mouse iPSC formation. We confirmed that Zfp296 was specifically expressed in pluripotent stem cells and germ cells. Zfp296 in combination with OSKM induced iPSC formation earlier and more efficiently than OSKM alone. Through mouse chimera and teratoma formation, we demonstrated that the resultant iPSCs were pluripotent. We showed that Zfp296 activates transcription of the Oct4 gene via the germ cell-specific conserved region 4 (CR4, and when overexpressed in mouse ESCs leads to upregulation of Nanog expression and downregulation of the expression of differentiation markers, including Sox17, Eomes, and T, which is consistent with the observation that Zfp296 enhances the efficiency of reprogramming. In contrast, knockdown of Zfp296 in ESCs leads to the expression of differentiation markers. Finally, we demonstrated that expression of Zfp296 in ESCs inhibits, but does not block, differentiation into neural cells.

  19. Developmental Programming of Renal Function and Re-Programming Approaches.

    Science.gov (United States)

    Nüsken, Eva; Dötsch, Jörg; Weber, Lutz T; Nüsken, Kai-Dietrich

    2018-01-01

    Chronic kidney disease affects more than 10% of the population. Programming studies have examined the interrelationship between environmental factors in early life and differences in morbidity and mortality between individuals. A number of important principles has been identified, namely permanent structural modifications of organs and cells, long-lasting adjustments of endocrine regulatory circuits, as well as altered gene transcription. Risk factors include intrauterine deficiencies by disturbed placental function or maternal malnutrition, prematurity, intrauterine and postnatal stress, intrauterine and postnatal overnutrition, as well as dietary dysbalances in postnatal life. This mini-review discusses critical developmental periods and long-term sequelae of renal programming in humans and presents studies examining the underlying mechanisms as well as interventional approaches to "re-program" renal susceptibility toward disease. Clinical manifestations of programmed kidney disease include arterial hypertension, proteinuria, aggravation of inflammatory glomerular disease, and loss of kidney function. Nephron number, regulation of the renin-angiotensin-aldosterone system, renal sodium transport, vasomotor and endothelial function, myogenic response, and tubuloglomerular feedback have been identified as being vulnerable to environmental factors. Oxidative stress levels, metabolic pathways, including insulin, leptin, steroids, and arachidonic acid, DNA methylation, and histone configuration may be significantly altered by adverse environmental conditions. Studies on re-programming interventions focused on dietary or anti-oxidative approaches so far. Further studies that broaden our understanding of renal programming mechanisms are needed to ultimately develop preventive strategies. Targeted re-programming interventions in animal models focusing on known mechanisms will contribute to new concepts which finally will have to be translated to human application. Early

  20. Targeting Lipid Metabolic Reprogramming as Anticancer Therapeutics

    OpenAIRE

    Cha, Ji-Young; Lee, Ho-Jae

    2016-01-01

    Cancer cells rewire their metabolism to satisfy the demands of growth and survival, and this metabolic reprogramming has been recognized as an emerging hallmark of cancer. Lipid metabolism is pivotal in cellular process that converts nutrients into energy, building blocks for membrane biogenesis and the generation of signaling molecules. Accumulating evidence suggests that cancer cells show alterations in different aspects of lipid metabolism. The changes in lipid metabolism of cancer cells c...

  1. Direct Reprogramming of Adult Human Somatic Stem Cells Into Functional Neurons Using Sox2, Ascl1, and Neurog2

    Directory of Open Access Journals (Sweden)

    Jessica Alves de Medeiros Araújo

    2018-06-01

    Full Text Available Reprogramming of somatic cells into induced pluripotent stem cells (iPS or directly into cells from a different lineage, including neurons, has revolutionized research in regenerative medicine in recent years. Mesenchymal stem cells are good candidates for lineage reprogramming and autologous transplantation, since they can be easily isolated from accessible sources in adult humans, such as bone marrow and dental tissues. Here, we demonstrate that expression of the transcription factors (TFs SRY (sex determining region Y-box 2 (Sox2, Mammalian achaete-scute homolog 1 (Ascl1, or Neurogenin 2 (Neurog2 is sufficient for reprogramming human umbilical cord mesenchymal stem cells (hUCMSC into induced neurons (iNs. Furthermore, the combination of Sox2/Ascl1 or Sox2/Neurog2 is sufficient to reprogram up to 50% of transfected hUCMSCs into iNs showing electrical properties of mature neurons and establishing synaptic contacts with co-culture primary neurons. Finally, we show evidence supporting the notion that different combinations of TFs (Sox2/Ascl1 and Sox2/Neurog2 may induce multiple and overlapping neuronal phenotypes in lineage-reprogrammed iNs, suggesting that neuronal fate is determined by a combination of signals involving the TFs used for reprogramming but also the internal state of the converted cell. Altogether, the data presented here contribute to the advancement of techniques aiming at obtaining specific neuronal phenotypes from lineage-converted human somatic cells to treat neurological disorders.

  2. Regulation of the DNA Methylation Landscape in Human Somatic Cell Reprogramming by the miR-29 Family

    Directory of Open Access Journals (Sweden)

    Eriona Hysolli

    2016-07-01

    Full Text Available Reprogramming to pluripotency after overexpression of OCT4, SOX2, KLF4, and MYC is accompanied by global genomic and epigenomic changes. Histone modification and DNA methylation states in induced pluripotent stem cells (iPSCs have been shown to be highly similar to embryonic stem cells (ESCs. However, epigenetic differences still exist between iPSCs and ESCs. In particular, aberrant DNA methylation states found in iPSCs are a major concern when using iPSCs in a clinical setting. Thus, it is critical to find factors that regulate DNA methylation states in reprogramming. Here, we found that the miR-29 family is an important epigenetic regulator during human somatic cell reprogramming. Our global DNA methylation and hydroxymethylation analysis shows that DNA demethylation is a major event mediated by miR-29a depletion during early reprogramming, and that iPSCs derived from miR-29a depletion are epigenetically closer to ESCs. Our findings uncover an important miRNA-based approach to generate clinically robust iPSCs.

  3. A systematic evaluation of integration free reprogramming methods for deriving clinically relevant patient specific induced pluripotent stem (iPS cells.

    Directory of Open Access Journals (Sweden)

    Pollyanna A Goh

    Full Text Available A systematic evaluation of three different methods for generating induced pluripotent stem (iPS cells was performed using the same set of parental cells in our quest to develop a feeder independent and xeno-free method for somatic cell reprogramming that could be transferred into a GMP environment. When using the BJ fibroblast cell line, the highest reprogramming efficiency (1.89% of starting cells was observed with the mRNA based method which was almost 20 fold higher than that observed with the retrovirus (0.2% and episomal plasmid (0.10% methods. Standard characterisation tests did not reveal any differences in an array of pluripotency markers between the iPS lines derived using the various methods. However, when the same methods were used to reprogram three different primary fibroblasts lines, two derived from patients with rapid onset parkinsonism dystonia and one from an elderly healthy volunteer, we consistently observed higher reprogramming efficiencies with the episomal plasmid method, which was 4 fold higher when compared to the retroviral method and over 50 fold higher than the mRNA method. Additionally, with the plasmid reprogramming protocol, recombinant vitronectin and synthemax® could be used together with commercially available, fully defined, xeno-free essential 8 medium without significantly impacting the reprogramming efficiency. To demonstrate the robustness of this protocol, we reprogrammed a further 2 primary patient cell lines, one with retinosa pigmentosa and the other with Parkinsons disease. We believe that we have optimised a simple and reproducible method which could be used as a starting point for developing GMP protocols, a prerequisite for generating clinically relevant patient specific iPS cells.

  4. An Algorithmic Information Calculus for Causal Discovery and Reprogramming Systems

    KAUST Repository

    Zenil, Hector

    2017-09-08

    We introduce a conceptual framework and an interventional calculus to steer and manipulate systems based on their intrinsic algorithmic probability using the universal principles of the theory of computability and algorithmic information. By applying sequences of controlled interventions to systems and networks, we estimate how changes in their algorithmic information content are reflected in positive/negative shifts towards and away from randomness. The strong connection between approximations to algorithmic complexity (the size of the shortest generating mechanism) and causality induces a sequence of perturbations ranking the network elements by the steering capabilities that each of them is capable of. This new dimension unmasks a separation between causal and non-causal components providing a suite of powerful parameter-free algorithms of wide applicability ranging from optimal dimension reduction, maximal randomness analysis and system control. We introduce methods for reprogramming systems that do not require the full knowledge or access to the system\\'s actual kinetic equations or any probability distributions. A causal interventional analysis of synthetic and regulatory biological networks reveals how the algorithmic reprogramming qualitatively reshapes the system\\'s dynamic landscape. For example, during cellular differentiation we find a decrease in the number of elements corresponding to a transition away from randomness and a combination of the system\\'s intrinsic properties and its intrinsic capabilities to be algorithmically reprogrammed can reconstruct an epigenetic landscape. The interventional calculus is broadly applicable to predictive causal inference of systems such as networks and of relevance to a variety of machine and causal learning techniques driving model-based approaches to better understanding and manipulate complex systems.

  5. Cell reprogramming by 3D bioprinting of human fibroblasts in polyurethane hydrogel for fabrication of neural-like constructs.

    Science.gov (United States)

    Ho, Lin; Hsu, Shan-Hui

    2018-04-01

    3D bioprinting is a technique which enables the direct printing of biodegradable materials with cells into 3D tissue. So far there is no cell reprogramming in situ performed with the 3D bioprinting process. Forkhead box D3 (FoxD3) is a transcription factor and neural crest marker, which was reported to reprogram human fibroblasts into neural crest stem-like cells. In this study, we synthesized a new biodegradable thermo-responsive waterborne polyurethane (PU) gel as a bioink. FoxD3 plasmids and human fibroblasts were co-extruded with the PU hydrogel through the syringe needle tip for cell reprogramming. The rheological properties of the PU hydrogel including the modulus, gelation time, and shear thinning were optimized for the transfection effect of FoxD3 in situ. The corresponding shear rate and shear stress were examined. Results showed that human fibroblasts could be reprogrammed into neural crest stem-like cells with high cell viability during the extrusion process under an average shear stress ∼190 Pa. We further translated the method to the extrusion-based 3D bioprinting, and demonstrated that human fibroblasts co-printed with FoxD3 in the thermo-responsive PU hydrogel could be reprogrammed and differentiated into a neural-tissue like construct at 14 days after induction. The neural-like tissue construct produced by 3D bioprinting from human fibroblasts may be applied to personalized drug screening or neuroregeneration. There is no study so far on cell reprogramming in situ with 3D bioprinting. In this manuscript, a new thermoresponsive polyurethane bioink was developed and employed to deliver FoxD3 plasmid into human fibroblasts by the extrusion-based bioprinting. When the polyurethane gel was extruded through the syringe tip, the shear stress generated may have caused the transient membrane permeability for transfection. The shear stress was optimized for transfection in situ by 3D bioprinting. We demonstrated that human fibroblasts could be

  6. Glycometabolic reprogramming associated with the initiation of human dental pulp stem cell differentiation.

    Science.gov (United States)

    Wang, Linyan; Cheng, Li; Wang, Huning; Pan, Hongying; Yang, Hui; Shao, Meiying; Hu, Tao

    2016-03-01

    Glycometabolism, particularly mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis, plays a central role in cell life activities. Glycometabolism can be reprogrammed to maintain the stemness or to induce the differentiation of stem cells, thereby regulating tissue repair and regeneration. However, research on the glycometabolism of human dental pulp stem cells (hDPSCs) remains scarce. Here, we investigated the relationship between glycometabolic reprogramming and initiation of hDPSC differentiation. We found the differentiation of hDPSCs commenced on day 3 when cells were cultured in mineralized medium. When cell differentiation commenced, mitochondria became elongated with well-developed cristae, and the oxygen consumption rate of mitochondria was enhanced, manifested as an increase in basal respiration, mitochondrial ATP production, and maximal respiration. Interestingly, glycolytic enzyme activities, glycolysis capacity, and glycolysis reserve were also upregulated at this time to match the powerful bioenergetic demands. More importantly, hDPSCs derived from different donors or cultured in various oxygen environments showed similar glycometabolic changes when they began to differentiate. Thus, glycometabolic reprogramming accompanies initiation of hDPSC differentiation and could potentially play a role in the regulation of dental pulp repair. © 2015 International Federation for Cell Biology.

  7. Generation of induced neurons by direct reprogramming in the mammalian cochlea.

    Science.gov (United States)

    Nishimura, K; Weichert, R M; Liu, W; Davis, R L; Dabdoub, A

    2014-09-05

    Primary auditory neurons (ANs) in the mammalian cochlea play a critical role in hearing as they transmit auditory information in the form of electrical signals from mechanosensory cochlear hair cells in the inner ear to the brainstem. Their progressive degeneration is associated with disease conditions, excessive noise exposure and aging. Replacement of ANs, which lack the ability to regenerate spontaneously, would have a significant impact on research and advancement in cochlear implants in addition to the amelioration of hearing impairment. The aim of this study was to induce a neuronal phenotype in endogenous non-neural cells in the cochlea, which is the essential organ of hearing. Overexpression of a neurogenic basic helix-loop-helix transcription factor, Ascl1, in the cochlear non-sensory epithelial cells induced neurons at high efficiency at embryonic, postnatal and juvenile stages. Moreover, induced neurons showed typical properties of neuron morphology, gene expression and electrophysiology. Our data indicate that Ascl1 alone or Ascl1 and NeuroD1 is sufficient to reprogram cochlear non-sensory epithelial cells into functional neurons. Generation of neurons from non-neural cells in the cochlea is an important step for the regeneration of ANs in the mature mammalian cochlea. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  8. Perspective for special Gurdon issue for differentiation: can cell fusion inform nuclear reprogramming?

    Science.gov (United States)

    Burns, David; Blau, Helen M

    2014-07-01

    Nuclear reprogramming was first shown to be possible by Sir John Gurdon over a half century ago. The process has been revolutionized by the production of induced pluripotent cells by overexpression of the four transcription factors discovered by Shinya Yamanaka, which now enables mammalian applications. Yet, reprogramming by a few transcription factors remains incomplete and inefficient, whether to pluripotent or differentiated cells. We propose that a better understanding of mechanistic insights based on developmental principles gained from heterokaryon studies may inform the process of directing cell fate, fundamentally and clinically. Copyright © 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  9. Growth Factor-Activated Stem Cell Circuits and Stromal Signals Cooperatively Accelerate Non-Integrated iPSC Reprogramming of Human Myeloid Progenitors

    Science.gov (United States)

    Park, Tea Soon; Huo, Jeffrey S.; Peters, Ann; Talbot, C. Conover; Verma, Karan; Zimmerlin, Ludovic; Kaplan, Ian M.; Zambidis, Elias T.

    2012-01-01

    Nonviral conversion of skin or blood cells into clinically useful human induced pluripotent stem cells (hiPSC) occurs in only rare fractions (∼0.001%–0.5%) of donor cells transfected with non-integrating reprogramming factors. Pluripotency induction of developmentally immature stem-progenitors is generally more efficient than differentiated somatic cell targets. However, the nature of augmented progenitor reprogramming remains obscure, and its potential has not been fully explored for improving the extremely slow pace of non-integrated reprogramming. Here, we report highly optimized four-factor reprogramming of lineage-committed cord blood (CB) myeloid progenitors with bulk efficiencies of ∼50% in purified episome-expressing cells. Lineage-committed CD33+CD45+CD34− myeloid cells and not primitive hematopoietic stem-progenitors were the main targets of a rapid and nearly complete non-integrated reprogramming. The efficient conversion of mature myeloid populations into NANOG+TRA-1-81+ hiPSC was mediated by synergies between hematopoietic growth factor (GF), stromal activation signals, and episomal Yamanaka factor expression. Using a modular bioinformatics approach, we demonstrated that efficient myeloid reprogramming correlated not to increased proliferation or endogenous Core factor expressions, but to poised expression of GF-activated transcriptional circuits that commonly regulate plasticity in both hematopoietic progenitors and embryonic stem cells (ESC). Factor-driven conversion of myeloid progenitors to a high-fidelity pluripotent state was further accelerated by soluble and contact-dependent stromal signals that included an implied and unexpected role for Toll receptor-NFκB signaling. These data provide a paradigm for understanding the augmented reprogramming capacity of somatic progenitors, and reveal that efficient induced pluripotency in other cell types may also require extrinsic activation of a molecular framework that commonly regulates self

  10. Reprogramming of Mouse Calvarial Osteoblasts into Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Yinxiang Wang

    2018-01-01

    Full Text Available Previous studies have demonstrated the ability of reprogramming endochondral bone into induced pluripotent stem (iPS cells, but whether similar phenomenon occurs in intramembranous bone remains to be determined. Here we adopted fluorescence-activated cell sorting-based strategy to isolate homogenous population of intramembranous calvarial osteoblasts from newborn transgenic mice carrying both Osx1-GFP::Cre and Oct4-EGFP transgenes. Following retroviral transduction of Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc, enriched population of osteoblasts underwent silencing of Osx1-GFP::Cre expression at early stage of reprogramming followed by late activation of Oct4-EGFP expression in the resulting iPS cells. These osteoblast-derived iPS cells exhibited gene expression profiles akin to embryonic stem cells and were pluripotent as demonstrated by their ability to form teratomas comprising tissues from all germ layers and also contribute to tail tissue in chimera embryos. These data demonstrate that iPS cells can be generated from intramembranous osteoblasts.

  11. Drug discovery for Diamond-Blackfan anemia using reprogrammed hematopoietic progenitors

    Science.gov (United States)

    Doulatov, Sergei; Vo, Linda T.; Macari, Elizabeth R.; Wahlster, Lara; Kinney, Melissa A.; Taylor, Alison M.; Barragan, Jessica; Gupta, Manav; McGrath, Katherine; Lee, Hsiang-Ying; Humphries, Jessica M.; DeVine, Alex; Narla, Anupama; Alter, Blanche P.; Beggs, Alan H.; Agarwal, Suneet; Ebert, Benjamin L.; Gazda, Hanna T.; Lodish, Harvey F.; Sieff, Colin A.; Schlaeger, Thorsten M.; Zon, Leonard I.; Daley, George Q.

    2017-01-01

    Diamond-Blackfan anemia (DBA) is a congenital disorder characterized by the failure of erythroid progenitor differentiation, severely curtailing red blood cell production. Because many DBA patients fail to respond to corticosteroid therapy, there is considerable need for therapeutics for this disorder. Identifying therapeutics for DBA requires circumventing the paucity of primary patient blood stem and progenitor cells. To this end, we adopted a reprogramming strategy to generate expandable hematopoietic progenitor cells from induced pluripotent stem cells (iPSCs) from DBA patients. Reprogrammed DBA progenitors recapitulate defects in erythroid differentiation, which were rescued by gene complementation. Unbiased chemical screens identified SMER28, a small-molecule inducer of autophagy, which enhanced erythropoiesis in a range of in vitro and in vivo models of DBA. SMER28 acted through autophagy factor ATG5 to stimulate erythropoiesis and up-regulate expression of globin genes. These findings present an unbiased drug screen for hematological disease using iPSCs and identify autophagy as a therapeutic pathway in DBA. PMID:28179501

  12. Chemical strategies for pancreatic β cell differentiation, reprogramming, and regeneration.

    Science.gov (United States)

    Ma, Xiaojie; Zhu, Saiyong

    2017-04-01

    Generation of unlimited functional pancreatic β cells is critical for the study of pancreatic biology and treatment of diabetes mellitus. Recent advances have suggested several promising directions, including directed differentiation of pancreatic β cells from pluripotent stem cells, reprogramming of pancreatic β cells from other types of somatic cells, and stimulated proliferation and enhanced functions of existing pancreatic β cells. Small molecules are useful in generating unlimited numbers of functional pancreatic cells in vitro and could be further developed as drugs to stimulate endogenous pancreatic regeneration. Here, we provide an updated summary of recent major achievements in pancreatic β cell differentiation, reprogramming, proliferation, and function. These studies will eventually lead to significant advances in the field of pancreatic biology and regeneration. © The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. Impaired imprinted X chromosome inactivation is responsible for the skewed sex ratio following in vitro fertilization

    Science.gov (United States)

    Tan, Kun; An, Lei; Miao, Kai; Ren, Likun; Hou, Zhuocheng; Tao, Li; Zhang, Zhenni; Wang, Xiaodong; Xia, Wei; Liu, Jinghao; Wang, Zhuqing; Xi, Guangyin; Gao, Shuai; Sui, Linlin; Zhu, De-Sheng; Wang, Shumin; Wu, Zhonghong; Bach, Ingolf; Chen, Dong-bao; Tian, Jianhui

    2016-01-01

    Dynamic epigenetic reprogramming occurs during normal embryonic development at the preimplantation stage. Erroneous epigenetic modifications due to environmental perturbations such as manipulation and culture of embryos during in vitro fertilization (IVF) are linked to various short- or long-term consequences. Among these, the skewed sex ratio, an indicator of reproductive hazards, was reported in bovine and porcine embryos and even human IVF newborns. However, since the first case of sex skewing reported in 1991, the underlying mechanisms remain unclear. We reported herein that sex ratio is skewed in mouse IVF offspring, and this was a result of female-biased peri-implantation developmental defects that were originated from impaired imprinted X chromosome inactivation (iXCI) through reduced ring finger protein 12 (Rnf12)/X-inactive specific transcript (Xist) expression. Compensation of impaired iXCI by overexpression of Rnf12 to up-regulate Xist significantly rescued female-biased developmental defects and corrected sex ratio in IVF offspring. Moreover, supplementation of an epigenetic modulator retinoic acid in embryo culture medium up-regulated Rnf12/Xist expression, improved iXCI, and successfully redeemed the skewed sex ratio to nearly 50% in mouse IVF offspring. Thus, our data show that iXCI is one of the major epigenetic barriers for the developmental competence of female embryos during preimplantation stage, and targeting erroneous epigenetic modifications may provide a potential approach for preventing IVF-associated complications. PMID:26951653

  14. Cancer progression by reprogrammed BCAA metabolism in myeloid leukaemia.

    Science.gov (United States)

    Hattori, Ayuna; Tsunoda, Makoto; Konuma, Takaaki; Kobayashi, Masayuki; Nagy, Tamas; Glushka, John; Tayyari, Fariba; McSkimming, Daniel; Kannan, Natarajan; Tojo, Arinobu; Edison, Arthur S; Ito, Takahiro

    2017-05-25

    Reprogrammed cellular metabolism is a common characteristic observed in various cancers. However, whether metabolic changes directly regulate cancer development and progression remains poorly understood. Here we show that BCAT1, a cytosolic aminotransferase for branched-chain amino acids (BCAAs), is aberrantly activated and functionally required for chronic myeloid leukaemia (CML) in humans and in mouse models of CML. BCAT1 is upregulated during progression of CML and promotes BCAA production in leukaemia cells by aminating the branched-chain keto acids. Blocking BCAT1 gene expression or enzymatic activity induces cellular differentiation and impairs the propagation of blast crisis CML both in vitro and in vivo. Stable-isotope tracer experiments combined with nuclear magnetic resonance-based metabolic analysis demonstrate the intracellular production of BCAAs by BCAT1. Direct supplementation with BCAAs ameliorates the defects caused by BCAT1 knockdown, indicating that BCAT1 exerts its oncogenic function through BCAA production in blast crisis CML cells. Importantly, BCAT1 expression not only is activated in human blast crisis CML and de novo acute myeloid leukaemia, but also predicts disease outcome in patients. As an upstream regulator of BCAT1 expression, we identified Musashi2 (MSI2), an oncogenic RNA binding protein that is required for blast crisis CML. MSI2 is physically associated with the BCAT1 transcript and positively regulates its protein expression in leukaemia. Taken together, this work reveals that altered BCAA metabolism activated through the MSI2-BCAT1 axis drives cancer progression in myeloid leukaemia.

  15. MAPK-triggered chromatin reprogramming by histone deacetylase in plant innate immunity

    KAUST Repository

    Latrasse, David; Jé gu, Teddy; Li, Huchen; Zé licourt, Axel de; Raynaud, Cé cile; Legras, Sté phanie; Gust, Andrea; Samajova, Olga; Veluchamy, Alaguraj; Rayapuram, Naganand; Ramirez Prado, Juan Sebastian; Kulikova, Olga; Colcombet, Jean; Bigeard, Jean; Genot, Baptiste; Bisseling, Ton; Benhamed, Moussa; Hirt, Heribert

    2017-01-01

    Microbial-associated molecular patterns activate several MAP kinases, which are major regulators of the innate immune response in Arabidopsis thaliana that induce large-scale changes in gene expression. Here, we determine whether microbial-associated molecular pattern-triggered gene expression involves modifications at the chromatin level.Histone acetylation and deacetylation are major regulators of microbial-associated molecular pattern-triggered gene expression and implicate the histone deacetylase HD2B in the reprogramming of defence gene expression and innate immunity. The MAP kinase MPK3 directly interacts with and phosphorylates HD2B, thereby regulating the intra-nuclear compartmentalization and function of the histone deacetylase.By studying a number of gene loci that undergo microbial-associated molecular pattern-dependent activation or repression, our data reveal a mechanistic model for how protein kinase signaling directly impacts chromatin reprogramming in plant defense.

  16. MAPK-triggered chromatin reprogramming by histone deacetylase in plant innate immunity

    KAUST Repository

    Latrasse, David

    2017-07-06

    Microbial-associated molecular patterns activate several MAP kinases, which are major regulators of the innate immune response in Arabidopsis thaliana that induce large-scale changes in gene expression. Here, we determine whether microbial-associated molecular pattern-triggered gene expression involves modifications at the chromatin level.Histone acetylation and deacetylation are major regulators of microbial-associated molecular pattern-triggered gene expression and implicate the histone deacetylase HD2B in the reprogramming of defence gene expression and innate immunity. The MAP kinase MPK3 directly interacts with and phosphorylates HD2B, thereby regulating the intra-nuclear compartmentalization and function of the histone deacetylase.By studying a number of gene loci that undergo microbial-associated molecular pattern-dependent activation or repression, our data reveal a mechanistic model for how protein kinase signaling directly impacts chromatin reprogramming in plant defense.

  17. Lin28b stimulates the reprogramming of rat Müller glia to retinal progenitors

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Chen; Tao, Zui; Xue, Langyue; Zeng, Yuxiao [Southwest Hospital/Southwest Eye Hospital, Third Military Medical University, Chongqing 400038 (China); Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Chongqing 400038 (China); Wang, Yi, E-mail: wangyieye@aliyun.com [Southwest Hospital/Southwest Eye Hospital, Third Military Medical University, Chongqing 400038 (China); Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Chongqing 400038 (China); Xu, Haiwei, E-mail: haiweixu2001@163.com [Southwest Hospital/Southwest Eye Hospital, Third Military Medical University, Chongqing 400038 (China); Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Chongqing 400038 (China); Yin, Zheng Qin, E-mail: qinzyin@aliyun.com [Southwest Hospital/Southwest Eye Hospital, Third Military Medical University, Chongqing 400038 (China); Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Chongqing 400038 (China)

    2017-03-01

    In lower-order vertebrates, Müller glia exhibit characteristics of retinal progenitor cells, while in higher vertebrates, such as mammals, the regenerative capacity of Müller glia is limited. Recently, we reported that Lin28b promoted the trans-differentiation of Müller cells to rod photoreceptor and bipolar cells in the retina of retinitis pigmentosa rat model, whereas it is unclear whether Lin28b can stimulate the reprogramming of Müller glia in vitro for transplantation into a damaged retina. In the present study, Long-Evens rat Müller glia were infected with Adeno-Lin28b or Adeno-GFP. Over-expression of Lin28b in isolated rat Müller glia resulted in the suppression of GFAP expression, enhancement of cell proliferation and a significant increase of the expression of retinal progenitor markers 5 days after infection. Moreover, Lin28b caused a significant reduction of the Let-7 family of microRNAs. Following sub-retinal space transplantation, Müller glia-derived retinal progenitors improved b-wave amplification of 30d Royal College of Surgeons retinitis pigmentosa model (RCS-P+) rats, as detected by electroretinography (ERG) recordings. Taken together, these data suggest that the up-regulation of Lin28b expression facilitated the reprogramming of Müller cells toward characteristics of retinal progenitors. - Highlights: • Lin28b reprograms Müller glia to retinal progenitors. • Let-7 micrRNAs are suppressed by Lin28b. • Transplantation of reprogrammed Müller glia restores retinal function.

  18. Lin28b stimulates the reprogramming of rat Müller glia to retinal progenitors

    International Nuclear Information System (INIS)

    Zhao, Chen; Tao, Zui; Xue, Langyue; Zeng, Yuxiao; Wang, Yi; Xu, Haiwei; Yin, Zheng Qin

    2017-01-01

    In lower-order vertebrates, Müller glia exhibit characteristics of retinal progenitor cells, while in higher vertebrates, such as mammals, the regenerative capacity of Müller glia is limited. Recently, we reported that Lin28b promoted the trans-differentiation of Müller cells to rod photoreceptor and bipolar cells in the retina of retinitis pigmentosa rat model, whereas it is unclear whether Lin28b can stimulate the reprogramming of Müller glia in vitro for transplantation into a damaged retina. In the present study, Long-Evens rat Müller glia were infected with Adeno-Lin28b or Adeno-GFP. Over-expression of Lin28b in isolated rat Müller glia resulted in the suppression of GFAP expression, enhancement of cell proliferation and a significant increase of the expression of retinal progenitor markers 5 days after infection. Moreover, Lin28b caused a significant reduction of the Let-7 family of microRNAs. Following sub-retinal space transplantation, Müller glia-derived retinal progenitors improved b-wave amplification of 30d Royal College of Surgeons retinitis pigmentosa model (RCS-P+) rats, as detected by electroretinography (ERG) recordings. Taken together, these data suggest that the up-regulation of Lin28b expression facilitated the reprogramming of Müller cells toward characteristics of retinal progenitors. - Highlights: • Lin28b reprograms Müller glia to retinal progenitors. • Let-7 micrRNAs are suppressed by Lin28b. • Transplantation of reprogrammed Müller glia restores retinal function.

  19. MicroRNA in Metabolic Re-Programming and Their Role in Tumorigenesis

    Czech Academy of Sciences Publication Activity Database

    Tomasetti, M.; Amati, M.; Santarelli, L.; Neužil, Jiří

    2016-01-01

    Roč. 17, č. 5 (2016), č. článku 754. E-ISSN 1422-0067 Institutional support: RVO:86652036 Keywords : miRNAs * tumorigenesis * miR-126 and cancer-stroma environment * metabolic reprogramming Subject RIV: EB - Genetics ; Molecular Biology

  20. Data in support of metabolic reprogramming in transformed mouse cortical astrocytes: A proteomic study

    Directory of Open Access Journals (Sweden)

    Azeddine Bentaib

    2015-03-01

    Full Text Available 2D-DIGE analysis coupled with mass spectrometry is a global, without a priori, comparative proteomic approach particularly suited to identify and quantify enzymes isoforms and structural proteins, thus making it an efficient tool for the characterization of the changes in cell phenotypes that occur in physiological and pathological conditions. In this data article in support of the research article entitled “Metabolic reprogramming in transformed mouse cortical astrocytes: a proteomic study” [1] we illustrate the changes in protein profile that occur during the metabolic reprogramming undergone by cultured mouse astrocytes in a model of in-vitro cancerous transformation [2].

  1. Early Trypanosoma cruzi Infection Reprograms Human Epithelial Cells

    Directory of Open Access Journals (Sweden)

    María Laura Chiribao

    2014-01-01

    Full Text Available Trypanosoma cruzi, the causative agent of Chagas disease, has the peculiarity, when compared with other intracellular parasites, that it is able to invade almost any type of cell. This property makes Chagas a complex parasitic disease in terms of prophylaxis and therapeutics. The identification of key host cellular factors that play a role in the T. cruzi invasion is important for the understanding of disease pathogenesis. In Chagas disease, most of the focus is on the response of macrophages and cardiomyocytes, since they are responsible for host defenses and cardiac lesions, respectively. In the present work, we studied the early response to infection of T. cruzi in human epithelial cells, which constitute the first barrier for establishment of infection. These studies identified up to 1700 significantly altered genes regulated by the immediate infection. The global analysis indicates that cells are literally reprogrammed by T. cruzi, which affects cellular stress responses (neutrophil chemotaxis, DNA damage response, a great number of transcription factors (including the majority of NFκB family members, and host metabolism (cholesterol, fatty acids, and phospholipids. These results raise the possibility that early host cell reprogramming is exploited by the parasite to establish the initial infection and posterior systemic dissemination.

  2. Systemic evaluation of cellular reprogramming processes exploiting a novel R-tool: eegc.

    Science.gov (United States)

    Zhou, Xiaoyuan; Meng, Guofeng; Nardini, Christine; Mei, Hongkang

    2017-08-15

    Cells derived by cellular engineering, i.e. differentiation of induced pluripotent stem cells and direct lineage reprogramming, carry a tremendous potential for medical applications and in particular for regenerative therapies. These approaches consist in the definition of lineage-specific experimental protocols that, by manipulation of a limited number of biological cues-niche mimicking factors, (in)activation of transcription factors, to name a few-enforce the final expression of cell-specific (marker) molecules. To date, given the intricate complexity of biological pathways, these approaches still present imperfect reprogramming fidelity, with uncertain consequences on the functional properties of the resulting cells. We propose a novel tool eegc to evaluate cellular engineering processes, in a systemic rather than marker-based fashion, by integrating transcriptome profiling and functional analysis. Our method clusters genes into categories representing different states of (trans)differentiation and further performs functional and gene regulatory network analyses for each of the categories of the engineered cells, thus offering practical indications on the potential lack of the reprogramming protocol. eegc R package is released under the GNU General Public License within the Bioconductor project, freely available at https://bioconductor.org/packages/eegc/. christine.nardini.rsrc@gmail.com or hongkang.k.mei@gsk.com. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

  3. Reprogrammed chondrocytes engineered to produce IL-12 provide novel ex vivo immune-gene therapy for cancer.

    Science.gov (United States)

    Tada, Hiroyuki; Kishida, Tsunao; Fujiwara, Hitoshi; Kosuga, Toshiyuki; Konishi, Hirotaka; Komatsu, Shuhei; Shiozaki, Atsushi; Ichikawa, Daisuke; Okamoto, Kazuma; Otsuji, Eigo; Mazda, Osam

    2017-03-01

    The somatic cell reprogramming technology was applied to a novel and promising ex vivo immune-gene therapy strategy for cancer. To establish a novel ex vivo cytokine gene therapy of cancer using the somatic cell reprogramming procedures. Mouse fibroblasts were converted into chondrocytes and subsequently transduced with IL-12 gene. The resultant IL-12 induced chondrogenic cells were irradiated with x-ray and inoculated into mice bearing CT26 colon cancer. The irradiation at 20 Gy or higher totally eliminated the proliferative potential of the cells, while less significantly influencing the IL-12 production from the cells. An inoculation of the irradiated IL-12 induced chondrogenic cells significantly suppressed tumor by inducing tumor-specific cytotoxic T lymphocytes, enhancing natural killer tumoricidal activity and inhibiting tumor neoangiogenesis in the mice. The somatic cell reprogramming procedures may provide a novel and effective means to treat malignancies.

  4. Xenopatients 2.0: reprogramming the epigenetic landscapes of patient-derived cancer genomes.

    Science.gov (United States)

    Menendez, Javier A; Alarcón, Tomás; Corominas-Faja, Bruna; Cuyàs, Elisabet; López-Bonet, Eugeni; Martin, Angel G; Vellon, Luciano

    2014-01-01

    In the science-fiction thriller film Minority Report, a specialized police department called "PreCrime" apprehends criminals identified in advance based on foreknowledge provided by 3 genetically altered humans called "PreCogs". We propose that Yamanaka stem cell technology can be similarly used to (epi)genetically reprogram tumor cells obtained directly from cancer patients and create self-evolving personalized translational platforms to foresee the evolutionary trajectory of individual tumors. This strategy yields a large stem cell population and captures the cancer genome of an affected individual, i.e., the PreCog-induced pluripotent stem (iPS) cancer cells, which are immediately available for experimental manipulation, including pharmacological screening for personalized "stemotoxic" cancer drugs. The PreCog-iPS cancer cells will re-differentiate upon orthotopic injection into the corresponding target tissues of immunodeficient mice (i.e., the PreCrime-iPS mouse avatars), and this in vivo model will run through specific cancer stages to directly explore their biological properties for drug screening, diagnosis, and personalized treatment in individual patients. The PreCog/PreCrime-iPS approach can perform sets of comparisons to directly observe changes in the cancer-iPS cell line vs. a normal iPS cell line derived from the same human genetic background. Genome editing of PreCog-iPS cells could create translational platforms to directly investigate the link between genomic expression changes and cellular malignization that is largely free from genetic and epigenetic noise and provide proof-of-principle evidence for cutting-edge "chromosome therapies" aimed against cancer aneuploidy. We might infer the epigenetic marks that correct the tumorigenic nature of the reprogrammed cancer cell population and normalize the malignant phenotype in vivo. Genetically engineered models of conditionally reprogrammable mice to transiently express the Yamanaka stemness factors

  5. Restoration of Mitochondrial NAD+ Levels Delays Stem Cell Senescence and Facilitates Reprogramming of Aged Somatic Cells.

    Science.gov (United States)

    Son, Myung Jin; Kwon, Youjeong; Son, Taekwon; Cho, Yee Sook

    2016-12-01

    The fundamental tenet that aging is irreversible has been challenged by the development of reprogramming technology that can restore molecular and cellular age by reversing the progression of aging. The use of cells from aged individuals as sources for reprogramming or transplantation creates a major barrier in stem cell therapy with respect to cell quality and quantity. Here, we investigated the molecular features underlying senescence and rejuvenation during aged cell reprogramming and identified novel factors that can overcome age-associated barriers. Enzymes, such as nicotinamide nucleotide transhydrogenase (NNT) and nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3), that control mitochondrial NAD + levels appear to be susceptible to aging. In aged cells, mitochondrial NAD + levels decrease, accompanied by reduced SIRT3 activity; these changes severely impede cell fate transition. However, in cells collected from aged p16 knockout mice, which exhibit delayed cellular senescence, no changes in NNT or NMNAT3 expression were found. Importantly, restoring mitochondrial NAD + levels by overexpressing NNT and NMNAT3 enhanced reprogramming efficiency of aged somatic cells and extended the lifespan of human mesenchymal stem cells by delaying replicative senescence. These results demonstrate that maintenance of mitochondrial NAD + levels is critical for reversing the mechanisms of aging and ensuring that cells collected from aged individuals are of high quality. Stem Cells 2016;34:2840-2851. © 2016 AlphaMed Press.

  6. The Power and the Promise of Cell Reprogramming: Personalized Autologous Body Organ and Cell Transplantation

    Directory of Open Access Journals (Sweden)

    Ana Belen Alvarez Palomo

    2014-04-01

    Full Text Available Reprogramming somatic cells to induced pluripotent stem cells (iPSCs or direct reprogramming to desired cell types are powerful and new in vitro methods for the study of human disease, cell replacement therapy, and drug development. Both methods to reprogram cells are unconstrained by the ethical and social questions raised by embryonic stem cells. iPSC technology promises to enable personalized autologous cell therapy and has the potential to revolutionize cell replacement therapy and regenerative medicine. Potential applications of iPSC technology are rapidly increasing in ambition from discrete cell replacement applications to the iPSC assisted bioengineering of body organs for personalized autologous body organ transplant. Recent work has demonstrated that the generation of organs from iPSCs is a future possibility. The development of embryonic-like organ structures bioengineered from iPSCs has been achieved, such as an early brain structure (cerebral organoids, bone, optic vesicle-like structures (eye, cardiac muscle tissue (heart, primitive pancreas islet cells, a tooth-like structure (teeth, and functional liver buds (liver. Thus, iPSC technology offers, in the future, the powerful and unique possibility to make body organs for transplantation removing the need for organ donation and immune suppressing drugs. Whilst it is clear that iPSCs are rapidly becoming the lead cell type for research into cell replacement therapy and body organ transplantation strategies in humans, it is not known whether (1 such transplants will stimulate host immune responses; and (2 whether this technology will be capable of the bioengineering of a complete and fully functional human organ. This review will not focus on reprogramming to iPSCs, of which a plethora of reviews can be found, but instead focus on the latest developments in direct reprogramming of cells, the bioengineering of body organs from iPSCs, and an analysis of the immune response induced by i

  7. Nucleosome organizations in induced pluripotent stem cells reprogrammed from somatic cells belonging to three different germ layers.

    Science.gov (United States)

    Tao, Yu; Zheng, Weisheng; Jiang, Yonghua; Ding, Guitao; Hou, Xinfeng; Tang, Yitao; Li, Yueying; Gao, Shuai; Chang, Gang; Zhang, Xiaobai; Liu, Wenqiang; Kou, Xiaochen; Wang, Hong; Jiang, Cizhong; Gao, Shaorong

    2014-12-21

    Nucleosome organization determines the chromatin state, which in turn controls gene expression or silencing. Nucleosome remodeling occurs during somatic cell reprogramming, but it is still unclear to what degree the re-established nucleosome organization of induced pluripotent stem cells (iPSCs) resembles embryonic stem cells (ESCs), and whether the iPSCs inherit some residual gene expression from the parental fibroblast cells. We generated genome-wide nucleosome maps in mouse ESCs and in iPSCs reprogrammed from somatic cells belonging to three different germ layers using a secondary reprogramming system. Pairwise comparisons showed that the nucleosome organizations in the iPSCs, regardless of the iPSCs' tissue of origin, were nearly identical to the ESCs, but distinct from mouse embryonic fibroblasts (MEF). There is a canonical nucleosome arrangement of -1, nucleosome depletion region, +1, +2, +3, and so on nucleosomes around the transcription start sites of active genes whereas only a nucleosome occupies silent transcriptional units. Transcription factor binding sites possessed characteristic nucleosomal architecture, such that their access was governed by the rotational and translational settings of the nucleosome. Interestingly, the tissue-specific genes were highly expressed only in the parental somatic cells of the corresponding iPS cell line before reprogramming, but had a similar expression level in all the resultant iPSCs and ESCs. The re-established nucleosome landscape during nuclear reprogramming provides a conserved setting for accessibility of DNA sequences in mouse pluripotent stem cells. No persistent residual expression program or nucleosome positioning of the parental somatic cells that reflected their tissue of origin was passed on to the resulting mouse iPSCs.

  8. Targeted gene therapy and cell reprogramming in Fanconi anemia

    Science.gov (United States)

    Rio, Paula; Baños, Rocio; Lombardo, Angelo; Quintana-Bustamante, Oscar; Alvarez, Lara; Garate, Zita; Genovese, Pietro; Almarza, Elena; Valeri, Antonio; Díez, Begoña; Navarro, Susana; Torres, Yaima; Trujillo, Juan P; Murillas, Rodolfo; Segovia, Jose C; Samper, Enrique; Surralles, Jordi; Gregory, Philip D; Holmes, Michael C; Naldini, Luigi; Bueren, Juan A

    2014-01-01

    Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies. PMID:24859981

  9. Highly efficient reprogramming to pluripotency and directed differentiation of human cells using synthetic modified mRNA

    OpenAIRE

    Warren, Luigi; Manos, Philip D.; Ahfeldt, Tim; Loh, Yuin-Han; Li, Hu; Lau, Frank; Ebina, Wataru; Mandal, Pankaj; Smith, Zachary D.; Meissner, Alexander; Daley, George Q.; Brack, Andrew S.; Collins, James J.; Cowan, Chad; Schlaeger, Thorsten M.

    2010-01-01

    Clinical application of induced pluripotent stem (iPS) cells is limited by the low efficiency of iPS derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPS cells towards clinically useful cell types are lacking. Here we describe a simple, non-integrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate anti-viral re...

  10. Lapatinib Resistance in Breast Cancer Cells Is Accompanied by Phosphorylation-Mediated Reprogramming of Glycolysis.

    Science.gov (United States)

    Ruprecht, Benjamin; Zaal, Esther A; Zecha, Jana; Wu, Wei; Berkers, Celia R; Kuster, Bernhard; Lemeer, Simone

    2017-04-15

    HER2/ERBB2-overexpressing breast cancers targeted effectively by the small-molecule kinase inhibitor lapatinib frequently acquire resistance to this drug. In this study, we employed explorative mass spectrometry to profile proteome, kinome, and phosphoproteome changes in an established model of lapatinib resistance to systematically investigate initial inhibitor response and subsequent reprogramming in resistance. The resulting dataset, which collectively contains quantitative data for >7,800 proteins, >300 protein kinases, and >15,000 phosphopeptides, enabled deep insight into signaling recovery and molecular reprogramming upon resistance. Our data-driven approach confirmed previously described mechanisms of resistance (e.g., AXL overexpression and PIK3 reactivation), revealed novel pharmacologically actionable targets, and confirmed the expectation of significant heterogeneity in molecular resistance drivers inducing distinct phenotypic changes. Furthermore, our approach identified an extensive and exclusively phosphorylation-mediated reprogramming of glycolytic activity, supported additionally by widespread changes of corresponding metabolites and an increased sensitivity towards glycolysis inhibition. Collectively, our multi-omic analysis offers deeper perspectives on cancer drug resistance and suggests new biomarkers and treatment options for lapatinib-resistant cancers. Cancer Res; 77(8); 1842-53. ©2017 AACR . ©2017 American Association for Cancer Research.

  11. System-Wide Hypersensitive Response-Associated Transcriptome and Metabolome Reprogramming in Tomato

    NARCIS (Netherlands)

    Etalo, D.W.; Stulemeijer, I.J.E.; Esse, van H.P.; Vos, de R.C.H.; Bouwmeester, H.J.; Joosten, M.H.A.J.

    2013-01-01

    The hypersensitive response (HR) is considered to be the hallmark of the resistance response of plants to pathogens. To study HR-associated transcriptome and metabolome reprogramming in tomato (Solanum lycopersicum), we used plants that express both a resistance gene to Cladosporium fulvum and the

  12. Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors

    Directory of Open Access Journals (Sweden)

    Ahmed Kamel El-Sayed

    2014-11-01

    Full Text Available Reprogramming of somatic cells has great potential to provide therapeutic treatments for a number of diseases as well as provide insight into mechanisms underlying early embryonic development. Improvement of induced Pluripotent Stem Cells (iPSCs generation through mRNA-based methods is currently an area of intense research. This approach provides a number of advantages over previously used methods such as DNA integration and insertional mutagenesis. Using transfection of specifically synthesized mRNAs of various pluripotency factors, we generated iPSCs from mouse embryonic fibroblast (MEF cells. The genetic, epigenetic and functional properties of the iPSCs were evaluated at different times during the reprogramming process. We successfully introduced synthesized mRNAs, which localized correctly inside the cells and exhibited efficient and stable translation into proteins. Our work demonstrated a robust up-regulation and a gradual promoter de-methylation of the pluripotency markers, including non-transfected factors such as Nanog, SSEA-1 (stage-specific embryonic antigen 1 and Rex-1 (ZFP-42, zinc finger protein 42. Using embryonic stem cells (ESCs conditions to culture the iPS cells resulted in formation of ES-like colonies after approximately 12 days with only five daily repeated transfections. The colonies were positive for alkaline phosphatase and pluripotency-specific markers associated with ESCs. This study revealed the ability of pluripotency induction and generation of mouse mRNA induced pluripotent stem cells (mRNA iPSCs using transfection of specifically synthesized mRNAs of various pluripotency factors into mouse embryonic fibroblast (MEF cells. These generated iPSCs exhibited molecular and functional properties similar to ESCs, which indicate that this method is an efficient and viable alternative to ESCs and can be used for further biological, developmental and therapeutic investigations.

  13. A hit and run approach to inducible direct reprogramming of astrocytes to neural stem cells

    Directory of Open Access Journals (Sweden)

    Maria ePoulou

    2016-04-01

    Full Text Available Temporal and spatial control of gene expression can be achieved using an inducible system as a fundamental tool for regulated transcription in basic, applied and eventually in clinical research. We describe a novel hit and run inducible direct reprogramming approach. In a single step, two days post-transfection, transiently transfected Sox2FLAG under the Leu3p-αIPM inducible control (iSox2 triggers the activation of endogenous Sox2, redirecting primary astrocytes into abundant distinct nestin-positive radial glia cells. This technique introduces a unique novel tool for safe, rapid and efficient reprogramming amendable to regenerative medicine.

  14. Reprogramming of B cells into macrophages: mechanistic insights

    OpenAIRE

    Di Tullio, Alessandro, 1982-

    2012-01-01

    Our earlier work has shown that pre-B cells can be converted into macrophages by the transcription factor C/EBPα at very high frequencies and also that a clonal pre-B cell line with an inducible form of C/EBPα can be converted into macrophage-like cells. Using these systems we have performed a systematic analysis of the questions whether during transdifferentiation the cells retrodifferentiate to a precursor cell state and whether cell cycle is required for reprogramming. As for the first ...

  15. Successful pregnancy in a female patient with congenital chloride diarrhea (CLD) and renal impairment.

    Science.gov (United States)

    Shimizu, Yoshio; Kamoda, Tomohiro; Nagata, Michio; Yoh, Keigyo; Hashimoto, Yuko; Matsui, Akira; Yoshikawa, Hiroyuki; Yamagata, Kunihiro; Koyama, Akio

    2009-01-01

    We report a successful case of pregnancy in a female patient with congenital chloride diarrhea (CLD) and reduced renal function due to interruption of treatment. CLD is an autosomal recessive disorder of intestinal electrolyte absorption caused by mutations in the solute carrier family 26, member 3 (SLC26A3) gene, and continuous production of watery diarrhea induces dehydration, metabolic alkalosis and many kinds of electrolyte disturbances in CLD patients. The patient in our case was a 24-year-old female CLD patient with moderate renal impairment; a renal biopsy specimen showed minimal glomerular changes, but tubulointerstitial damage by crystal formation, consistent with renal function data. One year after our initial examination and reinstitution of therapy, the patient got married and soon conceived. There were no major problems during the course of pregnancy, and the patient successfully delivered a healthy full-term infant vaginally. The symptoms and clinical course of the patient were particularly mild, and we discuss possible reasons for these observations from a perspective of genotype, phenotype and environmental conditions.

  16. Climbing Mount Efficiency--small steps, not giant leaps towards higher cloning success in farm animals.

    Science.gov (United States)

    Oback, Björn

    2008-07-01

    Despite more than a decade of research efforts, farm animal cloning by somatic cell nuclear transfer (SCNT) is still frustratingly inefficient. Inefficiency manifests itself at different levels, which are currently not well integrated. At the molecular level, it leads to widespread genetic, epigenetic and transcriptional aberrations in cloned embryos. At the organismal level, these genome-wide abnormalities compromise development of cloned foetuses and offspring. Specific molecular defects need to be causally linked to specific cloned phenotypes, in order to design specific treatments to correct them. Cloning efficiency depends on the ability of the nuclear donor cell to be fully reprogrammed into an embryonic state and the ability of the enucleated recipient cell to carry out the reprogramming reactions. It has been postulated that reprogrammability of the somatic donor cell epigenome is influenced by its differentiation status. However, direct comparisons between cells of divergent differentiation status within several somatic lineages have found no conclusive evidence for this. Choosing somatic stem cells as donors has not improved cloning efficiency, indicating that donor cell type may be less critical for cloning success. Different recipient cells, on the other hand, vary in their reprogramming ability. In bovine, using zygotes instead of oocytes has increased cloning success. Other improvements in livestock cloning efficiency include better coordinating donor cell type with cell cycle stage and aggregating cloned embryos. In the future, it will be important to demonstrate if these small increases at every step are cumulative, adding up to an integrated cloning protocol with greatly improved efficiency.

  17. Cellular Programming and Reprogramming: Sculpting Cell Fate for the Production of Dopamine Neurons for Cell Therapy

    Directory of Open Access Journals (Sweden)

    Julio C. Aguila

    2012-01-01

    success of clinical applications depends on our ability to steer pluripotent stem cells towards the right neuronal identity. In Parkinson disease, the loss of dopamine neurons is more pronounced in the ventrolateral population that projects to the sensorimotor striatum. Because synapses are highly specific, only neurons with this precise identity will contribute, upon transplantation, to the synaptic reconstruction of the dorsal striatum. Thus, understanding the developmental cell program of the mesostriatal dopamine neurons is critical for the identification of the extrinsic signals and cell-intrinsic factors that instruct and, ultimately, determine cell identity. Here, we review how extrinsic signals and transcription factors act together during development to shape midbrain cell fates. Further, we discuss how these same factors can be applied in vitro to induce, select, and reprogram cells to the mesostriatal dopamine fate.

  18. Endogenous retinal neural stem cell reprogramming for neuronal regeneration

    Directory of Open Access Journals (Sweden)

    Romain Madelaine

    2017-01-01

    Full Text Available In humans, optic nerve injuries and associated neurodegenerative diseases are often followed by permanent vision loss. Consequently, an important challenge is to develop safe and effective methods to replace retinal neurons and thereby restore neuronal functions and vision. Identifying cellular and molecular mechanisms allowing to replace damaged neurons is a major goal for basic and translational research in regenerative medicine. Contrary to mammals, the zebrafish has the capacity to fully regenerate entire parts of the nervous system, including retina. This regenerative process depends on endogenous retinal neural stem cells, the Müller glial cells. Following injury, zebrafish Müller cells go back into cell cycle to proliferate and generate new neurons, while mammalian Müller cells undergo reactive gliosis. Recently, transcription factors and microRNAs have been identified to control the formation of new neurons derived from zebrafish and mammalian Müller cells, indicating that cellular reprogramming can be an efficient strategy to regenerate human retinal neurons. Here we discuss recent insights into the use of endogenous neural stem cell reprogramming for neuronal regeneration, differences between zebrafish and mammalian Müller cells, and the need to pursue the identification and characterization of new molecular factors with an instructive and potent function in order to develop theurapeutic strategies for eye diseases.

  19. Cell fate reprogramming by control of intracellular network dynamics

    Science.gov (United States)

    Zanudo, Jorge G. T.; Albert, Reka

    Identifying control strategies for biological networks is paramount for practical applications that involve reprogramming a cell's fate, such as disease therapeutics and stem cell reprogramming. Although the topic of controlling the dynamics of a system has a long history in control theory, most of this work is not directly applicable to intracellular networks. Here we present a network control method that integrates the structural and functional information available for intracellular networks to predict control targets. Formulated in a logical dynamic scheme, our control method takes advantage of certain function-dependent network components and their relation to steady states in order to identify control targets, which are guaranteed to drive any initial state to the target state with 100% effectiveness and need to be applied only transiently for the system to reach and stay in the desired state. We illustrate our method's potential to find intervention targets for cancer treatment and cell differentiation by applying it to a leukemia signaling network and to the network controlling the differentiation of T cells. We find that the predicted control targets are effective in a broad dynamic framework. Moreover, several of the predicted interventions are supported by experiments. This work was supported by NSF Grant PHY 1205840.

  20. Pluripotent stem cells and reprogrammed cells in farm animals.

    Science.gov (United States)

    Nowak-Imialek, Monika; Kues, Wilfried; Carnwath, Joseph W; Niemann, Heiner

    2011-08-01

    Pluripotent cells are unique because of their ability to differentiate into the cell lineages forming the entire organism. True pluripotent stem cells with germ line contribution have been reported for mice and rats. Human pluripotent cells share numerous features of pluripotentiality, but confirmation of their in vivo capacity for germ line contribution is impossible due to ethical and legal restrictions. Progress toward derivation of embryonic stem cells from domestic species has been made, but the derived cells were not able to produce germ line chimeras and thus are termed embryonic stem-like cells. However, domestic animals, in particular the domestic pig (Sus scrofa), are excellent large animals models, in which the clinical potential of stem cell therapies can be studied. Reprogramming technologies for somatic cells, including somatic cell nuclear transfer, cell fusion, in vitro culture in the presence of cell extracts, in vitro conversion of adult unipotent spermatogonial stem cells into germ line derived pluripotent stem cells, and transduction with reprogramming factors have been developed with the goal of obtaining pluripotent, germ line competent stem cells from domestic animals. This review summarizes the present state of the art in the derivation and maintenance of pluripotent stem cells in domestic animals.

  1. Targeted gene therapy and cell reprogramming in Fanconi anemia.

    Science.gov (United States)

    Rio, Paula; Baños, Rocio; Lombardo, Angelo; Quintana-Bustamante, Oscar; Alvarez, Lara; Garate, Zita; Genovese, Pietro; Almarza, Elena; Valeri, Antonio; Díez, Begoña; Navarro, Susana; Torres, Yaima; Trujillo, Juan P; Murillas, Rodolfo; Segovia, Jose C; Samper, Enrique; Surralles, Jordi; Gregory, Philip D; Holmes, Michael C; Naldini, Luigi; Bueren, Juan A

    2014-06-01

    Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

  2. Aberrant epigenetic reprogramming of imprinted microRNA-127 and Rtl1 in cloned mouse embryos

    International Nuclear Information System (INIS)

    Cui Xiangshun; Zhang Dingxiao; Ko, Yoeung-Gyu; Kim, Nam-Hyung

    2009-01-01

    The microRNA (miRNA) genes mir-127 and mir-136 are located near two CpG islands in the imprinted mouse retrotransposon-like gene Rtl1, a key gene involved in placenta formation. These miRNAs appear to be involved in regulating the imprinting of Rtl1. To obtain insights into the epigenetic reprogramming of cloned embryos, we compared the expression levels of mir-127 and mir-136 in fertilized mouse embryos, parthenotes, androgenotes and cloned embryos developing in vitro. We also examined the DNA methylation status of the promoter regions of Rtl1 and mir-127 in these embryos. Our data showed that mir-127 and mir-136 were highly expressed in parthenotes, but rarely expressed in androgenotes. Interestingly, the expression levels of mir-127 and mir-136 in parthenotes were almost twice that seen in the fertilized embryos, but were much lower in the cloned embryos. The Rtl1 promoter region was hyper-methylated in blastocyst stage parthenotes (75.0%), moderately methylated (32.4%) in the fertilized embryos and methylated to a much lower extent (∼10%) in the cloned embryos. Conversely, the promoter region of mir-127 was hypo-methylated in parthenogenetically activated embryos (0.4%), moderately methylated (30.0%) in fertilized embryos and heavily methylated in cloned blastocysts (63-70%). These data support a role for mir-127 and mir-136 in the epigenetic reprogramming of the Rtl1 imprinting process. Analysis of the aberrant epigenetic reprogramming of mir-127 and Rtl1 in cloned embryos may help to explain the nuclear reprogramming procedures that occur in donor cells following somatic cell nuclear transfer (SCNT).

  3. Identification of SSEA-1 expressing enhanced reprogramming (SEER) cells in porcine embryonic fibroblasts

    DEFF Research Database (Denmark)

    Li, Dong; Secher, Jan Ole Bertelsen; Juhl, Morten

    2017-01-01

    Previous research has shown that a subpopulation of cells within cultured human dermal fibroblasts, termed multilineage-differentiating stress enduring (Muse) cells, are preferentially reprogrammed into induced pluripotent stem cells. However, controversy exists over whether these cells...... are the only cells capable of being reprogrammed from a heterogeneous population of fibroblasts. Similarly, there is little research to suggest such cells may exist in embryonic tissues or other species. To address if such a cell population exists in pigs, we investigated porcine embryonic fibroblast...... populations (pEFs) and identified heterogeneous expression of several key cell surface markers. Strikingly, we discovered a small population of stage-specific embryonic antigen 1 positive cells (SSEA-1+) in Danish Landrace and Göttingen minipig pEFs, which were absent in the Yucatan pEFs. Furthermore...

  4. Metabolic Reprogramming in Thyroid Carcinoma

    Directory of Open Access Journals (Sweden)

    Raquel Guimaraes Coelho

    2018-03-01

    Full Text Available Among all the adaptations of cancer cells, their ability to change metabolism from the oxidative to the glycolytic phenotype is a hallmark called the Warburg effect. Studies on tumor metabolism show that improved glycolysis and glutaminolysis are necessary to maintain rapid cell proliferation, tumor progression, and resistance to cell death. Thyroid neoplasms are common endocrine tumors that are more prevalent in women and elderly individuals. The incidence of thyroid cancer has increased in the Past decades, and recent findings describing the metabolic profiles of thyroid tumors have emerged. Currently, several drugs are in development or clinical trials that target the altered metabolic pathways of tumors are undergoing. We present a review of the metabolic reprogramming in cancerous thyroid tissues with a focus on the factors that promote enhanced glycolysis and the possible identification of promising metabolic targets in thyroid cancer.

  5. Metabolic Reprogramming in Thyroid Carcinoma

    Science.gov (United States)

    Coelho, Raquel Guimaraes; Fortunato, Rodrigo S.; Carvalho, Denise P.

    2018-01-01

    Among all the adaptations of cancer cells, their ability to change metabolism from the oxidative to the glycolytic phenotype is a hallmark called the Warburg effect. Studies on tumor metabolism show that improved glycolysis and glutaminolysis are necessary to maintain rapid cell proliferation, tumor progression, and resistance to cell death. Thyroid neoplasms are common endocrine tumors that are more prevalent in women and elderly individuals. The incidence of thyroid cancer has increased in the Past decades, and recent findings describing the metabolic profiles of thyroid tumors have emerged. Currently, several drugs are in development or clinical trials that target the altered metabolic pathways of tumors are undergoing. We present a review of the metabolic reprogramming in cancerous thyroid tissues with a focus on the factors that promote enhanced glycolysis and the possible identification of promising metabolic targets in thyroid cancer. PMID:29629339

  6. Optical reprogramming of human somatic cells using ultrashort Bessel-shaped near-infrared femtosecond laser pulses

    Science.gov (United States)

    Uchugonova, Aisada; Breunig, Hans Georg; Batista, Ana; König, Karsten

    2015-11-01

    We report a virus-free optical approach to human cell reprogramming into induced pluripotent stem cells with low-power nanoporation using ultrashort Bessel-shaped laser pulses. Picojoule near-infrared sub-20 fs laser pulses at a high 85 MHz repetition frequency are employed to generate transient nanopores in the membrane of dermal fibroblasts for the introduction of four transcription factors to induce the reprogramming process. In contrast to conventional approaches which utilize retro- or lentiviruses to deliver genes or transcription factors into the host genome, the laser method is virus-free; hence, the risk of virus-induced cancer generation limiting clinical application is avoided.

  7. The CSR-1 endogenous RNAi pathway ensures accurate transcriptional reprogramming during the oocyte-to-embryo transition in Caenorhabditis elegans.

    Science.gov (United States)

    Fassnacht, Christina; Tocchini, Cristina; Kumari, Pooja; Gaidatzis, Dimos; Stadler, Michael B; Ciosk, Rafal

    2018-03-01

    Endogenous RNAi (endoRNAi) is a conserved mechanism for fine-tuning gene expression. In the nematode Caenorhabditis elegans, several endoRNAi pathways are required for the successful development of reproductive cells. The CSR-1 endoRNAi pathway promotes germ cell development, primarily by facilitating the expression of germline genes. In this study, we report a novel function for the CSR-1 pathway in preventing premature activation of embryonic transcription in the developing oocytes, which is accompanied by a general Pol II activation. This CSR-1 function requires its RNase activity, suggesting that, by controlling the levels of maternal mRNAs, CSR-1-dependent endoRNAi contributes to an orderly reprogramming of transcription during the oocyte-to-embryo transition.

  8. Role of SIRT6 in Metabolic Reprogramming During Colorectal Carcinoma

    Science.gov (United States)

    2014-09-01

    pathway. To test this possibility, we analyzed the activation of oncogenic signaling pathways in SIRT6-deficient cells. Because deregulation of most...that SIRT6 sits at a critical metabolic node, modulating both glycolytic metabolism and ribosome biosynthesis (Figure 7L). SIRT6 deficiency deregulates ...C. Metabolic reprogramming: driving tumorigenesis from the origin In 1966, at the meeting of Nobel-Laureates at Lindau, Germany , Otto Warburg

  9. Hierarchical mechanisms for transcription factor-mediated reprogramming of fibroblasts to neurons

    Science.gov (United States)

    Wapinski, Orly L.; Vierbuchen, Thomas; Qu, Kun; Lee, Qian Yi; Chanda, Soham; Fuentes, Daniel R.; Giresi, Paul G.; Ng, Yi Han; Marro, Samuele; Neff, Norma F.; Drechsel, Daniela; Martynoga, Ben; Castro, Diogo S.; Webb, Ashley E.; Brunet, Anne; Guillemot, Francois; Chang, Howard Y.; Wernig, Marius

    2013-01-01

    SUMMARY Direct lineage reprogramming is a promising approach for human disease modeling and regenerative medicine with poorly understood mechanisms. Here we reveal a hierarchical mechanism in the direct conversion of fibroblasts into induced neuronal (iN) cells mediated by the transcription factors Ascl1, Brn2, and Myt1l. Ascl1 acts as an “on target” pioneer factor by immediately occupying most cognate genomic sites in fibroblasts. In contrast, Brn2 and Myt1l do not access fibroblast chromatin productively on their own; instead Ascl1 recruits Brn2 to Ascl1 sites genome-wide. A unique trivalent chromatin signature in the host cells predicts the permissiveness for Ascl1 pioneering activity among different cell types. Finally, we identified Zfp238 as a key Ascl1 target gene that can partially substitute for Ascl1 during iN cell reprogramming. Thus, precise match between pioneer factor and the chromatin context at key target genes is determinative for trans-differentiation to neurons and likely other cell types. PMID:24243019

  10. SIRT3 opposes reprogramming of cancer cell metabolism through HIF1α destabilization

    Science.gov (United States)

    Finley, Lydia W.S.; Carracedo, Arkaitz; Lee, Jaewon; Souza, Amanda; Egia, Ainara; Zhang, Jiangwen; Teruya-Feldstein, Julie; Moreira, Paula I.; Cardoso, Sandra M.; Clish, Clary B.; Pandolfi, Pier Paolo; Haigis, Marcia C.

    2011-01-01

    Summary Tumor cells exhibit aberrant metabolism characterized by high glycolysis even in the presence of oxygen. This metabolic reprogramming, known as the Warburg effect, provides tumor cells with the substrates required for biomass generation. Here, we show that the mitochondrial NAD-dependent deacetylase SIRT3 is a crucial regulator of the Warburg effect. Mechanistically, SIRT3 mediates metabolic reprogramming by destabilizing hypoxia-inducible factor-1α (HIF1α), a transcription factor that controls glycolytic gene expression. SIRT3 loss increases reactive oxygen species production, leading to HIF1α stabilization. SIRT3 expression is reduced in human breast cancers, and its loss correlates with the upregulation of HIF1α target genes. Finally, we find that SIRT3 overexpression represses glycolysis and proliferation in breast cancer cells, providing a metabolic mechanism for tumor suppression. PMID:21397863

  11. Analysis of nuclear reprogramming in cloned miniature pig embryos by expression of Oct-4 and Oct-4 related genes

    International Nuclear Information System (INIS)

    Lee, Eugine; Lee, So Hyun; Kim, Sue

    2006-01-01

    Xenotransplantation is a rapidly expanding field of research and cloned miniature pigs have been considered as a model animal for it. However, the efficiency of somatic cell nuclear transfer (SCNT) is extremely low, with most clones resulting in early lethality and several kinds of aberrant development. A possible explanation for the developmental failure of SCNT embryos is insufficient reprogramming of the somatic cell nucleus by the oocyte. In order to test this, we analyzed the reprogramming capacity of differentiated fibroblast cell nuclei and embryonic germ cell nuclei with Oct-4 and Oct-4 related genes (Ndp5211, Dppa2, Dppa3, and Dppa5), which are important for embryonic development, Hand1 and GATA-4, which are important for placental development, as molecular markers using RT-PCR. The Oct-4 expression level was significantly lower (P < 0.05) in cloned hatched blastocysts derived from fibroblasts and many of fibroblast-derived clones failed to reactivate at least one of the tested genes, while most of the germ cell clones and control embryos correctly expressed these genes. In conclusion, our results suggest that the reprogramming of fibroblast-derived cloned embryos is highly aberrant and this improper reprogramming could be one reason of the early lethality and post-implantation anomalies of somatic cell-derived clones

  12. Generation of Induced Neuronal Cells by the Single Reprogramming Factor ASCL1

    Directory of Open Access Journals (Sweden)

    Soham Chanda

    2014-08-01

    Full Text Available Direct conversion of nonneural cells to functional neurons holds great promise for neurological disease modeling and regenerative medicine. We previously reported rapid reprogramming of mouse embryonic fibroblasts (MEFs into mature induced neuronal (iN cells by forced expression of three transcription factors: ASCL1, MYT1L, and BRN2. Here, we show that ASCL1 alone is sufficient to generate functional iN cells from mouse and human fibroblasts and embryonic stem cells, indicating that ASCL1 is the key driver of iN cell reprogramming in different cell contexts and that the role of MYT1L and BRN2 is primarily to enhance the neuronal maturation process. ASCL1-induced single-factor neurons (1F-iN expressed mature neuronal markers, exhibited typical passive and active intrinsic membrane properties, and formed functional pre- and postsynaptic structures. Surprisingly, ASCL1-induced iN cells were predominantly excitatory, demonstrating that ASCL1 is permissive but alone not deterministic for the inhibitory neuronal lineage.

  13. Term amniotic fluid: an unexploited reserve of mesenchymal stromal cells for reprogramming and potential cell therapy applications.

    Science.gov (United States)

    Moraghebi, Roksana; Kirkeby, Agnete; Chaves, Patricia; Rönn, Roger E; Sitnicka, Ewa; Parmar, Malin; Larsson, Marcus; Herbst, Andreas; Woods, Niels-Bjarne

    2017-08-25

    Mesenchymal stromal cells (MSCs) are currently being evaluated in numerous pre-clinical and clinical cell-based therapy studies. Furthermore, there is an increasing interest in exploring alternative uses of these cells in disease modelling, pharmaceutical screening, and regenerative medicine by applying reprogramming technologies. However, the limited availability of MSCs from various sources restricts their use. Term amniotic fluid has been proposed as an alternative source of MSCs. Previously, only low volumes of term fluid and its cellular constituents have been collected, and current knowledge of the MSCs derived from this fluid is limited. In this study, we collected amniotic fluid at term using a novel collection system and evaluated amniotic fluid MSC content and their characteristics, including their feasibility to undergo cellular reprogramming. Amniotic fluid was collected at term caesarean section deliveries using a closed catheter-based system. Following fluid processing, amniotic fluid was assessed for cellularity, MSC frequency, in-vitro proliferation, surface phenotype, differentiation, and gene expression characteristics. Cells were also reprogrammed to the pluripotent stem cell state and differentiated towards neural and haematopoietic lineages. The average volume of term amniotic fluid collected was approximately 0.4 litres per donor, containing an average of 7 million viable mononuclear cells per litre, and a CFU-F content of 15 per 100,000 MNCs. Expanded CFU-F cultures showed similar surface phenotype, differentiation potential, and gene expression characteristics to MSCs isolated from traditional sources, and showed extensive expansion potential and rapid doubling times. Given the high proliferation rates of these neonatal source cells, we assessed them in a reprogramming application, where the derived induced pluripotent stem cells showed multigerm layer lineage differentiation potential. The potentially large donor base from caesarean section

  14. Peptide-enhanced mRNA transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards cardiomyocyte-like cells

    Directory of Open Access Journals (Sweden)

    Lee K

    2015-03-01

    Full Text Available Kunwoo Lee,1,2 Pengzhi Yu,3 Nithya Lingampalli,1 Hyun Jin Kim,1 Richard Tang,1 Niren Murthy1,2 1Department of Bioengineering, University of California, Berkeley, CA, USA; 2UC Berkeley and UCSF Joint Graduate Program in Bioengineering, Berkeley/San Francisco, CA, USA; 3Gladstone Institute of Cardiovascular Disease, San Francisco, CA, USA Abstract: The treatment of myocardial infarction is a major challenge in medicine due to the inability of heart tissue to regenerate. Direct reprogramming of endogenous cardiac fibroblasts into functional cardiomyocytes via the delivery of transcription factor mRNAs has the potential to regenerate cardiac tissue and to treat heart failure. Even though mRNA delivery to cardiac fibroblasts has the therapeutic potential, mRNA transfection in cardiac fibroblasts has been challenging. Herein, we develop an efficient mRNA transfection in cultured mouse cardiac fibroblasts via a polyarginine-fused heart-targeting peptide and lipofectamine complex, termed C-Lipo and demonstrate the partial direct reprogramming of cardiac fibroblasts towards cardiomyocyte cells. C-Lipo enabled the mRNA-induced direct cardiac reprogramming due to its efficient transfection with low toxicity, which allowed for multiple transfections of Gata4, Mef2c, and Tbx5 (GMT mRNAs for a period of 2 weeks. The induced cardiomyocyte-like cells had α-MHC promoter-driven GFP expression and striated cardiac muscle structure from a-actinin immunohistochemistry. GMT mRNA transfection of cultured mouse cardiac fibroblasts via C-Lipo significantly increased expression of the cardiomyocyte marker genes, Actc1, Actn2, Gja1, Hand2, and Tnnt2, after 2 weeks of transfection. Moreover, this study provides the first direct evidence that the stoichiometry of the GMT reprogramming factors influence the expression of cardiomyocyte marker genes. Our results demonstrate that mRNA delivery is a potential approach for cardiomyocyte generation. Keywords: direct cardiac

  15. The thioredoxin-1 system is essential for fueling DNA synthesis during T-cell metabolic reprogramming and proliferation.

    Science.gov (United States)

    Muri, Jonathan; Heer, Sebastian; Matsushita, Mai; Pohlmeier, Lea; Tortola, Luigi; Fuhrer, Tobias; Conrad, Marcus; Zamboni, Nicola; Kisielow, Jan; Kopf, Manfred

    2018-05-10

    The thioredoxin-1 (Trx1) system is an important contributor to cellular redox balance and is a sensor of energy and glucose metabolism. Here we show critical c-Myc-dependent activation of the Trx1 system during thymocyte and peripheral T-cell proliferation, but repression during T-cell quiescence. Deletion of thioredoxin reductase-1 (Txnrd1) prevents expansion the CD4 - CD8 - thymocyte population, whereas Txnrd1 deletion in CD4 + CD8 + thymocytes does not affect further maturation and peripheral homeostasis of αβT cells. However, Txnrd1 is critical for expansion of the activated T-cell population during viral and parasite infection. Metabolomics show that TrxR1 is essential for the last step of nucleotide biosynthesis by donating reducing equivalents to ribonucleotide reductase. Impaired availability of 2'-deoxyribonucleotides induces the DNA damage response and cell cycle arrest of Txnrd1-deficient T cells. These results uncover a pivotal function of the Trx1 system in metabolic reprogramming of thymic and peripheral T cells and provide a rationale for targeting Txnrd1 in T-cell leukemia.

  16. Metabolic Reprogramming: A Cancer Hallmark Even Warburg Did Not Anticipate

    OpenAIRE

    Ward, Patrick S.; Thompson, Craig B.

    2012-01-01

    Cancer metabolism has long been equated with aerobic glycolysis, seen by early biochemists as primitive and inefficient. Despite these early beliefs, the metabolic signatures of cancer cells are not passive responses to damaged mitochondria, but result from oncogene-directed metabolic reprogramming required to support anabolic growth. Recent evidence suggests that metabolites themselves can be oncogenic by altering cell signaling and blocking cellular differentiation. No longer can cancer-ass...

  17. Secure Rateless Deluge: Pollution-Resistant Reprogramming and Data Dissemination for Wireless Sensor Networks

    NARCIS (Netherlands)

    Law, Y.W.; Zhang, Yu; Jin meifang, J.; Palaniswami, Marimuthu; Havinga, Paul J.M.

    A network reprogramming protocol is made for updating the firmware of a wireless sensor network (WSN) in situ. For security reasons, every firmware update must be authenticated to prevent an attacker from installing its code in the network. While existing schemes can provide authentication services,

  18. Reprogramming somatic cell differentiation and the Hayflick Limit: contrasting two modern molecular bioengineering aims and their impact on the future of mankind.

    Science.gov (United States)

    Sills, E S; Takeuchi, T; Rosenwaks, Z; Palermo, G D

    2001-08-01

    The molecular biology of human cloning and aging research depend on the closely related laboratory techniques supported by a thorough understanding of cell-signaling processes. Unfortunately, the link between these two research fields has received only marginal attention in the lay press. Cloning is possible when somatic cell differentiation is successfully reprogrammed, and clinical control of cellular senescence depends on a proper reconfiguration of the predetermined number of divisions permitted during the cell life-cycle (the so-called "Hayflick Limit"). In this paper, we discuss these two concepts and compare the impact likely to be associated with bioengineering studies that facilitate both human cloning and longevity therapy.

  19. Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions.

    Science.gov (United States)

    Slamecka, Jaroslav; Salimova, Lilia; McClellan, Steven; van Kelle, Mathieu; Kehl, Debora; Laurini, Javier; Cinelli, Paolo; Owen, Laurie; Hoerstrup, Simon P; Weber, Benedikt

    2016-01-01

    Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or β-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.

  20. Opposing regulation of PROX1 by interleukin-3 receptor and NOTCH directs differential host cell fate reprogramming by Kaposi sarcoma herpes virus.

    Directory of Open Access Journals (Sweden)

    Jaehyuk Yoo

    Full Text Available Lymphatic endothelial cells (LECs are differentiated from blood vascular endothelial cells (BECs during embryogenesis and this physiological cell fate specification is controlled by PROX1, the master regulator for lymphatic development. When Kaposi sarcoma herpes virus (KSHV infects host cells, it activates the otherwise silenced embryonic endothelial differentiation program and reprograms their cell fates. Interestingly, previous studies demonstrated that KSHV drives BECs to acquire a partial lymphatic phenotype by upregulating PROX1 (forward reprogramming, but stimulates LECs to regain some BEC-signature genes by downregulating PROX1 (reverse reprogramming. Despite the significance of this KSHV-induced bidirectional cell fate reprogramming in KS pathogenesis, its underlying molecular mechanism remains undefined. Here, we report that IL3 receptor alpha (IL3Rα and NOTCH play integral roles in the host cell type-specific regulation of PROX1 by KSHV. In BECs, KSHV upregulates IL3Rα and phosphorylates STAT5, which binds and activates the PROX1 promoter. In LECs, however, PROX1 was rather downregulated by KSHV-induced NOTCH signal via HEY1, which binds and represses the PROX1 promoter. Moreover, PROX1 was found to be required to maintain HEY1 expression in LECs, establishing a reciprocal regulation between PROX1 and HEY1. Upon co-activation of IL3Rα and NOTCH, PROX1 was upregulated in BECs, but downregulated in LECs. Together, our study provides the molecular mechanism underlying the cell type-specific endothelial fate reprogramming by KSHV.

  1. An integrative analysis of reprogramming in human isogenic system identified a clone selection criterion.

    Science.gov (United States)

    Shutova, Maria V; Surdina, Anastasia V; Ischenko, Dmitry S; Naumov, Vladimir A; Bogomazova, Alexandra N; Vassina, Ekaterina M; Alekseev, Dmitry G; Lagarkova, Maria A; Kiselev, Sergey L

    2016-01-01

    The pluripotency of newly developed human induced pluripotent stem cells (iPSCs) is usually characterized by physiological parameters; i.e., by their ability to maintain the undifferentiated state and to differentiate into derivatives of the 3 germ layers. Nevertheless, a molecular comparison of physiologically normal iPSCs to the "gold standard" of pluripotency, embryonic stem cells (ESCs), often reveals a set of genes with different expression and/or methylation patterns in iPSCs and ESCs. To evaluate the contribution of the reprogramming process, parental cell type, and fortuity in the signature of human iPSCs, we developed a complete isogenic reprogramming system. We performed a genome-wide comparison of the transcriptome and the methylome of human isogenic ESCs, 3 types of ESC-derived somatic cells (fibroblasts, retinal pigment epithelium and neural cells), and 3 pairs of iPSC lines derived from these somatic cells. Our analysis revealed a high input of stochasticity in the iPSC signature that does not retain specific traces of the parental cell type and reprogramming process. We showed that 5 iPSC clones are sufficient to find with 95% confidence at least one iPSC clone indistinguishable from their hypothetical isogenic ESC line. Additionally, on the basis of a small set of genes that are characteristic of all iPSC lines and isogenic ESCs, we formulated an approach of "the best iPSC line" selection and confirmed it on an independent dataset.

  2. High-fat diet reprograms the epigenome of rat spermatozoa and transgenerationally affects metabolism of the offspring

    Directory of Open Access Journals (Sweden)

    Thais de Castro Barbosa

    2016-03-01

    Conclusion: Our results provide insight into mechanisms by which HFD transgenerationally reprograms the epigenome of sperm cells, thereby affecting metabolic tissues of offspring throughout two generations.

  3. Excision of a viral reprogramming cassette by delivery of synthetic Cre mRNA

    Science.gov (United States)

    Loh, Yuin-Han; Yang, Jimmy Chen; De Los Angeles, Alejandro; Guo, Chunguang; Cherry, Anne; Rossi, Derrick J.; Park, In-Hyun; Daley, George Q.

    2012-01-01

    The generation of patient-specific induced pluripotent stem (iPS) cells provides an invaluable resource for cell therapy, in vitro modeling of human disease, and drug screening. To date, most human iPS cells have been generated with integrating retro- and lenti-viruses and are limited in their potential utility because residual transgene expression may alter their differentiation potential or induce malignant transformation. Alternatively, transgene-free methods using adenovirus and protein transduction are limited by low efficiency. This report describes a protocol for the generation of transgene-free human induced pluripotent stem cells using retroviral transfection of a single vector, which includes the coding sequences of human OCT4, SOX2, KLF4, and cMYC linked with picornaviral 2A plasmids. Moreover, after reprogramming has been achieved, this cassette can be removed using mRNA transfection of Cre recombinase. The method described herein to excise reprogramming factors with ease and efficiency facilitates the experimental generation and use of transgene-free human iPS cells. PMID:22605648

  4. Switch-like reprogramming of gene expression after fusion of multinucleate plasmodial cells of two Physarum polycephalum sporulation mutants

    Energy Technology Data Exchange (ETDEWEB)

    Walter, Pauline; Hoffmann, Xenia-Katharina; Ebeling, Britta; Haas, Markus; Marwan, Wolfgang, E-mail: wolfgang.marwan@ovgu.de

    2013-05-24

    Highlights: •We investigate reprogramming of gene expression in multinucleate single cells. •Cells of two differentiation control mutants are fused. •Fused cells proceed to alternative gene expression patterns. •The population of nuclei damps stochastic fluctuations in gene expression. •Dynamic processes of cellular reprogramming can be observed by repeated sampling of a cell. -- Abstract: Nonlinear dynamic processes involving the differential regulation of transcription factors are considered to impact the reprogramming of stem cells, germ cells, and somatic cells. Here, we fused two multinucleate plasmodial cells of Physarum polycephalum mutants defective in different sporulation control genes while being in different physiological states. The resulting heterokaryons established one of two significantly different expression patterns of marker genes while the plasmodial halves that were fused to each other synchronized spontaneously. Spontaneous synchronization suggests that switch-like control mechanisms spread over and finally control the entire plasmodium as a result of cytoplasmic mixing. Regulatory molecules due to the large volume of the vigorously streaming cytoplasm will define concentrations in acting on the population of nuclei and in the global setting of switches. Mixing of a large cytoplasmic volume is expected to damp stochasticity when individual nuclei deliver certain RNAs at low copy number into the cytoplasm. We conclude that spontaneous synchronization, the damping of molecular noise in gene expression by the large cytoplasmic volume, and the option to take multiple macroscopic samples from the same plasmodium provide unique options for studying the dynamics of cellular reprogramming at the single cell level.

  5. Switch-like reprogramming of gene expression after fusion of multinucleate plasmodial cells of two Physarum polycephalum sporulation mutants

    International Nuclear Information System (INIS)

    Walter, Pauline; Hoffmann, Xenia-Katharina; Ebeling, Britta; Haas, Markus; Marwan, Wolfgang

    2013-01-01

    Highlights: •We investigate reprogramming of gene expression in multinucleate single cells. •Cells of two differentiation control mutants are fused. •Fused cells proceed to alternative gene expression patterns. •The population of nuclei damps stochastic fluctuations in gene expression. •Dynamic processes of cellular reprogramming can be observed by repeated sampling of a cell. -- Abstract: Nonlinear dynamic processes involving the differential regulation of transcription factors are considered to impact the reprogramming of stem cells, germ cells, and somatic cells. Here, we fused two multinucleate plasmodial cells of Physarum polycephalum mutants defective in different sporulation control genes while being in different physiological states. The resulting heterokaryons established one of two significantly different expression patterns of marker genes while the plasmodial halves that were fused to each other synchronized spontaneously. Spontaneous synchronization suggests that switch-like control mechanisms spread over and finally control the entire plasmodium as a result of cytoplasmic mixing. Regulatory molecules due to the large volume of the vigorously streaming cytoplasm will define concentrations in acting on the population of nuclei and in the global setting of switches. Mixing of a large cytoplasmic volume is expected to damp stochasticity when individual nuclei deliver certain RNAs at low copy number into the cytoplasm. We conclude that spontaneous synchronization, the damping of molecular noise in gene expression by the large cytoplasmic volume, and the option to take multiple macroscopic samples from the same plasmodium provide unique options for studying the dynamics of cellular reprogramming at the single cell level

  6. Hacker Within! Ehrlichia chaffeensis Effector Driven Phagocyte Reprogramming Strategy

    Directory of Open Access Journals (Sweden)

    Taslima Taher Lina

    2016-05-01

    Full Text Available Ehrlichia chaffeensis is a small, gram negative, obligately intracellular bacterium that preferentially infects mononuclear phagocytes. It is the etiologic agent of human monocytotropic ehrlichiosis (HME, an emerging life-threatening tick-borne zoonosis. Mechanisms by which E. chaffeensis establishes intracellular infection, and avoids host defenses are not well understood, but involve functionally relevant host-pathogen interactions associated with tandem and ankyrin repeat effector proteins. In this review, we discuss the recent advances in our understanding of the molecular and cellular mechanisms that underlie Ehrlichia host cellular reprogramming strategies that enable intracellular survival.

  7. Transient p53 suppression increases reprogramming of human fibroblasts without affecting apoptosis and DNA damage

    DEFF Research Database (Denmark)

    Rasmussen, Mikkel Aabech; Holst, Bjørn; Tümer, Zeynep

    2014-01-01

    The discovery of human-induced pluripotent stem cells (iPSCs) has sparked great interest in the potential treatment of patients with their own in vitro differentiated cells. Recently, knockout of the Tumor Protein 53 (p53) gene was reported to facilitate reprogramming but unfortunately also led...... to genomic instability. Here, we report that transient suppression of p53 during nonintegrative reprogramming of human fibroblasts leads to a significant increase in expression of pluripotency markers and overall number of iPSC colonies, due to downstream suppression of p21, without affecting apoptosis...... and DNA damage. Stable iPSC lines generated with or without p53 suppression showed comparable expression of pluripotency markers and methylation patterns, displayed normal karyotypes, contained between 0 and 5 genomic copy number variations and produced functional neurons in vitro. In conclusion...

  8. Integrating Gene Correction in the Reprogramming and Transdifferentiation Processes: A One-Step Strategy to Overcome Stem Cell-Based Gene Therapy Limitations

    Directory of Open Access Journals (Sweden)

    Seo-Young Lee

    2016-01-01

    Full Text Available The recent advent of induced pluripotent stem cells (iPSCs and gene therapy tools has raised the possibility of autologous cell therapy for rare genetic diseases. However, cellular reprogramming is inefficient in certain diseases such as ataxia telangiectasia, Fanconi anemia, LIG4 syndrome, and fibrodysplasia ossificans progressiva syndrome, owing to interference of the disease-related genes. To overcome these therapeutic limitations, it is necessary to fundamentally correct the abnormal gene during or prior to the reprogramming process. In addition, as genetic etiology of Parkinson’s disease, it has been well known that induced neural stem cells (iNSCs were progressively depleted by LRRK2 gene mutation, LRRK2 (G2019S. Thus, to maintain the induced NSCs directly derived from PD patient cells harboring LRRK2 (G2019S, it would be ideal to simultaneously treat the LRRK2 (G2019S fibroblast during the process of TD. Therefore, simultaneous reprogramming (or TD and gene therapy would provide the solution for therapeutic limitation caused by vulnerability of reprogramming or TD, in addition to being suitable for general application to the generation of autologous cell-therapy products for patients with genetic defects, thereby obviating the need for the arduous processes currently required.

  9. Telomere Length Reprogramming in Embryos and Stem Cells

    Directory of Open Access Journals (Sweden)

    Keri Kalmbach

    2014-01-01

    Full Text Available Telomeres protect and cap linear chromosome ends, yet these genomic buffers erode over an organism’s lifespan. Short telomeres have been associated with many age-related conditions in humans, and genetic mutations resulting in short telomeres in humans manifest as syndromes of precocious aging. In women, telomere length limits a fertilized egg’s capacity to develop into a healthy embryo. Thus, telomere length must be reset with each subsequent generation. Although telomerase is purportedly responsible for restoring telomere DNA, recent studies have elucidated the role of alternative telomeres lengthening mechanisms in the reprogramming of early embryos and stem cells, which we review here.

  10. Left Dorsal Premotor Cortex and Supramarginal Gyrus Complement Each Other during Rapid Action Reprogramming

    DEFF Research Database (Denmark)

    Hartwigsen, Gesa; Bestmann, Sven; Ward, Nick S

    2012-01-01

    The ability to discard a prepared action plan in favor of an alternative action is critical when facing sudden environmental changes. We tested whether the functional contribution of left supramarginal gyrus (SMG) during action reprogramming depends on the functional integrity of left dorsal...... premotor cortex (PMd). Adopting a dual-site repetitive transcranial magnetic stimulation (rTMS) strategy, we first transiently disrupted PMd with "off-line" 1 Hz rTMS and then applied focal "on-line" rTMS to SMG while human subjects performed a spatially precued reaction time (RT) task. Effective on-line r......TMS of SMG but not sham rTMS of SMG increased errors when subjects had to reprogram their action in response to an invalid precue regardless of the type of preceding off-line rTMS. This suggests that left SMG primarily contributes to the on-line updating of actions by suppressing invalidly prepared responses...

  11. Hepatic glucose-6-phosphatase-α deficiency leads to metabolic reprogramming in glycogen storage disease type Ia.

    Science.gov (United States)

    Cho, Jun-Ho; Kim, Goo-Young; Mansfield, Brian C; Chou, Janice Y

    2018-04-15

    Glycogen storage disease type Ia (GSD-Ia) is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC), a key enzyme in endogenous glucose production. This autosomal recessive disorder is characterized by impaired glucose homeostasis and long-term complications of hepatocellular adenoma/carcinoma (HCA/HCC). We have shown that hepatic G6Pase-α deficiency-mediated steatosis leads to defective autophagy that is frequently associated with carcinogenesis. We now show that hepatic G6Pase-α deficiency also leads to enhancement of hepatic glycolysis and hexose monophosphate shunt (HMS) that can contribute to hepatocarcinogenesis. The enhanced hepatic glycolysis is reflected by increased lactate accumulation, increased expression of many glycolytic enzymes, and elevated expression of c-Myc that stimulates glycolysis. The increased HMS is reflected by increased glucose-6-phosphate dehydrogenase activity and elevated production of NADPH and the reduced glutathione. We have previously shown that restoration of hepatic G6Pase-α expression in G6Pase-α-deficient liver corrects metabolic abnormalities, normalizes autophagy, and prevents HCA/HCC development in GSD-Ia. We now show that restoration of hepatic G6Pase-α expression normalizes both glycolysis and HMS in GSD-Ia. Moreover, the HCA/HCC lesions in L-G6pc-/- mice exhibit elevated levels of hexokinase 2 (HK2) and the M2 isoform of pyruvate kinase (PKM2) which play an important role in aerobic glycolysis and cancer cell proliferation. Taken together, hepatic G6Pase-α deficiency causes metabolic reprogramming, leading to enhanced glycolysis and elevated HMS that along with impaired autophagy can contribute to HCA/HCC development in GSD-Ia. Published by Elsevier Inc.

  12. Identification of secretaglobin Scgb2a1 as a target for developmental reprogramming by BPA in the rat prostate.

    Science.gov (United States)

    Wong, Rebecca Lee Yean; Wang, Quan; Treviño, Lindsey S; Bosland, Maarten C; Chen, Jing; Medvedovic, Mario; Prins, Gail S; Kannan, Kurunthachalam; Ho, Shuk-Mei; Walker, Cheryl Lyn

    2015-01-01

    Secretoglobins are a superfamily of secreted proteins thought to participate in inflammation, tissue repair, and tumorigenesis. Secretoglobin family 2A member 1 (Scgb2a1) is a component of prostatein, a major androgen-binding protein secreted by the rat prostate. Using a rat model for developmental reprogramming of susceptibility to prostate carcinogenesis, we identified, by RNA-seq, that Scgb2a1 is significantly upregulated (>100-fold) in the prostate of adult rats neonatally exposed to bisphenol A (BPA), with increased gene expression confirmed by quantitative RT-PCR and chromatin immunoprecipitation for histone H3 lysine 9 acetylation. Bisulfite analysis of both CpG islands located within 10 kb of the Scgb2a1 promoter identified significant hypomethylation of the CpG island upstream of the transcription start site of this gene in the reprogrammed prostate. These data suggest that expression of Scgb2a1 in the adult prostate could be epigenetically reprogrammed by BPA exposure during prostate development, with potential implications for cancer risk and response to chemotherapeutics associated with prostatein binding.

  13. Cellular programming and reprogramming: sculpting cell fate for the production of dopamine neurons for cell therapy.

    Science.gov (United States)

    Aguila, Julio C; Hedlund, Eva; Sanchez-Pernaute, Rosario

    2012-01-01

    Pluripotent stem cells are regarded as a promising cell source to obtain human dopamine neurons in sufficient amounts and purity for cell replacement therapy. Importantly, the success of clinical applications depends on our ability to steer pluripotent stem cells towards the right neuronal identity. In Parkinson disease, the loss of dopamine neurons is more pronounced in the ventrolateral population that projects to the sensorimotor striatum. Because synapses are highly specific, only neurons with this precise identity will contribute, upon transplantation, to the synaptic reconstruction of the dorsal striatum. Thus, understanding the developmental cell program of the mesostriatal dopamine neurons is critical for the identification of the extrinsic signals and cell-intrinsic factors that instruct and, ultimately, determine cell identity. Here, we review how extrinsic signals and transcription factors act together during development to shape midbrain cell fates. Further, we discuss how these same factors can be applied in vitro to induce, select, and reprogram cells to the mesostriatal dopamine fate.

  14. A deterministic method for estimating free energy genetic network landscapes with applications to cell commitment and reprogramming paths.

    Science.gov (United States)

    Olariu, Victor; Manesso, Erica; Peterson, Carsten

    2017-06-01

    Depicting developmental processes as movements in free energy genetic landscapes is an illustrative tool. However, exploring such landscapes to obtain quantitative or even qualitative predictions is hampered by the lack of free energy functions corresponding to the biochemical Michaelis-Menten or Hill rate equations for the dynamics. Being armed with energy landscapes defined by a network and its interactions would open up the possibility of swiftly identifying cell states and computing optimal paths, including those of cell reprogramming, thereby avoiding exhaustive trial-and-error simulations with rate equations for different parameter sets. It turns out that sigmoidal rate equations do have approximate free energy associations. With this replacement of rate equations, we develop a deterministic method for estimating the free energy surfaces of systems of interacting genes at different noise levels or temperatures. Once such free energy landscape estimates have been established, we adapt a shortest path algorithm to determine optimal routes in the landscapes. We explore the method on three circuits for haematopoiesis and embryonic stem cell development for commitment and reprogramming scenarios and illustrate how the method can be used to determine sequential steps for onsets of external factors, essential for efficient reprogramming.

  15. The phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2)-dependent Tup1 conversion (PIPTC) regulates metabolic reprogramming from glycolysis to gluconeogenesis.

    Science.gov (United States)

    Han, Bong-Kwan; Emr, Scott D

    2013-07-12

    Glucose/carbon metabolism is a fundamental cellular process in living cells. In response to varying environments, eukaryotic cells reprogram their glucose/carbon metabolism between aerobic or anaerobic glycolysis, oxidative phosphorylation, and/or gluconeogenesis. The distinct type of glucose/carbon metabolism that a cell carries out has significant effects on the cell's proliferation and differentiation. However, it is poorly understood how the reprogramming of glucose/carbon metabolism is regulated. Here, we report a novel endosomal PI(3,5)P2 lipid-dependent regulatory mechanism that is required for metabolic reprogramming from glycolysis to gluconeogenesis in Saccharomyces cerevisiae. Certain gluconeogenesis genes, such as FBP1 (encoding fructose-1,6-bisphosphatase 1) and ICL1 (encoding isocitrate lyase 1) are under control of the Mig1 repressor and Cyc8-Tup1 corepressor complex. We previously identified the PI(3,5)P2-dependent Tup1 conversion (PIPTC), a mechanism to convert Cyc8-Tup1 corepressor to Cti6-Cyc8-Tup1 coactivator. We demonstrate that the PIPTC plays a critical role for transcriptional activation of FBP1 and ICL1. Furthermore, without the PIPTC, the Cat8 and Sip4 transcriptional activators cannot be efficiently recruited to the promoters of FBP1 and ICL1, suggesting a key role for the PIPTC in remodulating the chromatin architecture at the promoters. Our findings expand our understanding of the regulatory mechanisms for metabolic reprogramming in eukaryotes to include key regulation steps outside the nucleus. Given that Tup1 and the metabolic enzymes that control PI(3,5)P2 are highly conserved among eukaryotes, our findings may provide important insights toward understanding glucose/carbon metabolic reprogramming in other eukaryotes, including humans.

  16. Comprehensive Identification of Krüppel-Like Factor Family Members Contributing to the Self-Renewal of Mouse Embryonic Stem Cells and Cellular Reprogramming.

    Directory of Open Access Journals (Sweden)

    Hyojung Jeon

    Full Text Available Pluripotency is maintained in mouse embryonic stem (ES cells and is induced from somatic cells by the activation of appropriate transcriptional regulatory networks. Krüppel-like factor gene family members, such as Klf2, Klf4 and Klf5, have important roles in maintaining the undifferentiated state of mouse ES cells as well as in cellular reprogramming, yet it is not known whether other Klf family members exert self-renewal and reprogramming functions when overexpressed. In this study, we examined whether overexpression of any representative Klf family member, such as Klf1-Klf10, would be sufficient for the self-renewal of mouse ES cells. We found that only Klf2, Klf4, and Klf5 produced leukemia inhibitory factor (LIF-independent self-renewal, although most KLF proteins, if not all, have the ability to occupy the regulatory regions of Nanog, a critical Klf target gene. We also examined whether overexpression of any of Klf1-Klf10 would be sufficient to convert epiblast stem cells into a naïve pluripotent state and found that Klf5 had such reprogramming ability, in addition to Klf2 and Klf4. We also delineated the functional domains of the Klf2 protein for LIF-independent self-renewal and reprogramming. Interestingly, we found that both the N-terminal transcriptional activation and C-terminal zinc finger domains were indispensable for this activity. Taken together, our comprehensive analysis provides new insight into the contribution of Klf family members to mouse ES self-renewal and cellular reprogramming.

  17. Comprehensive Identification of Krüppel-Like Factor Family Members Contributing to the Self-Renewal of Mouse Embryonic Stem Cells and Cellular Reprogramming.

    Science.gov (United States)

    Jeon, Hyojung; Waku, Tsuyoshi; Azami, Takuya; Khoa, Le Tran Phuc; Yanagisawa, Jun; Takahashi, Satoru; Ema, Masatsugu

    2016-01-01

    Pluripotency is maintained in mouse embryonic stem (ES) cells and is induced from somatic cells by the activation of appropriate transcriptional regulatory networks. Krüppel-like factor gene family members, such as Klf2, Klf4 and Klf5, have important roles in maintaining the undifferentiated state of mouse ES cells as well as in cellular reprogramming, yet it is not known whether other Klf family members exert self-renewal and reprogramming functions when overexpressed. In this study, we examined whether overexpression of any representative Klf family member, such as Klf1-Klf10, would be sufficient for the self-renewal of mouse ES cells. We found that only Klf2, Klf4, and Klf5 produced leukemia inhibitory factor (LIF)-independent self-renewal, although most KLF proteins, if not all, have the ability to occupy the regulatory regions of Nanog, a critical Klf target gene. We also examined whether overexpression of any of Klf1-Klf10 would be sufficient to convert epiblast stem cells into a naïve pluripotent state and found that Klf5 had such reprogramming ability, in addition to Klf2 and Klf4. We also delineated the functional domains of the Klf2 protein for LIF-independent self-renewal and reprogramming. Interestingly, we found that both the N-terminal transcriptional activation and C-terminal zinc finger domains were indispensable for this activity. Taken together, our comprehensive analysis provides new insight into the contribution of Klf family members to mouse ES self-renewal and cellular reprogramming.

  18. Cellular reprogramming by gram-positive bacterial components: a review.

    LENUS (Irish Health Repository)

    Buckley, Julliette M

    2012-02-03

    LPS tolerance has been the focus of extensive scientific and clinical research over the last several decades in an attempt to elucidate the sequence of changes that occur at a molecular level in tolerized cells. Tolerance to components of gram-positive bacterial cell walls such as bacterial lipoprotein and lipoteichoic acid is a much lesser studied, although equally important, phenomenon. This review will focus on cellular reprogramming by gram-positive bacterial components and examines the alterations in cell surface receptor expression, changes in intracellular signaling, gene expression and cytokine production, and the phenomenon of cross-tolerance.

  19. PKM2-dependent metabolic reprogramming in CD4+ T cells is crucial for hyperhomocysteinemia-accelerated atherosclerosis.

    Science.gov (United States)

    Lü, Silin; Deng, Jiacheng; Liu, Huiying; Liu, Bo; Yang, Juan; Miao, Yutong; Li, Jing; Wang, Nan; Jiang, Changtao; Xu, Qingbo; Wang, Xian; Feng, Juan

    2018-06-01

    Inflammation mediated by activated T cells plays an important role in the initiation and progression of hyperhomocysteinemia (HHcy)-accelerated atherosclerosis in ApoE -/- mice. Homocysteine (Hcy) activates T cells to secrete proinflammatory cytokines, especially interferon (IFN)-γ; however, the precise mechanisms remain unclear. Metabolic reprogramming is critical for T cell inflammatory activation and effector functions. Our previous study demonstrated that Hcy regulates T cell mitochondrial reprogramming by enhancing endoplasmic reticulum (ER)-mitochondria coupling. In this study, we further explored the important role of glycolysis-mediated metabolic reprogramming in Hcy-activated CD4 + T cells. Mechanistically, Hcy-activated CD4 + T cell increased the protein expression and activity of pyruvate kinase muscle isozyme 2 (PKM2), the final rate-limiting enzyme in glycolysis, via the phosphatidylinositol 3-kinase/AKT/mechanistic target of rapamycin signaling pathway. Knockdown of PKM2 by small interfering RNA reduced Hcy-induced CD4 + T cell IFN-γ secretion. Furthermore, we generated T cell-specific PKM2 knockout mice by crossing LckCre transgenic mice with PKM2 fl/fl mice and observed that Hcy-induced glycolysis and oxidative phosphorylation were both diminished in PKM2-deficient CD4 + T cells with reduced glucose and lipid metabolites, and subsequently reduced IFN-γ secretion. T cell-depleted apolipoprotein E-deficient (ApoE -/- ) mice adoptively transferred with PKM2-deficient CD4 + T cells, compared to mice transferred with control cells, showed significantly decreased HHcy-accelerated early atherosclerotic lesion formation. In conclusion, this work indicates that the PKM2-dependent glycolytic-lipogenic axis, a novel mechanism of metabolic regulation, is crucial for HHcy-induced CD4 + T cell activation to accelerate early atherosclerosis in ApoE -/- mice. Metabolic reprogramming is crucial for Hcy-induced CD4 + T cell inflammatory activation. Hcy activates

  20. Choices for Induction of Pluripotency: Recent Developments in Human Induced Pluripotent Stem Cell Reprogramming Strategies

    NARCIS (Netherlands)

    Brouwer, M.; Zhou, Huiqing; Nadif Kasri, N.

    2016-01-01

    The ability to generate human induced pluripotent stem cells (iPSCs) from somatic cells provides tremendous promises for regenerative medicine and its use has widely increased over recent years. However, reprogramming efficiencies remain low and chromosomal instability and tumorigenic potential are

  1. Cellular Reprogramming Using Protein and Cell-Penetrating Peptides

    Directory of Open Access Journals (Sweden)

    Bong Jong Seo

    2017-03-01

    Full Text Available Recently, stem cells have been suggested as invaluable tools for cell therapy because of their self-renewal and multilineage differentiation potential. Thus, scientists have developed a variety of methods to generate pluripotent stem cells, from nuclear transfer technology to direct reprogramming using defined factors, or induced pluripotent stem cells (iPSCs. Considering the ethical issues and efficiency, iPSCs are thought to be one of the most promising stem cells for cell therapy. Induced pluripotent stem cells can be generated by transduction with a virus, plasmid, RNA, or protein. Herein, we provide an overview of the current technology for iPSC generation and describe protein-based transduction technology in detail.

  2. A Survey of Basic Facilities and Service Provision for the Successful Inclusion of the Students with Hearing Impairment in Inklusive Education Setting in Cross River, Akwa IBOM & Rivers State of Nigeria

    Directory of Open Access Journals (Sweden)

    James Abua Ewa

    2014-12-01

    Full Text Available Obviously, since the challenges of life change with time, education should aim at bringing up children with hearing impairment to face these changes and make necessary adjustment in their lives for successful living. Hence education programs and service provisions should therefore be dynamic. To realise these important functions, programs and services designed for the students with hearing impairment in an inclusive education setting should not be on exemption, since the society itself is dynamic in many respects, its fundamental needs equally change with time which hitherto makes societal objective changes also, owing to advancement in scientific inventions, innovation in trends and practices of inclusive education. Bearing this in mind, this paper sought to ascertain the level of provision and availability of basic facilities and services necessary for the successful inclusion of students with hearing impairment. The survey focused on the availability and use of assistive technology devices by the hearing impaired students, recruitment and training of teaching and supportive staff, communication strategies adapted by the teachers, medical and socials services .Data were collected using a Likert-like questionnaire from the sampled secondary schools in the states under investigation. The researcher analysed the data using the Pearson Product Moment Correlation co-efficient. The results revealed that much is yet to be put in place in the states under study for the successful inclusion of students with hearing impairment into inclusive education setting.

  3. Engineering kidney cells: reprogramming and directed differentiation to renal tissues.

    Science.gov (United States)

    Kaminski, Michael M; Tosic, Jelena; Pichler, Roman; Arnold, Sebastian J; Lienkamp, Soeren S

    2017-07-01

    Growing knowledge of how cell identity is determined at the molecular level has enabled the generation of diverse tissue types, including renal cells from pluripotent or somatic cells. Recently, several in vitro protocols involving either directed differentiation or transcription-factor-based reprogramming to kidney cells have been established. Embryonic stem cells or induced pluripotent stem cells can be guided towards a kidney fate by exposing them to combinations of growth factors or small molecules. Here, renal development is recapitulated in vitro resulting in kidney cells or organoids that show striking similarities to mammalian embryonic nephrons. In addition, culture conditions are also defined that allow the expansion of renal progenitor cells in vitro. Another route towards the generation of kidney cells is direct reprogramming. Key transcription factors are used to directly impose renal cell identity on somatic cells, thus circumventing the pluripotent stage. This complementary approach to stem-cell-based differentiation has been demonstrated to generate renal tubule cells and nephron progenitors. In-vitro-generated renal cells offer new opportunities for modelling inherited and acquired renal diseases on a patient-specific genetic background. These cells represent a potential source for developing novel models for kidney diseases, drug screening and nephrotoxicity testing and might represent the first steps towards kidney cell replacement therapies. In this review, we summarize current approaches for the generation of renal cells in vitro and discuss the advantages of each approach and their potential applications.

  4. Second generation codon optimized minicircle (CoMiC) for nonviral reprogramming of human adult fibroblasts.

    Science.gov (United States)

    Diecke, Sebastian; Lisowski, Leszek; Kooreman, Nigel G; Wu, Joseph C

    2014-01-01

    The ability to induce pluripotency in somatic cells is one of the most important scientific achievements in the fields of stem cell research and regenerative medicine. This technique allows researchers to obtain pluripotent stem cells without the controversial use of embryos, providing a novel and powerful tool for disease modeling and drug screening approaches. However, using viruses for the delivery of reprogramming genes and transcription factors may result in integration into the host genome and cause random mutations within the target cell, thus limiting the use of these cells for downstream applications. To overcome this limitation, various non-integrating techniques, including Sendai virus, mRNA, minicircle, and plasmid-based methods, have recently been developed. Utilizing a newly developed codon optimized 4-in-1 minicircle (CoMiC), we were able to reprogram human adult fibroblasts using chemically defined media and without the need for feeder cells.

  5. In vitro epigenetic reprogramming of human cardiac mesenchymal stromal cells into functionally competent cardiovascular precursors.

    Directory of Open Access Journals (Sweden)

    Matteo Vecellio

    Full Text Available Adult human cardiac mesenchymal-like stromal cells (CStC represent a relatively accessible cell type useful for therapy. In this light, their conversion into cardiovascular precursors represents a potential successful strategy for cardiac repair. The aim of the present work was to reprogram CStC into functionally competent cardiovascular precursors using epigenetically active small molecules. CStC were exposed to low serum (5% FBS in the presence of 5 µM all-trans Retinoic Acid (ATRA, 5 µM Phenyl Butyrate (PB, and 200 µM diethylenetriamine/nitric oxide (DETA/NO, to create a novel epigenetically active cocktail (EpiC. Upon treatment the expression of markers typical of cardiac resident stem cells such as c-Kit and MDR-1 were up-regulated, together with the expression of a number of cardiovascular-associated genes including KDR, GATA6, Nkx2.5, GATA4, HCN4, NaV1.5, and α-MHC. In addition, profiling analysis revealed that a significant number of microRNA involved in cardiomyocyte biology and cell differentiation/proliferation, including miR 133a, 210 and 34a, were up-regulated. Remarkably, almost 45% of EpiC-treated cells exhibited a TTX-sensitive sodium current and, to a lower extent in a few cells, also the pacemaker I(f current. Mechanistically, the exposure to EpiC treatment introduced global histone modifications, characterized by increased levels of H3K4Me3 and H4K16Ac, as well as reduced H4K20Me3 and H3s10P, a pattern compatible with reduced proliferation and chromatin relaxation. Consistently, ChIP experiments performed with H3K4me3 or H3s10P histone modifications revealed the presence of a specific EpiC-dependent pattern in c-Kit, MDR-1, and Nkx2.5 promoter regions, possibly contributing to their modified expression. Taken together, these data indicate that CStC may be epigenetically reprogrammed to acquire molecular and biological properties associated with competent cardiovascular precursors.

  6. Characterization of primary human mammary epithelial cells isolated and propagated by conditional reprogrammed cell culture.

    Science.gov (United States)

    Jin, Liting; Qu, Ying; Gomez, Liliana J; Chung, Stacey; Han, Bingchen; Gao, Bowen; Yue, Yong; Gong, Yiping; Liu, Xuefeng; Amersi, Farin; Dang, Catherine; Giuliano, Armando E; Cui, Xiaojiang

    2018-02-20

    Conditional reprogramming methods allow for the inexhaustible in vitro proliferation of primary epithelial cells from human tissue specimens. This methodology has the potential to enhance the utility of primary cell culture as a model for mammary gland research. However, few studies have systematically characterized this method in generating in vitro normal human mammary epithelial cell models. We show that cells derived from fresh normal breast tissues can be propagated and exhibit heterogeneous morphologic features. The cultures are composed of CK18, desmoglein 3, and CK19-positive luminal cells and vimentin, p63, and CK14-positive myoepithelial cells, suggesting the maintenance of in vivo heterogeneity. In addition, the cultures contain subpopulations with different CD49f and EpCAM expression profiles. When grown in 3D conditions, cells self-organize into distinct structures that express either luminal or basal cell markers. Among these structures, CK8-positive cells enclosing a lumen are capable of differentiation into milk-producing cells in the presence of lactogenic stimulus. Furthermore, our short-term cultures retain the expression of ERα, as well as its ability to respond to estrogen stimulation. We have investigated conditionally reprogrammed normal epithelial cells in terms of cell type heterogeneity, cellular marker expression, and structural arrangement in two-dimensional (2D) and three-dimensional (3D) systems. The conditional reprogramming methodology allows generation of a heterogeneous culture from normal human mammary tissue in vitro . We believe that this cell culture model will provide a valuable tool to study mammary cell function and malignant transformation.

  7. The Phosphatidylinositol 3,5-Bisphosphate (PI(3,5)P2)-dependent Tup1 Conversion (PIPTC) Regulates Metabolic Reprogramming from Glycolysis to Gluconeogenesis*

    Science.gov (United States)

    Han, Bong-Kwan; Emr, Scott D.

    2013-01-01

    Glucose/carbon metabolism is a fundamental cellular process in living cells. In response to varying environments, eukaryotic cells reprogram their glucose/carbon metabolism between aerobic or anaerobic glycolysis, oxidative phosphorylation, and/or gluconeogenesis. The distinct type of glucose/carbon metabolism that a cell carries out has significant effects on the cell's proliferation and differentiation. However, it is poorly understood how the reprogramming of glucose/carbon metabolism is regulated. Here, we report a novel endosomal PI(3,5)P2 lipid-dependent regulatory mechanism that is required for metabolic reprogramming from glycolysis to gluconeogenesis in Saccharomyces cerevisiae. Certain gluconeogenesis genes, such as FBP1 (encoding fructose-1,6-bisphosphatase 1) and ICL1 (encoding isocitrate lyase 1) are under control of the Mig1 repressor and Cyc8-Tup1 corepressor complex. We previously identified the PI(3,5)P2-dependent Tup1 conversion (PIPTC), a mechanism to convert Cyc8-Tup1 corepressor to Cti6-Cyc8-Tup1 coactivator. We demonstrate that the PIPTC plays a critical role for transcriptional activation of FBP1 and ICL1. Furthermore, without the PIPTC, the Cat8 and Sip4 transcriptional activators cannot be efficiently recruited to the promoters of FBP1 and ICL1, suggesting a key role for the PIPTC in remodulating the chromatin architecture at the promoters. Our findings expand our understanding of the regulatory mechanisms for metabolic reprogramming in eukaryotes to include key regulation steps outside the nucleus. Given that Tup1 and the metabolic enzymes that control PI(3,5)P2 are highly conserved among eukaryotes, our findings may provide important insights toward understanding glucose/carbon metabolic reprogramming in other eukaryotes, including humans. PMID:23733183

  8. Developmental Programming of Renal Function and Re-Programming Approaches

    Science.gov (United States)

    Nüsken, Eva; Dötsch, Jörg; Weber, Lutz T.; Nüsken, Kai-Dietrich

    2018-01-01

    Chronic kidney disease affects more than 10% of the population. Programming studies have examined the interrelationship between environmental factors in early life and differences in morbidity and mortality between individuals. A number of important principles has been identified, namely permanent structural modifications of organs and cells, long-lasting adjustments of endocrine regulatory circuits, as well as altered gene transcription. Risk factors include intrauterine deficiencies by disturbed placental function or maternal malnutrition, prematurity, intrauterine and postnatal stress, intrauterine and postnatal overnutrition, as well as dietary dysbalances in postnatal life. This mini-review discusses critical developmental periods and long-term sequelae of renal programming in humans and presents studies examining the underlying mechanisms as well as interventional approaches to “re-program” renal susceptibility toward disease. Clinical manifestations of programmed kidney disease include arterial hypertension, proteinuria, aggravation of inflammatory glomerular disease, and loss of kidney function. Nephron number, regulation of the renin–angiotensin–aldosterone system, renal sodium transport, vasomotor and endothelial function, myogenic response, and tubuloglomerular feedback have been identified as being vulnerable to environmental factors. Oxidative stress levels, metabolic pathways, including insulin, leptin, steroids, and arachidonic acid, DNA methylation, and histone configuration may be significantly altered by adverse environmental conditions. Studies on re-programming interventions focused on dietary or anti-oxidative approaches so far. Further studies that broaden our understanding of renal programming mechanisms are needed to ultimately develop preventive strategies. Targeted re-programming interventions in animal models focusing on known mechanisms will contribute to new concepts which finally will have to be translated to human application

  9. Thermodynamic Aspects and Reprogramming Cellular Energy Metabolism during the Fibrosis Process

    Directory of Open Access Journals (Sweden)

    Alexandre Vallée

    2017-11-01

    Full Text Available Fibrosis is characterized by fibroblast proliferation and fibroblast differentiation into myofibroblasts, which generate a relaxation-free contraction mechanism associated with excessive collagen synthesis in the extracellular matrix, which promotes irreversible tissue retraction evolving towards fibrosis. From a thermodynamic point of view, the mechanisms leading to fibrosis are irreversible processes that can occur through changing the entropy production rate. The thermodynamic behaviors of metabolic enzymes involved in fibrosis are modified by the dysregulation of both transforming growth factor β (TGF-β signaling and the canonical WNT/β-catenin pathway, leading to aerobic glycolysis, called the Warburg effect. Molecular signaling pathways leading to fibrosis are considered dissipative structures that exchange energy or matter with their environment far from the thermodynamic equilibrium. The myofibroblastic cells arise from exergonic processes by switching the core metabolism from oxidative phosphorylation to glycolysis, which generates energy and reprograms cellular energy metabolism to induce the process of myofibroblast differentiation. Circadian rhythms are far-from-equilibrium thermodynamic processes. They directly participate in regulating the TGF-β and WNT/β-catenin pathways involved in energetic dysregulation and enabling fibrosis. The present review focusses on the thermodynamic implications of the reprogramming of cellular energy metabolism, leading to fibroblast differentiation into myofibroblasts through the positive interplay between TGF-β and WNT/β-catenin pathways underlying in fibrosis.

  10. CpG preconditioning regulates miRNA expression that modulates genomic reprogramming associated with neuroprotection against ischemic injury

    Science.gov (United States)

    Vartanian, Keri B; Mitchell, Hugh D; Stevens, Susan L; Conrad, Valerie K; McDermott, Jason E; Stenzel-Poore, Mary P

    2015-01-01

    Cytosine-phosphate-guanine (CpG) preconditioning reprograms the genomic response to stroke to protect the brain against ischemic injury. The mechanisms underlying genomic reprogramming are incompletely understood. MicroRNAs (miRNAs) regulate gene expression; however, their role in modulating gene responses produced by CpG preconditioning is unknown. We evaluated brain miRNA expression in response to CpG preconditioning before and after stroke using microarray. Importantly, we have data from previous gene microarrays under the same conditions, which allowed integration of miRNA and gene expression data to specifically identify regulated miRNA gene targets. CpG preconditioning did not significantly alter miRNA expression before stroke, indicating that miRNA regulation is not critical for the initiation of preconditioning-induced neuroprotection. However, after stroke, differentially regulated miRNAs between CpG- and saline-treated animals associated with the upregulation of several neuroprotective genes, implicating these miRNAs in genomic reprogramming that increases neuroprotection. Statistical analysis revealed that the miRNA targets were enriched in the gene population regulated in the setting of stroke, implying that miRNAs likely orchestrate this gene expression. These data suggest that miRNAs regulate endogenous responses to stroke and that manipulation of these miRNAs may have the potential to acutely activate novel neuroprotective processes that reduce damage. PMID:25388675

  11. MFG-E8 Reprogramming of Macrophages Promotes Wound Healing by Increased bFGF Production and Fibroblast Functions.

    Science.gov (United States)

    Laplante, Patrick; Brillant-Marquis, Frédéric; Brissette, Marie-Joëlle; Joannette-Pilon, Benjamin; Cayrol, Romain; Kokta, Victor; Cailhier, Jean-François

    2017-09-01

    Macrophages are essential for tissue repair. They have a crucial role in cutaneous wound healing, participating actively in the inflammation phase of the process. Unregulated macrophage activation may, however, represent a source of excessive inflammation, leading to abnormal wound healing and hypertrophic scars. Our research group has shown that apoptotic endothelial and epithelial cells secrete MFG-E8, which has the ability to reprogram macrophages from an M1 (proinflammatory) to an M2 (anti-inflammatory, pro-repair) phenotype. Hence, we tested whether modulation of macrophage reprogramming would promote tissue repair. Using a mouse model of wound healing, we showed that the presence and/or addition of MFG-E8 favors wound closure associated with an increase in CD206-positive cells and basic fibroblast growth factor production in healing tissues. More importantly, adoptive transfer of ex vivo MFG-E8-treated macrophages promoted wound closure. We also observed that MFG-E8-treated macrophages produced basic fibroblast growth factor that is responsible for fibroblast migration and proliferation. Taken together, our results strongly suggest that MFG-E8 plays a key role in macrophage reprogramming in tissue healing through induction of an anti-inflammatory M2 phenotype and basic fibroblast growth factor production, leading to fibroblast migration and wound closure. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  12. ATF4-Induced Metabolic Reprograming Is a Synthetic Vulnerability of the p62-Deficient Tumor Stroma.

    Science.gov (United States)

    Linares, Juan F; Cordes, Thekla; Duran, Angeles; Reina-Campos, Miguel; Valencia, Tania; Ahn, Christopher S; Castilla, Elias A; Moscat, Jorge; Metallo, Christian M; Diaz-Meco, Maria T

    2017-12-05

    Tumors undergo nutrient stress and need to reprogram their metabolism to survive. The stroma may play a critical role in this process by providing nutrients to support the epithelial compartment of the tumor. Here we show that p62 deficiency in stromal fibroblasts promotes resistance to glutamine deprivation by the direct control of ATF4 stability through its p62-mediated polyubiquitination. ATF4 upregulation by p62 deficiency in the stroma activates glucose carbon flux through a pyruvate carboxylase-asparagine synthase cascade that results in asparagine generation as a source of nitrogen for stroma and tumor epithelial proliferation. Thus, p62 directly targets nuclear transcription factors to control metabolic reprogramming in the microenvironment and repress tumorigenesis, and identifies ATF4 as a synthetic vulnerability in p62-deficient tumor stroma. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Epigenetic reprogramming of pericentromeric satellite DNA in premalignant and malignant lesions

    DEFF Research Database (Denmark)

    Brückmann, Nadine Heidi; Pedersen, Christina Bøg; Ditzel, Henrik Jørn

    2018-01-01

    on pericentromeric satellites in primary melanocytes. This suggests that polycomb bodies form in cancer cells with global DNA demethylation to control the stability of pericentromeric satellite DNA. These results reveal a novel epigenetic perturbation specific to premalignant and malignant cells thatmaybe used...... as an early diagnostic marker for detection of precancerous changes and a new therapeutic entry point. Implications: Pericentromeric satellite DNA is epigenetically reprogrammed into polycomb bodies as a premalignant event with implications for transcriptional activity and genomic stability. Mol Cancer Res...

  14. Epigenetic reprogramming in Mist1(-/- mice predicts the molecular response to cerulein-induced pancreatitis.

    Directory of Open Access Journals (Sweden)

    Rashid Mehmood

    Full Text Available Gene expression is affected by modifications to histone core proteins within chromatin. Changes in these modifications, or epigenetic reprogramming, can dictate cell fate and promote susceptibility to disease. The goal of this study was to determine the extent of epigenetic reprogramming in response to chronic stress that occurs following ablation of MIST1 (Mist1(-/- , which is repressed in pancreatic disease. Chromatin immunoprecipitation for trimethylation of lysine residue 4 on histone 3 (H3K4Me3 in purified acinar cells from wild type and Mist1(-/- mice was followed by Next Generation sequencing (ChIP-seq or ChIP-qPCR. H3K4Me3-enriched genes were assessed for expression by qRT-PCR in pancreatic tissue before and after induction of cerulein-induced pancreatitis. While most of H3K4Me3-enrichment is restricted to transcriptional start sites, >25% of enrichment sites are found within, downstream or between annotated genes. Less than 10% of these sites were altered in Mist1(-/- acini, with most changes in H3K4Me3 enrichment not reflecting altered gene expression. Ingenuity Pathway Analysis of genes differentially-enriched for H3K4Me3 revealed an association with pancreatitis and pancreatic ductal adenocarcinoma in Mist1(-/- tissue. Most of these genes were not differentially expressed but several were readily induced by acute experimental pancreatitis, with significantly increased expression in Mist1(-/- tissue relative to wild type mice. We suggest that the chronic cell stress observed in the absence of MIST1 results in epigenetic reprogramming of genes involved in promoting pancreatitis to a poised state, thereby increasing the sensitivity to events that promote disease.

  15. Reprogramming LCLs to iPSCs Results in Recovery of Donor-Specific Gene Expression Signature.

    Directory of Open Access Journals (Sweden)

    Samantha M Thomas

    2015-05-01

    Full Text Available Renewable in vitro cell cultures, such as lymphoblastoid cell lines (LCLs, have facilitated studies that contributed to our understanding of genetic influence on human traits. However, the degree to which cell lines faithfully maintain differences in donor-specific phenotypes is still debated. We have previously reported that standard cell line maintenance practice results in a loss of donor-specific gene expression signatures in LCLs. An alternative to the LCL model is the induced pluripotent stem cell (iPSC system, which carries the potential to model tissue-specific physiology through the use of differentiation protocols. Still, existing LCL banks represent an important source of starting material for iPSC generation, and it is possible that the disruptions in gene regulation associated with long-term LCL maintenance could persist through the reprogramming process. To address this concern, we studied the effect of reprogramming mature LCL cultures from six unrelated donors to iPSCs on the ensuing gene expression patterns within and between individuals. We show that the reprogramming process results in a recovery of donor-specific gene regulatory signatures, increasing the number of genes with a detectable donor effect by an order of magnitude. The proportion of variation in gene expression statistically attributed to donor increases from 6.9% in LCLs to 24.5% in iPSCs (P < 10-15. Since environmental contributions are unlikely to be a source of individual variation in our system of highly passaged cultured cell lines, our observations suggest that the effect of genotype on gene regulation is more pronounced in iPSCs than in LCLs. Our findings indicate that iPSCs can be a powerful model system for studies of phenotypic variation across individuals in general, and the genetic association with variation in gene regulation in particular. We further conclude that LCLs are an appropriate starting material for iPSC generation.

  16. System-Wide Hypersensitive Response-Associated Transcriptome and Metabolome Reprogramming in Tomato1[W][OA

    Science.gov (United States)

    Etalo, Desalegn W.; Stulemeijer, Iris J.E.; Peter van Esse, H.; de Vos, Ric C.H.; Bouwmeester, Harro J.; Joosten, Matthieu H.A.J.

    2013-01-01

    The hypersensitive response (HR) is considered to be the hallmark of the resistance response of plants to pathogens. To study HR-associated transcriptome and metabolome reprogramming in tomato (Solanum lycopersicum), we used plants that express both a resistance gene to Cladosporium fulvum and the matching avirulence gene of this pathogen. In these plants, massive reprogramming occurred, and we found that the HR and associated processes are highly energy demanding. Ubiquitin-dependent protein degradation, hydrolysis of sugars, and lipid catabolism are used as alternative sources of amino acids, energy, and carbon skeletons, respectively. We observed strong accumulation of secondary metabolites, such as hydroxycinnamic acid amides. Coregulated expression of WRKY transcription factors and genes known to be involved in the HR, in addition to a strong enrichment of the W-box WRKY-binding motif in the promoter sequences of the coregulated genes, point to WRKYs as the most prominent orchestrators of the HR. Our study has revealed several novel HR-related genes, and reverse genetics tools will allow us to understand the role of each individual component in the HR. PMID:23719893

  17. Positional information is reprogrammed in blastema cells of the regenerating limb of the axolotl (Ambystoma mexicanum).

    Science.gov (United States)

    McCusker, Catherine D; Gardiner, David M

    2013-01-01

    The regenerating region of an amputated salamander limb, known as the blastema, has the amazing capacity to replace exactly the missing structures. By grafting cells from different stages and regions of blastemas induced to form on donor animals expressing Green Fluorescent Protein (GFP), to non-GFP host animals, we have determined that the cells from early stage blastemas, as well as cells at the tip of late stage blastemas are developmentally labile such that their positional identity is reprogrammed by interactions with more proximal cells with stable positional information. In contrast, cells from the adjacent, more proximal stump tissues as well as the basal region of late bud blastemas are positionally stable, and thus form ectopic limb structures when grafted. Finally, we have found that a nerve is required to maintain the blastema cells in a positionally labile state, thus indicating a role for reprogramming cues in the blastema microenvironment.

  18. Developmental Programming of Renal Function and Re-Programming Approaches

    Directory of Open Access Journals (Sweden)

    Eva Nüsken

    2018-02-01

    Full Text Available Chronic kidney disease affects more than 10% of the population. Programming studies have examined the interrelationship between environmental factors in early life and differences in morbidity and mortality between individuals. A number of important principles has been identified, namely permanent structural modifications of organs and cells, long-lasting adjustments of endocrine regulatory circuits, as well as altered gene transcription. Risk factors include intrauterine deficiencies by disturbed placental function or maternal malnutrition, prematurity, intrauterine and postnatal stress, intrauterine and postnatal overnutrition, as well as dietary dysbalances in postnatal life. This mini-review discusses critical developmental periods and long-term sequelae of renal programming in humans and presents studies examining the underlying mechanisms as well as interventional approaches to “re-program” renal susceptibility toward disease. Clinical manifestations of programmed kidney disease include arterial hypertension, proteinuria, aggravation of inflammatory glomerular disease, and loss of kidney function. Nephron number, regulation of the renin–angiotensin–aldosterone system, renal sodium transport, vasomotor and endothelial function, myogenic response, and tubuloglomerular feedback have been identified as being vulnerable to environmental factors. Oxidative stress levels, metabolic pathways, including insulin, leptin, steroids, and arachidonic acid, DNA methylation, and histone configuration may be significantly altered by adverse environmental conditions. Studies on re-programming interventions focused on dietary or anti-oxidative approaches so far. Further studies that broaden our understanding of renal programming mechanisms are needed to ultimately develop preventive strategies. Targeted re-programming interventions in animal models focusing on known mechanisms will contribute to new concepts which finally will have to be translated

  19. Reprogramming tumor-infiltrating dendritic cells for CD103+CD8+ mucosal T cell differentiation and breast cancer rejection

    Science.gov (United States)

    Wu, Te-Chia; Xu, Kangling; Banchereau, Romain; Marches, Florentina; Yu, Chun I; Martinek, Jan; Anguiano, Esperanza; Pedroza-Gonzalez, Alexander; Snipes, G. Jackson; O’Shaughnessy, Joyce; Nishimura, Stephen; Liu, Yong-Jun; Pascual, Virginia; Banchereau, Jacques; Oh, Sangkon; Palucka, Karolina

    2014-01-01

    Our studies showed that tumor-infiltrating dendritic cells (DC) in breast cancer drive inflammatory T helper 2 (iTh2) cells and protumor inflammation. Here we show that intratumoral delivery of the β-glucan curdlan, a ligand of dectin-1, blocks the generation of iTh2 cells, and prevents breast cancer progression in vivo. Curdlan reprograms tumor-infiltrating DC via the ligation of dectin-1, enabling the DC to become resistant to cancer-derived thymic stromal lymphopoietin (TSLP), to produce IL12p70, and to favor the generation of T helper 1 (Th1) cells. DC activated via dectin-1, but not those activated with TLR-7/8 ligand or poly IC, induce CD8+ T cells to express CD103 (αE integrin), a ligand for cancer cells E-cadherin. Generation of these mucosal CD8+ T cells is regulated by DC-derived integrin αvβ8 and TGF-β activation in a dectin-1-dependent fashion. These CD103+CD8+ mucosal T cells accumulate in the tumors thereby increasing cancer necrosis and inhibiting cancer progression in vivo in a humanized mouse model of breast cancer. Importantly, CD103+CD8+ mucosal T cells elicited by reprogrammed DC can reject established cancer. Thus, reprogramming tumor-infiltrating DC represents a new strategy for cancer rejection. PMID:24795361

  20. Overexpression of the human DEK oncogene reprograms cellular metabolism and promotes glycolysis

    Science.gov (United States)

    Watanabe, Miki; Muraleedharan, Ranjithmenon; Lambert, Paul F.; Lane, Andrew N.; Romick-Rosendale, Lindsey E.; Wells, Susanne I.

    2017-01-01

    The DEK oncogene is overexpressed in many human malignancies including at early tumor stages. Our reported in vitro and in vivo models of squamous cell carcinoma have demonstrated that DEK contributes functionally to cellular and tumor survival and to proliferation. However, the underlying molecular mechanisms remain poorly understood. Based on recent RNA sequencing experiments, DEK expression was necessary for the transcription of several metabolic enzymes involved in anabolic pathways. This identified a possible mechanism whereby DEK may drive cellular metabolism to enable cell proliferation. Functional metabolic Seahorse analysis demonstrated increased baseline and maximum extracellular acidification rates, a readout of glycolysis, in DEK-overexpressing keratinocytes and squamous cell carcinoma cells. DEK overexpression also increased the maximum rate of oxygen consumption and therefore increased the potential for oxidative phosphorylation (OxPhos). To detect small metabolites that participate in glycolysis and the tricarboxylic acid cycle (TCA) that supplies substrate for OxPhos, we carried out NMR-based metabolomics studies. We found that high levels of DEK significantly reprogrammed cellular metabolism and altered the abundances of amino acids, TCA cycle intermediates and the glycolytic end products lactate, alanine and NAD+. Taken together, these data support a scenario whereby overexpression of the human DEK oncogene reprograms keratinocyte metabolism to fulfill energy and macromolecule demands required to enable and sustain cancer cell growth. PMID:28558019

  1. Reprogramming the Dynamin 2 mRNA by Spliceosome-mediated RNA Trans-splicing

    Directory of Open Access Journals (Sweden)

    Delphine Trochet

    2016-01-01

    Full Text Available Dynamin 2 (DNM2 is a large GTPase, ubiquitously expressed, involved in membrane trafficking and regulation of actin and microtubule cytoskeletons. DNM2 mutations cause autosomal dominant centronuclear myopathy which is a rare congenital myopathy characterized by skeletal muscle weakness and histopathological features including nuclear centralization in absence of regeneration. No curative treatment is currently available for the DNM2-related autosomal dominant centronuclear myopathy. In order to develop therapeutic strategy, we evaluated here the potential of Spliceosome-Mediated RNA Trans-splicing technology to reprogram the Dnm2-mRNA in vitro and in vivo in mice. We show that classical 3′-trans-splicing strategy cannot be considered as accurate therapeutic strategy regarding toxicity of the pre-trans-splicing molecules leading to low rate of trans-splicing in vivo. Thus, we tested alternative strategies devoted to prevent this toxicity and enhance frequency of trans-splicing events. We succeeded to overcome the toxicity through a 5′-trans-splicing strategy which also allows detection of trans-splicing events at mRNA and protein levels in vitro and in vivo. These results suggest that the Spliceosome-Mediated RNA Trans-splicing strategy may be used to reprogram mutated Dnm2-mRNA but highlight the potential toxicity linked to the molecular tools which have to be carefully investigated during preclinical development.

  2. Overexpression of the human DEK oncogene reprograms cellular metabolism and promotes glycolysis.

    Directory of Open Access Journals (Sweden)

    Marie C Matrka

    Full Text Available The DEK oncogene is overexpressed in many human malignancies including at early tumor stages. Our reported in vitro and in vivo models of squamous cell carcinoma have demonstrated that DEK contributes functionally to cellular and tumor survival and to proliferation. However, the underlying molecular mechanisms remain poorly understood. Based on recent RNA sequencing experiments, DEK expression was necessary for the transcription of several metabolic enzymes involved in anabolic pathways. This identified a possible mechanism whereby DEK may drive cellular metabolism to enable cell proliferation. Functional metabolic Seahorse analysis demonstrated increased baseline and maximum extracellular acidification rates, a readout of glycolysis, in DEK-overexpressing keratinocytes and squamous cell carcinoma cells. DEK overexpression also increased the maximum rate of oxygen consumption and therefore increased the potential for oxidative phosphorylation (OxPhos. To detect small metabolites that participate in glycolysis and the tricarboxylic acid cycle (TCA that supplies substrate for OxPhos, we carried out NMR-based metabolomics studies. We found that high levels of DEK significantly reprogrammed cellular metabolism and altered the abundances of amino acids, TCA cycle intermediates and the glycolytic end products lactate, alanine and NAD+. Taken together, these data support a scenario whereby overexpression of the human DEK oncogene reprograms keratinocyte metabolism to fulfill energy and macromolecule demands required to enable and sustain cancer cell growth.

  3. Elucidating the Metabolic Plasticity of Cancer: Mitochondrial Reprogramming and Hybrid Metabolic States

    Directory of Open Access Journals (Sweden)

    Dongya Jia

    2018-03-01

    Full Text Available Aerobic glycolysis, also referred to as the Warburg effect, has been regarded as the dominant metabolic phenotype in cancer cells for a long time. More recently, it has been shown that mitochondria in most tumors are not defective in their ability to carry out oxidative phosphorylation (OXPHOS. Instead, in highly aggressive cancer cells, mitochondrial energy pathways are reprogrammed to meet the challenges of high energy demand, better utilization of available fuels and macromolecular synthesis for rapid cell division and migration. Mitochondrial energy reprogramming is also involved in the regulation of oncogenic pathways via mitochondria-to-nucleus retrograde signaling and post-translational modification of oncoproteins. In addition, neoplastic mitochondria can engage in crosstalk with the tumor microenvironment. For example, signals from cancer-associated fibroblasts can drive tumor mitochondria to utilize OXPHOS, a process known as the reverse Warburg effect. Emerging evidence shows that cancer cells can acquire a hybrid glycolysis/OXPHOS phenotype in which both glycolysis and OXPHOS can be utilized for energy production and biomass synthesis. The hybrid glycolysis/OXPHOS phenotype facilitates metabolic plasticity of cancer cells and may be specifically associated with metastasis and therapy-resistance. Moreover, cancer cells can switch their metabolism phenotypes in response to external stimuli for better survival. Taking into account the metabolic heterogeneity and plasticity of cancer cells, therapies targeting cancer metabolic dependency in principle can be made more effective.

  4. Positional information is reprogrammed in blastema cells of the regenerating limb of the axolotl (Ambystoma mexicanum.

    Directory of Open Access Journals (Sweden)

    Catherine D McCusker

    Full Text Available The regenerating region of an amputated salamander limb, known as the blastema, has the amazing capacity to replace exactly the missing structures. By grafting cells from different stages and regions of blastemas induced to form on donor animals expressing Green Fluorescent Protein (GFP, to non-GFP host animals, we have determined that the cells from early stage blastemas, as well as cells at the tip of late stage blastemas are developmentally labile such that their positional identity is reprogrammed by interactions with more proximal cells with stable positional information. In contrast, cells from the adjacent, more proximal stump tissues as well as the basal region of late bud blastemas are positionally stable, and thus form ectopic limb structures when grafted. Finally, we have found that a nerve is required to maintain the blastema cells in a positionally labile state, thus indicating a role for reprogramming cues in the blastema microenvironment.

  5. Identification of Neuroprotective Factors Associated with Successful Ageing and Risk of Cognitive Impairment among Malaysia Older Adults

    Directory of Open Access Journals (Sweden)

    Huijin Lau

    2017-01-01

    Full Text Available The increase of ageing population has raised public attention on the concept of successful ageing. Studies have shown that vitamin D, telomere length, and brain-derived neurotrophic factor (BDNF have been associated with cognitive function. Therefore, this study aimed to identify neuroprotective factors for cognitive decline in different ageing groups. A total of 300 older adults aged 60 years and above were recruited in this population based cross-sectional study. Participants were categorized into three groups: mild cognitive impairment (MCI (n=100, usual ageing (UA (n=100, and successful ageing (SA (n=100. Dietary vitamin D intake was assessed through Diet History Questionnaire (DHQ. Out of the 300 participants, only 150 were subjected to fasting blood sample collection. These samples were used for serum vitamin D and plasma BDNF measurements. Whole blood telomere length was measured using RT-PCR method. The results show that the reduction of the risk of MCI was achieved by higher serum vitamin D level (OR: 0.96, 95% CI: 0.92–0.99, p<0.05, higher plasma BDNF level (OR: 0.51, 95% CI: 0.30–0.88,  p<0.05, and longer telomere (OR: 0.97, 95% CI: 0.95–0.99,  p<0.001. In conclusion, participants with higher vitamin D level, higher BDNF level, and longer telomere length were more likely to age successfully.

  6. Mitochondrial pyruvate carrier function determines cell stemness and metabolic reprogramming in cancer cells

    Science.gov (United States)

    Li, Xiaoran; Kan, Quancheng; Fan, Zhirui; Li, Yaqing; Ji, Yasai; Zhao, Jing; Zhang, Mingzhi; Grigalavicius, Mantas; Berge, Viktor; Goscinski, Mariusz Adam; M. Nesland, Jahn; Suo, Zhenhe

    2017-01-01

    One of the remarkable features of cancer cells is aerobic glycolysis, a phenomenon known as the “Warburg Effect”, in which cells rely preferentially on glycolysis instead of oxidative phosphorylation (OXPHOS) as the main energy source even in the presence of high oxygen tension. Cells with dysfunctional mitochondria are unable to generate sufficient ATP from mitochondrial OXPHOS, and then are forced to rely on glycolysis for ATP generation. Here we report our results in a prostate cancer cell line in which the mitochondrial pyruvate carrier 1 (MPC1) gene was knockout. It was discovered that the MPC1 gene knockout cells revealed a metabolism reprogramming to aerobic glycolysis with reduced ATP production, and the cells became more migratory and resistant to both chemotherapy and radiotherapy. In addition, the MPC1 knockout cells expressed significantly higher levels of the stemness markers Nanog, Hif1α, Notch1, CD44 and ALDH. To further verify the correlation of MPC gene function and cell stemness/metabolic reprogramming, MPC inhibitor UK5099 was applied in two ovarian cancer cell lines and similar results were obtained. Taken together, our results reveal that functional MPC may determine the fate of metabolic program and the stemness status of cancer cells in vitro. PMID:28624784

  7. Biological pacemaker created by minimally invasive somatic reprogramming in pigs with complete heart block

    Science.gov (United States)

    Hu, Yu-Feng; Dawkins, James Frederick; Cho, Hee Cheol; Marbán, Eduardo; Cingolani, Eugenio

    2016-01-01

    Somatic reprogramming by reexpression of the embryonic transcription factor T-box 18 (TBX18) converts cardiomyocytes into pacemaker cells. We hypothesized that this could be a viable therapeutic avenue for pacemaker-dependent patients afflicted with device-related complications, and therefore tested whether adenoviral TBX18 gene transfer could create biological pacemaker activity in vivo in a large-animal model of complete heart block. Biological pacemaker activity, originating from the intramyocardial injection site, was evident in TBX18-transduced animals starting at day 2 and persisted for the duration of the study (14 days) with minimal backup electronic pacemaker use. Relative to controls transduced with a reporter gene, TBX18-transduced animals exhibited enhanced autonomic responses and physiologically superior chronotropic support of physical activity. Induced sinoatrial node cells could be identified by their distinctive morphology at the site of injection in TBX18-transduced animals, but not in controls. No local or systemic safety concerns arose. Thus, minimally invasive TBX18 gene transfer creates physiologically relevant pacemaker activity in complete heart block, providing evidence for therapeutic somatic reprogramming in a clinically relevant disease model. PMID:25031269

  8. Metabolic reprogramming: a cancer hallmark even warburg did not anticipate.

    Science.gov (United States)

    Ward, Patrick S; Thompson, Craig B

    2012-03-20

    Cancer metabolism has long been equated with aerobic glycolysis, seen by early biochemists as primitive and inefficient. Despite these early beliefs, the metabolic signatures of cancer cells are not passive responses to damaged mitochondria but result from oncogene-directed metabolic reprogramming required to support anabolic growth. Recent evidence suggests that metabolites themselves can be oncogenic by altering cell signaling and blocking cellular differentiation. No longer can cancer-associated alterations in metabolism be viewed as an indirect response to cell proliferation and survival signals. We contend that altered metabolism has attained the status of a core hallmark of cancer. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. Reprogramming Antagonizes the Oncogenicity of HOXA13-Long Noncoding RNA HOTTIP Axis in Gastric Cancer Cells.

    Science.gov (United States)

    Wu, Deng-Chyang; Wang, Sophie S W; Liu, Chung-Jung; Wuputra, Kenly; Kato, Kohsuke; Lee, Yen-Liang; Lin, Ying-Chu; Tsai, Ming-Ho; Ku, Chia-Chen; Lin, Wen-Hsin; Wang, Shin-Wei; Kishikawa, Shotaro; Noguchi, Michiya; Wu, Chu-Chieh; Chen, Yi-Ting; Chai, Chee-Yin; Lin, Chen-Lung Steve; Kuo, Kung-Kai; Yang, Ya-Han; Miyoshi, Hiroyuki; Nakamura, Yukio; Saito, Shigeo; Nagata, Kyosuke; Lin, Chang-Shen; Yokoyama, Kazunari K

    2017-10-01

    Reprogramming of cancer cells into induced pluripotent stem cells (iPSCs) is a compelling idea for inhibiting oncogenesis, especially through modulation of homeobox proteins in this reprogramming process. We examined the role of various long noncoding RNAs (lncRNAs)-homeobox protein HOXA13 axis on the switching of the oncogenic function of bone morphogenetic protein 7 (BMP7), which is significantly lost in the gastric cancer cell derived iPS-like cells (iPSLCs). BMP7 promoter activation occurred through the corecruitment of HOXA13, mixed-lineage leukemia 1 lysine N-methyltransferase, WD repeat-containing protein 5, and lncRNA HoxA transcript at the distal tip (HOTTIP) to commit the epigenetic changes to the trimethylation of lysine 4 on histone H3 in cancer cells. By contrast, HOXA13 inhibited BMP7 expression in iPSLCs via the corecruitment of HOXA13, enhancer of zeste homolog 2, Jumonji and AT rich interactive domain 2, and lncRNA HoxA transcript antisense RNA (HOTAIR) to various cis-element of the BMP7 promoter. Knockdown experiments demonstrated that HOTTIP contributed positively, but HOTAIR regulated negatively to HOXA13-mediated BMP7 expression in cancer cells and iPSLCs, respectively. These findings indicate that the recruitment of HOXA13-HOTTIP and HOXA13-HOTAIR to different sites in the BMP7 promoter is crucial for the oncogenic fate of human gastric cells. Reprogramming with octamer-binding protein 4 and Jun dimerization protein 2 can inhibit tumorigenesis by switching off BMP7. Stem Cells 2017;35:2115-2128. © 2017 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  10. Electromagnetized gold nanoparticles mediate direct lineage reprogramming into induced dopamine neurons in vivo for Parkinson's disease therapy

    Science.gov (United States)

    Yoo, Junsang; Lee, Euiyeon; Kim, Hee Young; Youn, Dong-Ho; Jung, Junghyun; Kim, Hongwon; Chang, Yujung; Lee, Wonwoong; Shin, Jaein; Baek, Soonbong; Jang, Wonhee; Jun, Won; Kim, Soochan; Hong, Jongki; Park, Hi-Joon; Lengner, Christopher J.; Moh, Sang Hyun; Kwon, Youngeun; Kim, Jongpil

    2017-10-01

    Electromagnetic fields (EMF) are physical energy fields generated by electrically charged objects, and specific ranges of EMF can influence numerous biological processes, which include the control of cell fate and plasticity. In this study, we show that electromagnetized gold nanoparticles (AuNPs) in the presence of specific EMF conditions facilitate an efficient direct lineage reprogramming to induced dopamine neurons in vitro and in vivo. Remarkably, electromagnetic stimulation leads to a specific activation of the histone acetyltransferase Brd2, which results in histone H3K27 acetylation and a robust activation of neuron-specific genes. In vivo dopaminergic neuron reprogramming by EMF stimulation of AuNPs efficiently and non-invasively alleviated symptoms in mouse Parkinson's disease models. This study provides a proof of principle for EMF-based in vivo lineage conversion as a potentially viable and safe therapeutic strategy for the treatment of neurodegenerative disorders.

  11. DNA double-strand breaks in human induced pluripotent stem cell reprogramming and long-term in vitro culturing.

    Science.gov (United States)

    Simara, Pavel; Tesarova, Lenka; Rehakova, Daniela; Matula, Pavel; Stejskal, Stanislav; Hampl, Ales; Koutna, Irena

    2017-03-21

    Human induced pluripotent stem cells (hiPSCs) play roles in both disease modelling and regenerative medicine. It is critical that the genomic integrity of the cells remains intact and that the DNA repair systems are fully functional. In this article, we focused on the detection of DNA double-strand breaks (DSBs) by phosphorylated histone H2AX (known as γH2AX) and p53-binding protein 1 (53BP1) in three distinct lines of hiPSCs, their source cells, and one line of human embryonic stem cells (hESCs). We measured spontaneously occurring DSBs throughout the process of fibroblast reprogramming and during long-term in vitro culturing. To assess the variations in the functionality of the DNA repair system among the samples, the number of DSBs induced by γ-irradiation and the decrease over time was analysed. The foci number was detected by fluorescence microscopy separately for the G1 and S/G2 cell cycle phases. We demonstrated that fibroblasts contained a low number of non-replication-related DSBs, while this number increased after reprogramming into hiPSCs and then decreased again after long-term in vitro passaging. The artificial induction of DSBs revealed that the repair mechanisms function well in the source cells and hiPSCs at low passages, but fail to recognize a substantial proportion of DSBs at high passages. Our observations suggest that cellular reprogramming increases the DSB number but that the repair mechanism functions well. However, after prolonged in vitro culturing of hiPSCs, the repair capacity decreases.

  12. MiR-25 regulates Wwp2 and Fbxw7 and promotes reprogramming of mouse fibroblast cells to iPSCs.

    Directory of Open Access Journals (Sweden)

    Dong Lu

    Full Text Available miRNAs are a class of small non-coding RNAs that regulate gene expression and have critical functions in various biological processes. Hundreds of miRNAs have been identified in mammalian genomes but only a small number of them have been functionally characterized. Recent studies also demonstrate that some miRNAs have important roles in reprogramming somatic cells to induced pluripotent stem cells (iPSCs.We screened 52 miRNAs cloned in a piggybac (PB vector for their roles in reprogramming of mouse embryonic fibroblast cells to iPSCs. To identify targets of miRNAs, we made Dgcr8-deficient embryonic stem (ES cells and introduced miRNA mimics to these cells, which lack miRNA biogenesis. The direct target genes of miRNA were identified through global gene expression analysis and target validation.We found that over-expressing miR-25 or introducing miR-25 mimics enhanced production of iPSCs. We identified a number of miR-25 candidate gene targets. Of particular interest were two ubiquitin ligases, Wwp2 and Fbxw7, which have been proposed to regulate Oct4, c-Myc and Klf5, respectively. Our findings thus highlight the complex interplay between miRNAs and transcription factors involved in reprogramming, stem cell self-renewal and maintenance of pluripotency.

  13. Anakoinosis: Communicative Reprogramming of Tumor Systems - for Rescuing from Chemorefractory Neoplasia.

    Science.gov (United States)

    Hart, Christina; Vogelhuber, Martin; Wolff, Daniel; Klobuch, Sebastian; Ghibelli, Lina; Foell, Jürgen; Corbacioglu, Selim; Rehe, Klaus; Haegeman, Guy; Thomas, Simone; Herr, Wolfgang; Reichle, Albrecht

    2015-08-01

    Disruptive technologies, such as communicative reprogramming (anakoinosis) with cellular therapies in situ for treating refractory metastatic cancer allow patient care to accelerate along a totally new trajectory and highlight what may well become the next sea change in the care of patients with many types of advanced neoplasia. Cellular therapy in situ consisted of repurposed drugs, pioglitazone plus all-trans retinoic acid or dexamethasone or interferon-alpha (dual transcriptional modulation) combined with metronomic low-dose chemotherapy or low-dose 5-azacytidine, plus/minus classic targeted therapy. The novel therapeutic tools for specifically designing communication processes within tumor diseases focus on redirecting (1) rationalizations of cancer hallmarks (constitution of single cancer hallmarks), (2) modular events, (3) the 'metabolism' of evolutionary processes (the sum of therapeutically and intrinsically inducible evolutionary processes) and (4) the holistic communicative context, which determines validity and denotation of tumor promoting communication lines. Published data on cellular therapies in situ (6 histologic tumor types, 144 patients, age 0.9-83 years) in castration-resistant prostate cancer, pretreated renal clear cell carcinoma, chemorefractory acute myelocytic leukemia, multiple myeloma > second-line, chemorefractory Hodgkin lymphoma or multivisceral Langerhans cell histiocytosis, outline the possibility for treating refractory metastatic cancer with the hope that this type of reprogrammed communication will be scalable with minimal toxicity. Accessibility to anakoinosis is a tumor inherent feature, and cellular therapy in situ addresses extrinsic and intrinsic drug resistance, by redirecting convergent organized communication tools, while been supported by quite different pattern of (molecular-)genetic aberrations.

  14. Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Ulm, Ashley; Mayhew, Christopher N; Debley, Jason; Khurana Hershey, Gurjit K; Ji, Hong

    2016-03-10

    Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

  15. Transient Acquisition of Pluripotency During Somatic Cell Transdifferentiation with iPSC Reprogramming Factors

    OpenAIRE

    Maza, Itay; Caspi, Inbal; Zviran, Asaf; Chomsky, Elad; Rais, Yoach; Viukov, Sergey; Geula, Shay; Buenrostro, Jason D.; Weinberger, Leehee; Krupalnik, Vladislav; Hanna, Suhair; Zerbib, Mirie; Dutton, James R.; Greenleaf, William J.; Massarwa, Rada

    2015-01-01

    Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors 1,2 . Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote linea...

  16. Two new routes to make blood: Hematopoietic specification from pluripotent cell lines versus reprogramming of somatic cells.

    Science.gov (United States)

    Singbrant, Sofie; van Galen, Peter; Lucas, Daniel; Challen, Grant; Rossi, Derrick J; Daley, George Q

    2015-09-01

    Transplantation of hematopoietic stem cells (HSCs) to treat hematologic disorders is routinely used in the clinic. However, HSC therapy is hindered by the requirements of finding human leukocyte antigen (HLA)-matched donors and attaining sufficient numbers of long-term HSCs in the graft. Therefore, ex vivo expansion of transplantable HSCs remains one of the "holy grails" of hematology. Without the ability to maintain and expand human HSCs in vitro, two complementary approaches involving cellular reprogramming to generate transplantable HSCs have emerged. Reprogrammed HSCs represent a potentially inexhaustible supply of autologous tissue. On March 18th, 2015, Dr. George Q. Daley and Dr. Derrick J. Rossi, two pioneers in the field, presented and discussed their most recent research on these topics in a webinar organized by the International Society for Experimental Hematology (ISEH). Here, we summarize these seminars and discuss the possibilities and challenges in the field of hematopoietic specification. Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  17. Executive cognitive impairment detected by simple bedside testing ...

    African Journals Online (AJOL)

    Aims. Cognitive impairment in people with type 2 diabetes is a barrier to successful disease management. We sought to determine whether impaired executive function as detected by a battery of simple bedside cognitive tests of executive function was associated with inadequate glycaemic control. Methods. People with ...

  18. Enhancer Analysis Unveils Genetic Interactions between TLX and SOX2 in Neural Stem Cells and In Vivo Reprogramming.

    Science.gov (United States)

    Islam, Mohammed M; Smith, Derek K; Niu, Wenze; Fang, Sanhua; Iqbal, Nida; Sun, Guoqiang; Shi, Yanhong; Zhang, Chun-Li

    2015-11-10

    The orphan nuclear receptor TLX is a master regulator of postnatal neural stem cell (NSC) self-renewal and neurogenesis; however, it remains unclear how TLX expression is precisely regulated in these tissue-specific stem cells. Here, we show that a highly conserved cis-element within the Tlx locus functions to drive gene expression in NSCs. We demonstrate that the transcription factors SOX2 and MYT1 specifically interact with this genomic element to directly regulate Tlx enhancer activity in vivo. Knockdown experiments further reveal that SOX2 dominantly controls endogenous expression of TLX, whereas MYT1 only plays a modulatory role. Importantly, TLX is essential for SOX2-mediated in vivo reprogramming of astrocytes and itself is also sufficient to induce neurogenesis in the adult striatum. Together, these findings unveil functional genetic interactions among transcription factors that are critical to NSCs and in vivo cell reprogramming.

  19. Application of mitochondrial pyruvate carrier blocker UK5099 creates metabolic reprogram and greater stem-like properties in LnCap prostate cancer cells in vitro

    Science.gov (United States)

    Zhong, Yali; Li, Xiaoran; Yu, Dandan; Li, Xiaoli; Li, Yaqing; Long, Yuan; Yuan, Yuan; Ji, Zhenyu; Zhang, Mingzhi; Wen, Jian-Guo; Nesland, Jahn M.; Suo, Zhenhe

    2015-01-01

    Aerobic glycolysis is one of the important hallmarks of cancer cells and eukaryotic cells. In this study, we have investigated the relationship between blocking mitochondrial pyruvate carrier (MPC) with UK5099 and the metabolic alteration as well as stemness phenotype of prostatic cancer cells. It was found that blocking pyruvate transportation into mitochondrial attenuated mitochondrial oxidative phosphorylation (OXPHOS) and increased glycolysis. The UK5099 treated cells showed significantly higher proportion of side population (SP) fraction and expressed higher levels of stemness markers Oct3/4 and Nanog. Chemosensitivity examinations revealed that the UK5099 treated cells became more resistant to chemotherapy compared to the non-treated cells. These results demonstrate probably an intimate connection between metabolic reprogram and stem-like phenotype of LnCap cells in vitro. We propose that MPC blocker (UK5099) application may be an ideal model for Warburg effect studies, since it attenuates mitochondrial OXPHOS and increases aerobic glycolysis, a phenomenon typically reflected in the Warburg effect. We conclude that impaired mitochondrial OXPHOS and upregulated glycolysis are related with stem-like phenotype shift in prostatic cancer cells. PMID:26413751

  20. Human mesenchymal stromal cell-secreted lactate induces M2-macrophage differentiation by metabolic reprogramming

    OpenAIRE

    Selleri, Silvia; Bifsha, Panojot; Civini, Sara; Pacelli, Consiglia; Dieng, Mame Massar; Lemieux, William; Jin, Ping; Bazin, Ren?e; Patey, Natacha; Marincola, Francesco M.; Moldovan, Florina; Zaouter, Charlotte; Trudeau, Louis-Eric; Benabdhalla, Basma; Louis, Isabelle

    2016-01-01

    Human mesenchymal stromal cells (MSC) have been shown to dampen immune response and promote tissue repair, but the underlying mechanisms are still under investigation. Herein, we demonstrate that umbilical cord-derived MSC (UC-MSC) alter the phenotype and function of monocyte-derived dendritic cells (DC) through lactate-mediated metabolic reprogramming. UC-MSC can secrete large quantities of lactate and, when present during monocyte-to-DC differentiation, induce instead the acquisition of M2-...

  1. Reprogramming of enteroendocrine K cells to pancreatic β-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation in suspension culture

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Esder; Ryu, Gyeong Ryul; Moon, Sung-Dae; Ko, Seung-Hyun; Ahn, Yu-Bae; Song, Ki-Ho, E-mail: kihos@catholic.ac.kr

    2014-01-17

    Highlights: •K cells were selected from STC-1 cells, a heterogeneous enteroendocrine cell line. •K cells did not express Nkx6.1 and Neurogenin3. •Combined expression of Nkx6.1 and Neurogenin3 reprogrammed K cells to β-cells. •Reprogramming of K cells to β-cells was not complete. -- Abstract: Recent studies have demonstrated that adult cells such as pancreatic exocrine cells can be converted to pancreatic β-cells in a process called cell reprogramming. Enteroendocrine cells and β-cells share similar pathways of differentiation during embryonic development. Notably, enteroendocrine K cells express many of the key proteins found in β-cells. Thus, K cells could be reprogrammed to β-cells under certain conditions. However, there is no clear evidence on whether these cells convert to β-cells. K cells were selected from STC-1 cells, an enteroendocrine cell line expressing multiple hormones. K cells were found to express many genes of transcription factors crucial for islet development and differentiation except for Nkx6.1 and Neurogenin3. A K cell clone stably expressing Nkx6.1 (Nkx6.1{sup +}-K cells) was established. Induction of Neurogenin3 expression in Nkx6.1{sup +}-K cells, by either treatment with a γ-secretase inhibitor or infection with a recombinant adenovirus expressing Neurogenin3, led to a significant increase in Insulin1 mRNA expression. After infection with the adenovirus expressing Neurogenin3 and reaggregation in suspension culture, about 50% of Nkx6.1{sup +}-K cells expressed insulin as determined by immunostaining. The intracellular insulin content was increased markedly. Electron microscopy revealed the presence of insulin granules. However, glucose-stimulated insulin secretion was defective, and there was no glucose lowering effect after transplantation of these cells in diabetic mice. In conclusion, we demonstrated that K cells could be reprogrammed partially to β-cells through the combined expression of Nkx6.1 and Neurogenin3, and

  2. Reprogramming of enteroendocrine K cells to pancreatic β-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation in suspension culture

    International Nuclear Information System (INIS)

    Lee, Esder; Ryu, Gyeong Ryul; Moon, Sung-Dae; Ko, Seung-Hyun; Ahn, Yu-Bae; Song, Ki-Ho

    2014-01-01

    Highlights: •K cells were selected from STC-1 cells, a heterogeneous enteroendocrine cell line. •K cells did not express Nkx6.1 and Neurogenin3. •Combined expression of Nkx6.1 and Neurogenin3 reprogrammed K cells to β-cells. •Reprogramming of K cells to β-cells was not complete. -- Abstract: Recent studies have demonstrated that adult cells such as pancreatic exocrine cells can be converted to pancreatic β-cells in a process called cell reprogramming. Enteroendocrine cells and β-cells share similar pathways of differentiation during embryonic development. Notably, enteroendocrine K cells express many of the key proteins found in β-cells. Thus, K cells could be reprogrammed to β-cells under certain conditions. However, there is no clear evidence on whether these cells convert to β-cells. K cells were selected from STC-1 cells, an enteroendocrine cell line expressing multiple hormones. K cells were found to express many genes of transcription factors crucial for islet development and differentiation except for Nkx6.1 and Neurogenin3. A K cell clone stably expressing Nkx6.1 (Nkx6.1 + -K cells) was established. Induction of Neurogenin3 expression in Nkx6.1 + -K cells, by either treatment with a γ-secretase inhibitor or infection with a recombinant adenovirus expressing Neurogenin3, led to a significant increase in Insulin1 mRNA expression. After infection with the adenovirus expressing Neurogenin3 and reaggregation in suspension culture, about 50% of Nkx6.1 + -K cells expressed insulin as determined by immunostaining. The intracellular insulin content was increased markedly. Electron microscopy revealed the presence of insulin granules. However, glucose-stimulated insulin secretion was defective, and there was no glucose lowering effect after transplantation of these cells in diabetic mice. In conclusion, we demonstrated that K cells could be reprogrammed partially to β-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation

  3. Efficient Direct Reprogramming of Mature Amniotic Cells into Endothelial Cells by ETS Factors and TGFβ Suppression

    Science.gov (United States)

    Ginsberg, Michael; James, Daylon; Ding, Bi-Sen; Nolan, Daniel; Geng, Fuqiang; Butler, Jason M; Schachterle, William; Pulijaal, Venkat R; Mathew, Susan; Chasen, Stephen T; Xiang, Jenny; Rosenwaks, Zev; Shido, Koji; Elemento, Olivier; Rabbany, Sina Y; Rafii, Shahin

    2012-01-01

    ETS transcription factors ETV2, FLI1 and ERG1 specify pluripotent stem cells into endothelial cells (ECs). However, these ECs are unstable and drift towards non-vascular cell fates. We show that human mid-gestation c-Kit− lineage-committed amniotic cells (ACs) can be readily reprogrammed into induced vascular endothelial cells (iVECs). Transient ETV2 expression in ACs generated proliferative but immature iVECs, while co-expression with FLI1/ERG1 endowed iVECs with a vascular repertoire and morphology matching mature stable ECs. Brief TGFβ-inhibition functionalized VEGFR2 signaling, augmenting specification of ACs to iVECs. Genome-wide transcriptional analyses showed that iVECs are similar to adult ECs in which vascular-specific genes are turned on and non-vascular genes are silenced. Functionally, iVECs form long-lasting patent vasculature in Matrigel plugs and regenerating livers. Thus, short-term ETV2 expression and TGFβ-inhibition along with constitutive ERG1/FLI1 co-expression reprogram mature ACs into durable and functional iVECs with clinical-scale expansion potential. Public banking of HLA-typed iVECs would establish a vascular inventory for treatment of genetically diverse disorders. PMID:23084400

  4. Environmentally induced transgenerational epigenetic reprogramming of primordial germ cells and the subsequent germ line.

    Directory of Open Access Journals (Sweden)

    Michael K Skinner

    Full Text Available A number of environmental factors (e.g. toxicants have been shown to promote the epigenetic transgenerational inheritance of disease and phenotypic variation. Transgenerational inheritance requires the germline transmission of altered epigenetic information between generations in the absence of direct environmental exposures. The primary periods for epigenetic programming of the germ line are those associated with primordial germ cell development and subsequent fetal germline development. The current study examined the actions of an agricultural fungicide vinclozolin on gestating female (F0 generation progeny in regards to the primordial germ cell (PGC epigenetic reprogramming of the F3 generation (i.e. great-grandchildren. The F3 generation germline transcriptome and epigenome (DNA methylation were altered transgenerationally. Interestingly, disruptions in DNA methylation patterns and altered transcriptomes were distinct between germ cells at the onset of gonadal sex determination at embryonic day 13 (E13 and after cord formation in the testis at embryonic day 16 (E16. A larger number of DNA methylation abnormalities (epimutations and transcriptional alterations were observed in the E13 germ cells than in the E16 germ cells. These observations indicate that altered transgenerational epigenetic reprogramming and function of the male germline is a component of vinclozolin induced epigenetic transgenerational inheritance of disease. Insights into the molecular control of germline transmitted epigenetic inheritance are provided.

  5. Direct reprogramming of somatic cells into neural stem cells or neurons for neurological disorders.

    Science.gov (United States)

    Hou, Shaoping; Lu, Paul

    2016-01-01

    Direct reprogramming of somatic cells into neurons or neural stem cells is one of the most important frontier fields in current neuroscience research. Without undergoing the pluripotency stage, induced neurons or induced neural stem cells are a safer and timelier manner resource in comparison to those derived from induced pluripotent stem cells. In this prospective, we review the recent advances in generation of induced neurons and induced neural stem cells in vitro and in vivo and their potential treatments of neurological disorders.

  6. Enhancer Analysis Unveils Genetic Interactions between TLX and SOX2 in Neural Stem Cells and In Vivo Reprogramming

    Science.gov (United States)

    Islam, Mohammed M.; Smith, Derek K.; Niu, Wenze; Fang, Sanhua; Iqbal, Nida; Sun, Guoqiang; Shi, Yanhong; Zhang, Chun-Li

    2015-01-01

    Summary The orphan nuclear receptor TLX is a master regulator of postnatal neural stem cell (NSC) self-renewal and neurogenesis; however, it remains unclear how TLX expression is precisely regulated in these tissue-specific stem cells. Here, we show that a highly conserved cis-element within the Tlx locus functions to drive gene expression in NSCs. We demonstrate that the transcription factors SOX2 and MYT1 specifically interact with this genomic element to directly regulate Tlx enhancer activity in vivo. Knockdown experiments further reveal that SOX2 dominantly controls endogenous expression of TLX, whereas MYT1 only plays a modulatory role. Importantly, TLX is essential for SOX2-mediated in vivo reprogramming of astrocytes and itself is also sufficient to induce neurogenesis in the adult striatum. Together, these findings unveil functional genetic interactions among transcription factors that are critical to NSCs and in vivo cell reprogramming. PMID:26607952

  7. Enhancer Analysis Unveils Genetic Interactions between TLX and SOX2 in Neural Stem Cells and In Vivo Reprogramming

    Directory of Open Access Journals (Sweden)

    Mohammed M. Islam

    2015-11-01

    Full Text Available The orphan nuclear receptor TLX is a master regulator of postnatal neural stem cell (NSC self-renewal and neurogenesis; however, it remains unclear how TLX expression is precisely regulated in these tissue-specific stem cells. Here, we show that a highly conserved cis-element within the Tlx locus functions to drive gene expression in NSCs. We demonstrate that the transcription factors SOX2 and MYT1 specifically interact with this genomic element to directly regulate Tlx enhancer activity in vivo. Knockdown experiments further reveal that SOX2 dominantly controls endogenous expression of TLX, whereas MYT1 only plays a modulatory role. Importantly, TLX is essential for SOX2-mediated in vivo reprogramming of astrocytes and itself is also sufficient to induce neurogenesis in the adult striatum. Together, these findings unveil functional genetic interactions among transcription factors that are critical to NSCs and in vivo cell reprogramming.

  8. Epigenetic Reprogramming of Lineage-Committed Human Mammary Epithelial Cells Requires DNMT3A and Loss of DOT1L

    Directory of Open Access Journals (Sweden)

    Jerrica L. Breindel

    2017-09-01

    Full Text Available Organogenesis and tissue development occur through sequential stepwise processes leading to increased lineage restriction and loss of pluripotency. An exception to this appears in the adult human breast, where rare variant epithelial cells exhibit pluripotency and multilineage differentiation potential when removed from the signals of their native microenvironment. This phenomenon provides a unique opportunity to study mechanisms that lead to cellular reprogramming and lineage plasticity in real time. Here, we show that primary human mammary epithelial cells (HMECs lose expression of differentiated mammary epithelial markers in a manner dependent on paracrine factors and epigenetic regulation. Furthermore, we demonstrate that HMEC reprogramming is dependent on gene silencing by the DNA methyltransferase DNMT3A and loss of histone transcriptional marks following downregulation of the methyltransferase DOT1L. These results demonstrate that lineage commitment in adult tissues is context dependent and highlight the plasticity of somatic cells when removed from their native tissue microenvironment.

  9. Nanos promotes epigenetic reprograming of the germline by down-regulation of the THAP transcription factor LIN-15B.

    Science.gov (United States)

    Lee, Chih-Yung Sean; Lu, Tu; Seydoux, Geraldine

    2017-11-07

    Nanos RNA-binding proteins are required for germline development in metazoans, but the underlying mechanisms remain poorly understood. We have profiled the transcriptome of primordial germ cells (PGCs) lacking the nanos homologs nos-1 and nos-2 in C. elegans. nos-1nos-2 PGCs fail to silence hundreds of transcripts normally expressed in oocytes. We find that this misregulation is due to both delayed turnover of maternal transcripts and inappropriate transcriptional activation. The latter appears to be an indirect consequence of delayed turnover of the maternally-inherited transcription factor LIN-15B, a synMuvB class transcription factor known to antagonize PRC2 activity. PRC2 is required for chromatin reprogramming in the germline, and the transcriptome of PGCs lacking PRC2 resembles that of nos-1nos-2 PGCs. Loss of maternal LIN-15B restores fertility to nos-1nos-2 mutants. These findings suggest that Nanos promotes germ cell fate by downregulating maternal RNAs and proteins that would otherwise interfere with PRC2-dependent reprogramming of PGC chromatin.

  10. Long-term in vitro, cell-type-specific genome-wide reprogramming of gene expression

    International Nuclear Information System (INIS)

    Hakelien, Anne-Mari; Gaustad, Kristine G.; Taranger, Christel K.; Skalhegg, Bjorn S.; Kuentziger, Thomas; Collas, Philippe

    2005-01-01

    We demonstrate a cell extract-based, genome-wide and heritable reprogramming of gene expression in vitro. Kidney epithelial 293T cells have previously been shown to take on T cell properties following a brief treatment with an extract of Jurkat T cells. We show here that 293T cells exposed for 1 h to a Jurkat cell extract undergo genome-wide, target cell-type-specific and long-lasting transcriptional changes. Microarray analyses indicate that on any given week after extract treatment, ∼2500 genes are upregulated >3-fold, of which ∼900 are also expressed in Jurkat cells. Concomitantly, ∼1500 genes are downregulated or repressed, of which ∼500 are also downregulated in Jurkat cells. Gene expression changes persist for over 30 passages (∼80 population doublings) in culture. Target cell-type specificity of these changes is shown by the lack of activation or repression of Jurkat-specific genes by extracts of 293T cells or carcinoma cells. Quantitative RT-PCR analysis confirms the long-term transcriptional activation of genes involved in key T cell functions. Additionally, growth of cells in suspended aggregates, expression of CD3 and CD28 T cell surface markers, and interleukin-2 secretion by 293T cells treated with extract of adult peripheral blood T cells illustrate a functional nuclear reprogramming. Therefore, target cell-type-specific and heritable changes in gene expression, and alterations in cell function, can be promoted by extracts derived from transformed cells as well as from adult primary cells

  11. Response (re-)programming in aging: a kinematic analysis.

    Science.gov (United States)

    Bellgrove, M A; Phillips, J G; Bradshaw, J L; Gallucci, R M

    1998-05-01

    Age-related motor slowing may reflect either motor programming deficits, poorer movement execution, or mere strategic preferences for online guidance of movement. We controlled such preferences, limiting the extent to which movements could be programmed. Twenty-four young and 24 older adults performed a line drawing task that allowed movements to be prepared in advance in one case (i.e., cue initially available indicating target location) and not in another (i.e., no cue initially available as to target location). Participants connected large or small targets illuminated by light-emitting diodes upon a graphics tablet that sampled pen tip position at 200 Hz. Older adults had a disproportionate difficulty initiating movement when prevented from programming in advance. Older adults produced slower, less efficient movements, particularly when prevented from programming under greater precision requirements. The slower movements of older adults do not simply reflect a preference for online control, as older adults have less efficient movements when forced to reprogram their movements. Age-related motor slowing kinematically resembles that seen in patients with cerebellar dysfunction.

  12. Hacking cell differentiation: transcriptional rerouting in reprogramming, lineage infidelity and metaplasia.

    Science.gov (United States)

    Regalo, Gonçalo; Leutz, Achim

    2013-08-01

    Initiating neoplastic cell transformation events are of paramount importance for the comprehension of regeneration and vanguard oncogenic processes but are difficult to characterize and frequently clinically overlooked. In epithelia, pre-neoplastic transformation stages are often distinguished by the appearance of phenotypic features of another differentiated tissue, termed metaplasia. In haemato/lymphopoietic malignancies, cell lineage ambiguity is increasingly recorded. Both, metaplasia and biphenotypic leukaemia/lymphoma represent examples of dysregulated cell differentiation that reflect a history of trans-differentiation and/or epigenetic reprogramming. Here we compare the similarity between molecular events of experimental cell trans-differentiation as an emerging therapeutic concept, with lineage confusion, as in metaplasia and dysplasia forecasting tumour development. © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.

  13. Salmonella enterica serovar-specific transcriptional reprogramming of infected cells.

    Science.gov (United States)

    Hannemann, Sebastian; Galán, Jorge E

    2017-07-01

    Despite their high degree of genomic similarity, different Salmonella enterica serovars are often associated with very different clinical presentations. In humans, for example, the typhoidal S. enterica serovar Typhi causes typhoid fever, a life-threatening systemic disease. In contrast, the non-typhoidal S. enterica serovar Typhimurium causes self-limiting gastroenteritis. The molecular bases for these different clinical presentations are incompletely understood. The ability to re-program gene expression in host cells is an essential virulence factor for typhoidal and non-typhoidal S. enterica serovars. Here, we have compared the transcriptional profile of cultured epithelial cells infected with S. Typhimurium or S. Typhi. We found that both serovars stimulated distinct transcriptional responses in infected cells that are associated with the stimulation of specific signal transduction pathways. These specific responses were associated with the presence of a distinct repertoire of type III secretion effector proteins. These observations provide major insight into the molecular bases for potential differences in the pathogenic mechanisms of typhoidal and non-typhoidal S. enterica serovars.

  14. A systemic evaluation of cardiac differentiation from mRNA reprogrammed human induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Ashish Mehta

    Full Text Available Genetically unmodified cardiomyocytes mandated for cardiac regenerative therapy is conceivable by "foot-print free" reprogramming of somatic cells to induced pluripotent stem cells (iPSC. In this study, we report generation of foot-print free hiPSC through messenger RNA (mRNA based reprograming. Subsequently, we characterize cardiomyocytes derived from these hiPSC using molecular and electrophysiological methods to characterize their applicability for regenerative medicine. Our results demonstrate that mRNA-iPSCs differentiate ontogenetically into cardiomyocytes with increased expression of early commitment markers of mesoderm, cardiac mesoderm, followed by cardiac specific transcriptional and sarcomeric structural and ion channel genes. Furthermore, these cardiomyocytes stained positively for sarcomeric and ion channel proteins. Based on multi-electrode array (MEA recordings, these mRNA-hiPSC derived cardiomyocytes responded predictably to various pharmacologically active drugs that target adrenergic, sodium, calcium and potassium channels. The cardiomyocytes responded chronotropically to isoproterenol in a dose dependent manner, inotropic activity of nifidipine decreased spontaneous contractions. Moreover, Sotalol and E-4031 prolonged QT intervals, while TTX reduced sodium influx. Our results for the first time show a systemic evaluation based on molecular, structural and functional properties of cardiomyocytes differentiated from mRNA-iPSC. These results, coupled with feasibility of generating patient-specific iPSCs hold great promise for the development of large-scale generation of clinical grade cardiomyocytes for cardiac regenerative medicine.

  15. Metabolic Reprogramming of Macrophages Exposed to Silk, Poly(lactic-co-glycolic acid), and Silica Nanoparticles.

    Science.gov (United States)

    Saborano, Raquel; Wongpinyochit, Thidarat; Totten, John D; Johnston, Blair F; Seib, F Philipp; Duarte, Iola F

    2017-07-01

    Monitoring macrophage metabolism in response to nanoparticle exposure provides new insights into biological outcomes, such as inflammation or toxicity, and supports the design of tailored nanomedicines. This paper describes the metabolic signature of macrophages exposed to nanoparticles ranging in diameter from 100 to 125 nm and made from silk, poly(lactic-co-glycolic acid) or silica. Nanoparticles of this size and type are currently at various stages of preclinical and clinical development for drug delivery applications. 1 H NMR analysis of cell extracts and culture media is used to quantify the changes in the intracellular and extracellular metabolomes of macrophages in response to nanoparticle exposure. Increased glycolytic activity, an altered tricarboxylic acid cycle, and reduced ATP generation are consistent with a proinflammatory phenotype. Furthermore, amino acids possibly arising from autophagy, the creatine kinase/phosphocreatine system, and a few osmolytes and antioxidants emerge as important players in the metabolic reprogramming of macrophages exposed to nanoparticles. This metabolic signature is a common response to all nanoparticles tested; however, the direction and magnitude of some variations are clearly nanoparticle specific, indicating material-induced biological specificity. Overall, metabolic reprogramming of macrophages can be achieved with nanoparticle treatments, modulated through the choice of the material, and monitored using 1 H NMR metabolomics. © 2017 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Direct Neuronal Reprogramming for Disease Modeling Studies Using Patient-Derived Neurons: What Have We Learned?

    Directory of Open Access Journals (Sweden)

    Janelle Drouin-Ouellet

    2017-09-01

    Full Text Available Direct neuronal reprogramming, by which a neuron is formed via direct conversion from a somatic cell without going through a pluripotent intermediate stage, allows for the possibility of generating patient-derived neurons. A unique feature of these so-called induced neurons (iNs is the potential to maintain aging and epigenetic signatures of the donor, which is critical given that many diseases of the CNS are age related. Here, we review the published literature on the work that has been undertaken using iNs to model human brain disorders. Furthermore, as disease-modeling studies using this direct neuronal reprogramming approach are becoming more widely adopted, it is important to assess the criteria that are used to characterize the iNs, especially in relation to the extent to which they are mature adult neurons. In particular: i what constitutes an iN cell, ii which stages of conversion offer the earliest/optimal time to assess features that are specific to neurons and/or a disorder and iii whether generating subtype-specific iNs is critical to the disease-related features that iNs express. Finally, we discuss the range of potential biomedical applications that can be explored using patient-specific models of neurological disorders with iNs, and the challenges that will need to be overcome in order to realize these applications.

  17. Alkaline phosphatase and OCT-3/4 as useful markers for predicting susceptibility of human deciduous teeth-derived dental pulp cells to reprogramming factor-induced iPS cells.

    Science.gov (United States)

    Inada, Emi; Saitoh, Issei; Kubota, Naoko; Soda, Miki; Matsueda, Kazunari; Murakami, Tomoya; Sawami, Tadashi; Kagoshima, Akiko; Yamasaki, Youichi; Sato, Masahiro

    2017-11-01

    The aim of the present study was to prove that primary cells enriched with stem cells are more easily reprogrammed to generate induced pluripotent stem (iPS) cells than those with scarce numbers of stem cells. We surveyed the alkaline phosphatase (ALP) activity in five primarily-isolated human deciduous teeth-derived dental pulp cells (HDDPC) with cytochemical staining to examine the possible presence of stem cells. Next, the expression of stemness-specific factors, such as OCT(Octumer-binding transcription factor)3/4, NANOG, SOX2(SRY (sex determining region Y)-box 2), CD90, muscle segment homeodomain homeobox (MSX) 1, and MSX2, was assessed with a reverse transcription polymerase chain reaction method. Finally, these isolated HDDPC were transfected with plasmids carrying genes coding Yamanaka factors to determine whether these cells could be reprogrammed to generate iPS cells. Of the five primarily-isolated HDDPC, two (HDDPC-1 and -5) exhibited higher degrees of ALP activity. OCT-3/4 expression was also prominent in those two lines. Furthermore, these two lines proliferated faster than the other three lines. The transfection of HDDPC with Yamanaka factors resulted in the generation of iPS cells from HDDPC-1 and -5. The number of cells with the stemness property of HDDPC differs among individuals, which suggests that HDDPC showing an increased expression of both ALP and OCT-3/4 can be more easily reprogrammed to generate iPS cells after the forced expression of reprogramming factors. © 2016 John Wiley & Sons Australia, Ltd.

  18. Social Support and Successful Aging in Assisted Living Residents

    Science.gov (United States)

    Howie, Laura Odell; Troutman-Jordan, Meredith; Newman, Ann M.

    2014-01-01

    Successful aging has been associated with adequate social support. However, impaired functionality, increased dependence, multiple comorbidities, and reduced social interactions place older assisted living community (ALC) residents at risk for poorer social support and less successful aging. This cross-sectional descriptive study used the revised…

  19. The specification and global reprogramming of histone epigenetic marks during gamete formation and early embryo development in C. elegans.

    Directory of Open Access Journals (Sweden)

    Mark Samson

    2014-10-01

    Full Text Available In addition to the DNA contributed by sperm and oocytes, embryos receive parent-specific epigenetic information that can include histone variants, histone post-translational modifications (PTMs, and DNA methylation. However, a global view of how such marks are erased or retained during gamete formation and reprogrammed after fertilization is lacking. To focus on features conveyed by histones, we conducted a large-scale proteomic identification of histone variants and PTMs in sperm and mixed-stage embryo chromatin from C. elegans, a species that lacks conserved DNA methylation pathways. The fate of these histone marks was then tracked using immunostaining. Proteomic analysis found that sperm harbor ∼2.4 fold lower levels of histone PTMs than embryos and revealed differences in classes of PTMs between sperm and embryos. Sperm chromatin repackaging involves the incorporation of the sperm-specific histone H2A variant HTAS-1, a widespread erasure of histone acetylation, and the retention of histone methylation at sites that mark the transcriptional history of chromatin domains during spermatogenesis. After fertilization, we show HTAS-1 and 6 histone PTM marks distinguish sperm and oocyte chromatin in the new embryo and characterize distinct paternal and maternal histone remodeling events during the oocyte-to-embryo transition. These include the exchange of histone H2A that is marked by ubiquitination, retention of HTAS-1, removal of the H2A variant HTZ-1, and differential reprogramming of histone PTMs. This work identifies novel and conserved features of paternal chromatin that are specified during spermatogenesis and processed in the embryo. Furthermore, our results show that different species, even those with diverged DNA packaging and imprinting strategies, use conserved histone modification and removal mechanisms to reprogram epigenetic information.

  20. Polycomb group (PcG) proteins and Pax6 cooperate to inhibit in vivo reprogramming of the developing Drosophila eye.

    Science.gov (United States)

    Zhu, Jinjin; Ordway, Alison J; Weber, Lena; Buddika, Kasun; Kumar, Justin P

    2018-04-04

    How different cells and tissues commit to and determine their fates has been a central question in developmental biology since the seminal embryological experiments conducted by Wilhelm Roux and Hans Driesch in sea urchins and frogs. Here, we demonstrate that Polycomb group (PcG) proteins maintain Drosophila eye specification by suppressing the activation of alternative fate choices. The loss of PcG in the developing eye results in a cellular reprogramming event in which the eye is redirected to a wing fate. This fate transformation occurs with either the individual loss of Polycomb proteins or the simultaneous reduction of the Pleiohomeotic repressive complex and Pax6. Interestingly, the requirement for retinal selector genes is limited to Pax6, as the removal of more downstream members does not lead to the eye-wing transformation. We also show that distinct PcG complexes are required during different developmental windows throughout eye formation. These findings build on earlier observations that the eye can be reprogrammed to initiate head epidermis, antennal and leg development. © 2018. Published by The Company of Biologists Ltd.

  1. Reprogramming neutral lipid metabolism in mouse dendritic leucocytes hosting live Leishmania amazonensis amastigotes.

    Directory of Open Access Journals (Sweden)

    Hervé Lecoeur

    Full Text Available BACKGROUND: After loading with live Leishmania (L amazonensis amastigotes, mouse myeloid dendritic leucocytes/DLs are known to undergo reprogramming of their immune functions. In the study reported here, we investigated whether the presence of live L. amazonensis amastigotes in mouse bone marrow-derived DLs is able to trigger re-programming of DL lipid, and particularly neutral lipid metabolism. METHODOLOGY/PRINCIPAL FINDINGS: Affymetrix-based transcriptional profiles were determined in C57BL/6 and DBA/2 mouse bone marrow-derived DLs that had been sorted from cultures exposed or not to live L. amazonensis amastigotes. This showed that live amastigote-hosting DLs exhibited a coordinated increase in: (i long-chain fatty acids (LCFA and cholesterol uptake/transport, (ii LCFA and cholesterol (re-esterification to triacyl-sn-glycerol (TAG and cholesteryl esters (CE, respectively. As these neutral lipids are known to make up the lipid body (LB core, oleic acid was added to DL cultures and LB accumulation was compared in live amastigote-hosting versus amastigote-free DLs by epi-fluorescence and transmission electron microscopy. This showed that LBs were both significantly larger and more numerous in live amastigote-hosting mouse dendritic leucocytes. Moreover, many of the larger LB showed intimate contact with the membrane of the parasitophorous vacuoles hosting the live L. amazonensis amastigotes. CONCLUSIONS/SIGNIFICANCE: As leucocyte LBs are known to be more than simple neutral lipid repositories, we set about addressing two related questions. Could LBs provide lipids to live amastigotes hosted within the DL parasitophorous vacuole and also deliver? Could LBs impact either directly or indirectly on the persistence of L. amazonensis amastigotes in rodent skin?

  2. Strategies to overcome HBV-specific T cell exhaustion: checkpoint inhibitors and metabolic re-programming.

    Science.gov (United States)

    Fisicaro, Paola; Boni, Carolina; Barili, Valeria; Laccabue, Diletta; Ferrari, Carlo

    2018-01-29

    HBV-specific T cells play a key role in antiviral protection and failure to control HBV is associated with severely dysfunctional T cell responses. Therefore, functional T cell reconstitution represents a potential way to treat chronically infected patients. The growing understanding of the dysregulated transcriptional/epigenetic and metabolic programs underlying T cell exhaustion allows to envisage functional T cell reconstitution strategies based on the combined/sequential use of compounds able to induce decline of antigen load, checkpoint modulation, metabolic and epigenetic reprogramming with possible boosting of functionally restored responses by specific vaccines. Copyright © 2018 Elsevier B.V. All rights reserved.

  3. Reprogramming One-Carbon Metabolic Pathways To Decouple l-Serine Catabolism from Cell Growth in Corynebacterium glutamicum.

    Science.gov (United States)

    Zhang, Yun; Shang, Xiuling; Lai, Shujuan; Zhang, Yu; Hu, Qitiao; Chai, Xin; Wang, Bo; Liu, Shuwen; Wen, Tingyi

    2018-02-16

    l-Serine, the principal one-carbon source for DNA biosynthesis, is difficult for microorganisms to accumulate due to the coupling of l-serine catabolism and microbial growth. Here, we reprogrammed the one-carbon unit metabolic pathways in Corynebacterium glutamicum to decouple l-serine catabolism from cell growth. In silico model-based simulation showed a negative influence on glyA-encoding serine hydroxymethyltransferase flux with l-serine productivity. Attenuation of glyA transcription resulted in increased l-serine accumulation, and a decrease in purine pools, poor growth and longer cell shapes. The gcvTHP-encoded glycine cleavage (Gcv) system from Escherichia coli was introduced into C. glutamicum, allowing glycine-derived 13 CH 2 to be assimilated into intracellular purine synthesis, which resulted in an increased amount of one-carbon units. Gcv introduction not only restored cell viability and morphology but also increased l-serine accumulation. Moreover, comparative proteomic analysis indicated that abundance changes of the enzymes involved in one-carbon unit cycles might be responsible for maintaining one-carbon unit homeostasis. Reprogramming of the one-carbon metabolic pathways allowed cells to reach a comparable growth rate to accumulate 13.21 g/L l-serine by fed-batch fermentation in minimal medium. This novel strategy provides new insights into the regulation of cellular properties and essential metabolite accumulation by introducing an extrinsic pathway.

  4. Targeting Metabolic Reprogramming by Influenza Infection for Therapeutic Intervention

    Directory of Open Access Journals (Sweden)

    Heather S. Smallwood

    2017-05-01

    Full Text Available Influenza is a worldwide health and financial burden posing a significant risk to the immune-compromised, obese, diabetic, elderly, and pediatric populations. We identified increases in glucose metabolism in the lungs of pediatric patients infected with respiratory pathogens. Using quantitative mass spectrometry, we found metabolic changes occurring after influenza infection in primary human respiratory cells and validated infection-associated increases in c-Myc, glycolysis, and glutaminolysis. We confirmed these findings with a metabolic drug screen that identified the PI3K/mTOR inhibitor BEZ235 as a regulator of infectious virus production. BEZ235 treatment ablated the transient induction of c-Myc, restored PI3K/mTOR pathway homeostasis measured by 4E-BP1 and p85 phosphorylation, and reversed infection-induced changes in metabolism. Importantly, BEZ235 reduced infectious progeny but had no effect on the early stages of viral replication. BEZ235 significantly increased survival in mice, while reducing viral titer. We show metabolic reprogramming of host cells by influenza virus exposes targets for therapeutic intervention.

  5. Targeting Metabolic Reprogramming by Influenza Infection for Therapeutic Intervention

    Energy Technology Data Exchange (ETDEWEB)

    Smallwood, Heather S.; Duan, Susu; Morfouace, Marie; Rezinciuc, Svetlana; Shulkin, Barry L.; Shelat, Anang; Zink, Erika E.; Milasta, Sandra; Bajracharya, Resha; Oluwaseum, Ajayi J.; Roussel, Martine F.; Green, Douglas R.; Pasa-Tolic, Ljiljana; Thomas, Paul G.

    2017-05-01

    Influenza is a worldwide health and financial burden posing a significant risk to the immune-compromised, obese, diabetic, elderly, and pediatric populations. We identified increases in glucose metabolism in the lungs of pediatric patients infected with respiratory pathogens. Using quantitative mass spectrometry, we found metabolic changes occurring after influenza infection in primary human respiratory cells and validated infection-associated increases in c-Myc, glycolysis, and glutaminolysis. We confirmed these findings with a metabolic drug screen that identified the PI3K/mTOR inhibitor BEZ235 as a regulator of infectious virus production. BEZ235 treatment ablated the transient induction of c-Myc, restored PI3K/mTOR pathway homeostasis measured by 4E-BP1 and p85 phosphorylation, and reversed infection-induced changes in metabolism. Importantly, BEZ235 reduced infectious progeny but had no effect on the early stages of viral replication. BEZ235 significantly increased survival in mice, while reducing viral titer. We show metabolic reprogramming of host cells by influenza virus exposes targets for therapeutic intervention.

  6. Salmonella enterica serovar-specific transcriptional reprogramming of infected cells.

    Directory of Open Access Journals (Sweden)

    Sebastian Hannemann

    2017-07-01

    Full Text Available Despite their high degree of genomic similarity, different Salmonella enterica serovars are often associated with very different clinical presentations. In humans, for example, the typhoidal S. enterica serovar Typhi causes typhoid fever, a life-threatening systemic disease. In contrast, the non-typhoidal S. enterica serovar Typhimurium causes self-limiting gastroenteritis. The molecular bases for these different clinical presentations are incompletely understood. The ability to re-program gene expression in host cells is an essential virulence factor for typhoidal and non-typhoidal S. enterica serovars. Here, we have compared the transcriptional profile of cultured epithelial cells infected with S. Typhimurium or S. Typhi. We found that both serovars stimulated distinct transcriptional responses in infected cells that are associated with the stimulation of specific signal transduction pathways. These specific responses were associated with the presence of a distinct repertoire of type III secretion effector proteins. These observations provide major insight into the molecular bases for potential differences in the pathogenic mechanisms of typhoidal and non-typhoidal S. enterica serovars.

  7. Glimpse into Hox and tale regulation of cell differentiation and reprogramming.

    Science.gov (United States)

    Cerdá-Esteban, Nuria; Spagnoli, Francesca M

    2014-01-01

    During embryonic development, cells become gradually restricted in their developmental potential and start elaborating lineage-specific transcriptional networks to ultimately acquire a unique differentiated state. Hox genes play a central role in specifying regional identities, thereby providing the cell with critical information on positional value along its differentiation path. The exquisite DNA-binding specificity of the Hox proteins is frequently dependent upon their interaction with members of the TALE family of homeodomain proteins. In addition to their function as Hox-cofactors, TALE homeoproteins control multiple crucial developmental processes through Hox-independent mechanisms. Here, we will review recent findings on the function of both Hox and TALE proteins in cell differentiation, referring mostly to vertebrate species. In addition, we will discuss the direct implications of this knowledge on cell plasticity and cell reprogramming. Copyright © 2013 Wiley Periodicals, Inc.

  8. Bioenergetic reprogramming plasticity under nitrogen depletion by the unicellular green alga Scenedesmus obliquus.

    Science.gov (United States)

    Papazi, Aikaterini; Korelidou, Anna; Andronis, Efthimios; Parasyri, Athina; Stamatis, Nikolaos; Kotzabasis, Kiriakos

    2018-03-01

    Simultaneous nitrogen depletion and 3,4-dichlorophenol addition induce a bioenergetic microalgal reprogramming, through strong Cyt b 6 f synthesis, that quench excess electrons from dichlorophenol's biodegradation to an overactivated photosynthetic electron flow and H 2 -productivity. Cellular energy management includes "rational" planning and operation of energy production and energy consumption units. Microalgae seem to have the ability to calculate their energy reserves and select the most profitable bioenergetic pathways. Under oxygenic mixotrophic conditions, microalgae invest the exogenously supplied carbon source (glucose) to biomass increase. If 3,4-dichlorophenol is added in the culture medium, then glucose is invested more to biodegradation rather than to growth. The biodegradation yield is enhanced in nitrogen-depleted conditions, because of an increase in the starch accumulation and a delay in the establishment of oxygen-depleted conditions in a closed system. In nitrogen-depleted conditions, starch cannot be invested in PSII-dependent and PSII-independent pathways for H 2 -production, mainly because of a strong decrease of the cytochrome b 6 f complex of the photosynthetic electron flow. For this reason, it seems more profitable for the microalga under these conditions to direct the metabolism to the synthesis of lipids as cellular energy reserves. Nitrogen-depleted conditions with exogenously supplied 3,4-dichlorophenol induce reprogramming of the microalgal bioenergetic strategy. Cytochrome b 6 f is strongly synthesized (mainly through catabolism of polyamines) to manage the electron bypass from the dichlorophenol biodegradation procedure to the photosynthetic electron flow (at the level of PQ pool) and consequently through cytochrome b 6 f and PSI to hydrogenase and H 2 -production. All the above showed that the selection of the appropriate cultivation conditions is the key for the manipulation of microalgal bioenergetic strategy that leads to

  9. Circadian Reprogramming in the Liver Identifies Metabolic Pathways of Aging.

    Science.gov (United States)

    Sato, Shogo; Solanas, Guiomar; Peixoto, Francisca Oliveira; Bee, Leonardo; Symeonidi, Aikaterini; Schmidt, Mark S; Brenner, Charles; Masri, Selma; Benitah, Salvador Aznar; Sassone-Corsi, Paolo

    2017-08-10

    The process of aging and circadian rhythms are intimately intertwined, but how peripheral clocks involved in metabolic homeostasis contribute to aging remains unknown. Importantly, caloric restriction (CR) extends lifespan in several organisms and rewires circadian metabolism. Using young versus old mice, fed ad libitum or under CR, we reveal reprogramming of the circadian transcriptome in the liver. These age-dependent changes occur in a highly tissue-specific manner, as demonstrated by comparing circadian gene expression in the liver versus epidermal and skeletal muscle stem cells. Moreover, de novo oscillating genes under CR show an enrichment in SIRT1 targets in the liver. This is accompanied by distinct circadian hepatic signatures in NAD + -related metabolites and cyclic global protein acetylation. Strikingly, this oscillation in acetylation is absent in old mice while CR robustly rescues global protein acetylation. Our findings indicate that the clock operates at the crossroad between protein acetylation, liver metabolism, and aging. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Tributyltin impaired reproductive success in female zebrafish through disrupting oogenesis, reproductive behaviors and serotonin synthesis.

    Science.gov (United States)

    Xiao, Wei-Yang; Li, Ying-Wen; Chen, Qi-Liang; Liu, Zhi-Hao

    2018-07-01

    study demonstrated that TBT may impair the reproductive success of zebrafish females probably through disrupting oogenesis, disturbing reproductive behaviors and altering serotonin synthesis. The present study greatly extends our understanding on the reproductive toxicity of TBT on fish. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. PPARα inhibition modulates multiple reprogrammed metabolic pathways in kidney cancer and attenuates tumor growth.

    Science.gov (United States)

    Abu Aboud, Omran; Donohoe, Dallas; Bultman, Scott; Fitch, Mark; Riiff, Tim; Hellerstein, Marc; Weiss, Robert H

    2015-06-01

    Kidney cancer [renal cell carcinoma (RCC)] is the sixth-most-common cancer in the United States, and its incidence is increasing. The current progression-free survival for patients with advanced RCC rarely extends beyond 1-2 yr due to the development of therapeutic resistance. We previously identified peroxisome proliferator-activating receptor-α (PPARα) as a potential therapeutic target for this disease and showed that a specific PPARα antagonist, GW6471, induced apoptosis and cell cycle arrest at G0/G1 in RCC cell lines associated with attenuation of cell cycle regulatory proteins. We now extend that work and show that PPARα inhibition attenuates components of RCC metabolic reprogramming, capitalizing on the Warburg effect. The specific PPARα inhibitor GW6471, as well as a siRNA specific to PPARα, attenuates the enhanced fatty acid oxidation and oxidative phosphorylation associated with glycolysis inhibition, and PPARα antagonism also blocks the enhanced glycolysis that has been observed in RCC cells; this effect did not occur in normal human kidney epithelial cells. Such cell type-specific inhibition of glycolysis corresponds with changes in protein levels of the oncogene c-Myc and has promising clinical implications. Furthermore, we show that treatment with GW6471 results in RCC tumor growth attenuation in a xenograft mouse model, with minimal obvious toxicity, a finding associated with the expected on-target effects on c-Myc. These studies demonstrate that several pivotal cancer-relevant metabolic pathways are inhibited by PPARα antagonism. Our data support the concept that targeting PPARα, with or without concurrent inhibition of glycolysis, is a potential novel and effective therapeutic approach for RCC that targets metabolic reprogramming in this tumor.

  12. Dual Transcriptome Profiling of Leishmania-Infected Human Macrophages Reveals Distinct Reprogramming Signatures.

    Science.gov (United States)

    Fernandes, Maria Cecilia; Dillon, Laura A L; Belew, Ashton Trey; Bravo, Hector Corrada; Mosser, David M; El-Sayed, Najib M

    2016-05-10

    Macrophages are mononuclear phagocytes that constitute a first line of defense against pathogens. While lethal to many microbes, they are the primary host cells of Leishmania spp. parasites, the obligate intracellular pathogens that cause leishmaniasis. We conducted transcriptomic profiling of two Leishmania species and the human macrophage over the course of intracellular infection by using high-throughput RNA sequencing to characterize the global gene expression changes and reprogramming events that underlie the interactions between the pathogen and its host. A systematic exclusion of the generic effects of large-particle phagocytosis revealed a vigorous, parasite-specific response of the human macrophage early in the infection that was greatly tempered at later time points. An analogous temporal expression pattern was observed with the parasite, suggesting that much of the reprogramming that occurs as parasites transform into intracellular forms generally stabilizes shortly after entry. Following that, the parasite establishes an intracellular niche within macrophages, with minimal communication between the parasite and the host cell later during the infection. No significant difference was observed between parasite species transcriptomes or in the transcriptional response of macrophages infected with each species. Our comparative analysis of gene expression changes that occur as mouse and human macrophages are infected by Leishmania spp. points toward a general signature of the Leishmania-macrophage infectome. Little is known about the transcriptional changes that occur within mammalian cells harboring intracellular pathogens. This study characterizes the gene expression signatures of Leishmania spp. parasites and the coordinated response of infected human macrophages as the pathogen enters and persists within them. After accounting for the generic effects of large-particle phagocytosis, we observed a parasite-specific response of the human macrophages early in

  13. Digital gene expression profiling by 5'-end sequencing of cDNAs during reprogramming in the moss Physcomitrella patens.

    Directory of Open Access Journals (Sweden)

    Tomoaki Nishiyama

    Full Text Available Stem cells self-renew and repeatedly produce differentiated cells during development and growth. The differentiated cells can be converted into stem cells in some metazoans and land plants with appropriate treatments. After leaves of the moss Physcomitrella patens are excised, leaf cells reenter the cell cycle and commence tip growth, which is characteristic of stem cells called chloronema apical cells. To understand the underlying molecular mechanisms, a digital gene expression profiling method using mRNA 5'-end tags (5'-DGE was established. The 5'-DGE method produced reproducible data with a dynamic range of four orders that correlated well with qRT-PCR measurements. After the excision of leaves, the expression levels of 11% of the transcripts changed significantly within 6 h. Genes involved in stress responses and proteolysis were induced and those involved in metabolism, including photosynthesis, were reduced. The later processes of reprogramming involved photosynthesis recovery and higher macromolecule biosynthesis, including of RNA and proteins. Auxin and cytokinin signaling pathways, which are activated during stem cell formation via callus in flowering plants, are also activated during reprogramming in P. patens, although no exogenous phytohormone is applied in the moss system, suggesting that an intrinsic phytohormone regulatory system may be used in the moss.

  14. Effect of the time interval between fusion and activation on epigenetic reprogramming and development of bovine somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Liu, Jun; Wang, Yongsheng; Su, Jianmin; Wang, Lijun; Li, Ruizhe; Li, Qian; Wu, Yongyan; Hua, Song; Quan, Fusheng; Guo, Zekun; Zhang, Yong

    2013-04-01

    Previous studies have shown that the time interval between fusion and activation (FA interval) play an important role in nuclear remodeling and in vitro development of somatic cell nuclear transfer (SCNT) embryos. However, the effects of FA interval on the epigenetic reprogramming and in vivo developmental competence of SCNT embryos remain unknown. In the present study, the effects of different FA intervals (0 h, 2 h, and 4 h) on the epigenetic reprogramming and developmental competence of bovine SCNT embryos were assessed. The results demonstrated that H3 lysine 9 (H3K9ac) levels decreased rapidly after fusion in all three groups. H3K9ac was practically undetectable 2 h after fusion in the 2-h and 4-h FA interval groups. However, H3K9ac was still evidently detectable in the 0-h FA interval group. The H3K9ac levels increased 10 h after fusion in all three groups, but were higher in the 2-h and 4-h FA interval groups than that in the 0-h FA interval group. The methylation levels of the satellite I region in day-7 blastocysts derived from the 2-h or 4-h FA interval groups was similar to that of in vitro fertilization blastocysts and is significantly lower than that of the 0-h FA interval group. SCNT embryos derived from 2-h FA interval group showed higher developmental competence than those from the 0-h and 4-h FA interval groups in terms of cleavage rate, blastocyst formation rate, apoptosis index, and pregnancy and calving rates. Hence, the FA interval is an important factor influencing the epigenetic reprogramming and developmental competence of bovine SCNT embryos.

  15. Donor cell differentiation, reprogramming, and cloning efficiency: elusive or illusive correlation?

    Science.gov (United States)

    Oback, B; Wells, D N

    2007-05-01

    Compared to other assisted reproductive technologies, mammalian nuclear transfer (NT) cloning is inefficient in generating viable offspring. It has been postulated that nuclear reprogramming and cloning efficiency can be increased by choosing less differentiated cell types as nuclear donors. This hypothesis is mainly supported by comparative mouse cloning experiments using early blastomeres, embryonic stem (ES) cells, and terminally differentiated somatic donor cells. We have re-evaluated these comparisons, taking into account different NT procedures, the use of donor cells from different genetic backgrounds, sex, cell cycle stages, and the lack of robust statistical significance when post-blastocyst development is compared. We argue that while the reprogrammability of early blastomeres appears to be much higher than that of somatic cells, it has so far not been conclusively determined whether differentiation status affects cloning efficiency within somatic donor cell lineages. Copyright (c) 2006 Wiley-Liss, Inc.

  16. Reprogramming with Small Molecules instead of Exogenous Transcription Factors

    Directory of Open Access Journals (Sweden)

    Tongxiang Lin

    2015-01-01

    Full Text Available Induced pluripotent stem cells (iPSCs could be employed in the creation of patient-specific stem cells, which could subsequently be used in various basic and clinical applications. However, current iPSC methodologies present significant hidden risks with respect to genetic mutations and abnormal expression which are a barrier in realizing the full potential of iPSCs. A chemical approach is thought to be a promising strategy for safety and efficiency of iPSC generation. Many small molecules have been identified that can be used in place of exogenous transcription factors and significantly improve iPSC reprogramming efficiency and quality. Recent studies have shown that the use of small molecules results in the generation of chemically induced pluripotent stem cells from mouse embryonic fibroblast cells. These studies might lead to new areas of stem cell research and medical applications, not only human iPSC by chemicals alone, but also safe generation of somatic stem cells for cell based clinical trials and other researches. In this paper, we have reviewed the recent advances in small molecule approaches for the generation of iPSCs.

  17. Direct identification of the Meloidogyne incognita secretome reveals proteins with host cell reprogramming potential.

    Directory of Open Access Journals (Sweden)

    Stéphane Bellafiore

    2008-10-01

    Full Text Available The root knot nematode, Meloidogyne incognita, is an obligate parasite that causes significant damage to a broad range of host plants. Infection is associated with secretion of proteins surrounded by proliferating cells. Many parasites are known to secrete effectors that interfere with plant innate immunity, enabling infection to occur; they can also release pathogen-associated molecular patterns (PAMPs, e.g., flagellin that trigger basal immunity through the nematode stylet into the plant cell. This leads to suppression of innate immunity and reprogramming of plant cells to form a feeding structure containing multinucleate giant cells. Effectors have generally been discovered using genetics or bioinformatics, but M. incognita is non-sexual and its genome sequence has not yet been reported. To partially overcome these limitations, we have used mass spectrometry to directly identify 486 proteins secreted by M. incognita. These proteins contain at least segmental sequence identity to those found in our 3 reference databases (published nematode proteins; unpublished M. incognita ESTs; published plant proteins. Several secreted proteins are homologous to plant proteins, which they may mimic, and they contain domains that suggest known effector functions (e.g., regulating the plant cell cycle or growth. Others have regulatory domains that could reprogram cells. Using in situ hybridization we observed that most secreted proteins were produced by the subventral glands, but we found that phasmids also secreted proteins. We annotated the functions of the secreted proteins and classified them according to roles they may play in the development of root knot disease. Our results show that parasite secretomes can be partially characterized without cognate genomic DNA sequence. We observed that the M. incognita secretome overlaps the reported secretome of mammalian parasitic nematodes (e.g., Brugia malayi, suggesting a common parasitic behavior and a possible

  18. Molecular beacon nanosensors for live cell detection and tracking differentiation and reprogramming

    DEFF Research Database (Denmark)

    Ilieva, Mirolyuba

    2013-01-01

    open to closed state within living cells. Using MBs targeting pluripotent stem cell markers we demonstrated reverse into a more immature state of LUHMES induced by neurosphere-like growth conditions. Moreover, we have been able to trace localisation of this particular population during differentiation...... in separation of fluorophore from quencher and thereby emission of a fluorescent signal that can be detected. In this project the usability and applicability of MBs for live cell detection and tracing of gene expression was demonstrated. MBs library targeting gene markers for pluripotent stem cells as well...... and thus demonstrate the usability of MBs for monitoring cell behaviour within 3D clusters. Finally, MBs detection of expression of human pluripotent markers after reprograming of adult somatic cells with plasmid codding for mouse transcription factors was demonstrated. In conclusion, the method of using...

  19. Restricted mobility of Dnmt1 in preimplantation embryos: implications for epigenetic reprogramming

    Science.gov (United States)

    Grohmann, Maik; Spada, Fabio; Schermelleh, Lothar; Alenina, Natalia; Bader, Michael; Cardoso, M Cristina; Leonhardt, Heinrich

    2005-01-01

    Background Mouse preimplantation development is characterized by both active and passive genomic demethylation. A short isoform of the prevalent maintenance DNA methyltransferase (Dnmt1S) is found in the cytoplasm of preimplantation embryos and transiently enters the nucleus only at the 8-cell stage. Results Using GFP fusions we show that both the long and short isoforms of Dnmt1 localize to the nucleus of somatic cells and the cytoplasm of preimplantation embryos and that these subcellular localization properties are independent of phosphorylation. Importantly, photobleaching techniques and salt extraction revealed that Dnmt1S has a very restricted mobility in the cytoplasm, while it is highly mobile in the nucleus of preimplantation embryos. Conclusion The restricted mobility of Dnmt1S limits its access to DNA and likely contributes to passive demethylation and epigenetic reprogramming during preimplantationdevelopment. PMID:16120212

  20. Reprogramming of somatic cells induced by fusion of embryonic stem cells using hemagglutinating virus of Japan envelope (HVJ-E)

    International Nuclear Information System (INIS)

    Yue, Xiao-shan; Fujishiro, Masako; Toyoda, Masashi; Akaike, Toshihiro; Ito, Yoshihiro

    2010-01-01

    In this research, hemagglutinating virus of Japan envelope (HVJ-E) was used to reprogram somatic cells by fusion with mouse embryonic stem (ES) cells. Neomycin-resistant mouse embryonic fibroblasts (MEFs) were used as somatic cells. Nanog-overexpressing puromycin-resistant EB3 cells were used as mouse ES cells. These two cells were fused by exposing to HVJ-E and the generated fusion cells were selected by puromycin and G418 to get the stable fusion cell line. The fusion cells form colonies in feeder-free culture system. Microsatellite analysis of the fusion cells showed that they possessed genes from both ES cells and fibroblasts. The fusion cells were tetraploid, had alkali phosphatase activity, and expressed stem cell marker genes such as Pou5f1, Nanog, and Sox2, but not the fibroblast cell marker genes such as Col1a1 and Col1a2. The pluripotency of fusion cells was confirmed by their expression of marker genes for all the three germ layers after differentiation induction, and by their ability to form teratoma which contained all the three primary layers. Our results show that HVJ-E can be used as a fusion reagent for reprogramming of somatic cells.

  1. Reprogramming of somatic cells induced by fusion of embryonic stem cells using hemagglutinating virus of Japan envelope (HVJ-E)

    Energy Technology Data Exchange (ETDEWEB)

    Yue, Xiao-shan [Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Department of Biomolecular Engineering, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501 (Japan); Fujishiro, Masako [Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Toyoda, Masashi [Department of Reproductive Biology, National Institute for Child Health and Development, 2-10-1, Okura, Setagaya-ku, Tokyo 157-8535 (Japan); Akaike, Toshihiro [Department of Biomolecular Engineering, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501 (Japan); Ito, Yoshihiro, E-mail: y-ito@riken.jp [Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Department of Biomolecular Engineering, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501 (Japan)

    2010-04-16

    In this research, hemagglutinating virus of Japan envelope (HVJ-E) was used to reprogram somatic cells by fusion with mouse embryonic stem (ES) cells. Neomycin-resistant mouse embryonic fibroblasts (MEFs) were used as somatic cells. Nanog-overexpressing puromycin-resistant EB3 cells were used as mouse ES cells. These two cells were fused by exposing to HVJ-E and the generated fusion cells were selected by puromycin and G418 to get the stable fusion cell line. The fusion cells form colonies in feeder-free culture system. Microsatellite analysis of the fusion cells showed that they possessed genes from both ES cells and fibroblasts. The fusion cells were tetraploid, had alkali phosphatase activity, and expressed stem cell marker genes such as Pou5f1, Nanog, and Sox2, but not the fibroblast cell marker genes such as Col1a1 and Col1a2. The pluripotency of fusion cells was confirmed by their expression of marker genes for all the three germ layers after differentiation induction, and by their ability to form teratoma which contained all the three primary layers. Our results show that HVJ-E can be used as a fusion reagent for reprogramming of somatic cells.

  2. Posttranscriptional (Re)programming of Cell Fate: Examples in Stem Cells, Progenitor, and Differentiated Cells.

    Science.gov (United States)

    Kanellopoulou, Chrysi; Muljo, Stefan A

    2018-01-01

    How a single genome can give rise to many different transcriptomes and thus all the different cell lineages in the human body is a fundamental question in biology. While signaling pathways, transcription factors, and chromatin architecture, to name a few determinants, have been established to play critical roles, recently, there is a growing appreciation of the roles of non-coding RNAs and RNA-binding proteins in controlling cell fates posttranscriptionally. Thus, it is vital that these emerging players are also integrated into models of gene regulatory networks that underlie programs of cellular differentiation. Sometimes, we can leverage knowledge about such posttranscriptional circuits to reprogram patterns of gene expression in meaningful ways. Here, we review three examples from our work.

  3. Siblings of the Hearing Impaired: Perspectives for Parents.

    Science.gov (United States)

    Atkins, Dale V.

    1987-01-01

    The author describes the concerns and needs of siblings of hearing-impaired children, offering practical suggestions for parents, educators, and mental health and community service workers. Examples of successful programs for siblings that provide emotional support, training and information about their siblings' disability, and effective…

  4. New clues in the nucleus: Transcriptional reprogramming in effector-triggered immunity

    Directory of Open Access Journals (Sweden)

    SAIKAT eBHATTACHARJEE

    2013-09-01

    Full Text Available The robustness of plant effector-triggered immunity is correlated with massive alterations of the host transcriptome. Yet the molecular mechanisms that cause and underlie this reprogramming remain obscure. Here we will review recent advances in deciphering nuclear functions of plant immune receptors and of associated proteins. Important open questions remain, such as the identities of the primary transcription factors involved in control of effector-triggered immune responses, and indeed whether this can be generalized or whether particular effector-resistance protein interactions impinge on distinct sectors in the transcriptional response web. Multiple lines of evidence have implicated WRKY transcription factors at the core of responses to microbe-associated molecular patterns and in intersections with effector-triggered immunity. Recent findings from yeast two-hybrid studies suggest that members of the TCP transcription factor family are targets of several effectors from diverse pathogens. Additional transcription factor families that are directly or indirectly involved in effector-triggered immunity are likely to be identified.

  5. Reprogramming of rabbit induced pluripotent stem cells toward epiblast and chimeric competency using Krüppel-like factors

    Directory of Open Access Journals (Sweden)

    Yann Tapponnier

    2017-10-01

    Full Text Available Rabbit induced pluripotent stem cells (rbiPSCs possess the characteristic features of primed pluripotency as defined in rodents and primates. In the present study, we reprogrammed rbiPSCs using human Krüppel-like factors (KLFs 2 and 4 and cultured them in a medium supplemented with fetal calf serum and leukemia inhibitory factor. These cells (designated rbEKA were propagated by enzymatic dissociation for at least 30 passages, during which they maintained a normal karyotype. This new culturing protocol resulted in transcriptional and epigenetic reconfiguration, as substantiated by the expression of transcription factors and the presence of histone modifications associated with naïve pluripotency. Furthermore, microarray analysis of rbiPSCs, rbEKA cells, rabbit ICM cells, and rabbit epiblast showed that the global gene expression profile of the reprogrammed rbiPSCs was more similar to that of rabbit ICM and epiblast cells. Injection of rbEKA cells into 8-cell stage rabbit embryos resulted in extensive colonization of ICM in 9% early-blastocysts (E3.5, epiblast in 10% mid-blastocysts (E4.5, and embryonic disk in 1.4% pre-gastrulae (E6. Thus, these results indicate that KLF2 and KLF4 triggered the conversion of rbiPSCs into epiblast-like, embryo colonization-competent PSCs. Our results highlight some of the requirements to achieve bona fide chimeric competency.

  6. Application of Induced Pluripotent Stem Cells Reprogrammed from Dental Pulp Cells: a Novel Approach for Tooth Regeneration

    Directory of Open Access Journals (Sweden)

    Xiaoyan Zhou

    2011-03-01

    Full Text Available Introduction: Candidate human dental stem/progenitor cells have been isolated and charac-terized from dental tissues and shown to hold the capability to differentiate into tooth-generating cells. However, ad-vances in engineering a whole tooth by these stem cells are hindered by various factors, such as the poor availability of human primitive tooth bud stem cells, difficulties in isolating and purifying dental mesenchymal stem cells and ethical controversies when using embryonic oral epithelium. As a result it is meaningful to find other autologous dental cells for the purpose of reconstructing a tooth.The hypothesis: Previous studies demonstrated that somatic cells can be reprogrammed into induced pluripotent stem cells by ex-ogenous expression Oct-4 and Sox-2. On the basis of these findings we can reasonably hypothesize that when transfected with specific transcription factors Oct-4 and Sox-2, dental pulp cells, the main cell in pulp, could also be reprogrammed into induced pluripotent stem cells, which are considered to be of best potential to regenerate a whole tooth. Evaluation of the hypothesis: After transfection with Oct-4 and Sox-2 into human dental pulp cells, the positive colonies are isolated and then identified according to the characteristics of iPS cells. These cells are further investigated the capability in differentiating into ameloblasts and odontoblasts and finally seeded onto the sur-face of a tooth-shaped biodegradable polymer scaffold to detect the ability of constructing a bioengineered tooth.

  7. SUCCES AT SCHOOL IN VISUALLY IMPAIRED CHILDREN

    Directory of Open Access Journals (Sweden)

    Stanika DIKIC

    1998-04-01

    Full Text Available The research included 200 visually impaired children of primary school during the period from 1992 to 1996. By means of adequate instruments we have tested the relation between the success at school of partially seeing children and hyperkinetic behavior, active and passive vocabulary richness, visuo-motoric coordination and the maturity of handwriting. Besides the already known factors (intellectual level, specific learning disturbances, emotional and neurotic disturbances, cultural deprivation, the success in class depends very much on the intensity of hyperkinetic behavior as well as its features: unstable attention, impulsiveness and hyperactivity. Visual-motor coordination eye-hand and the maturity of handwriting have a strong influence on their success at school.

  8. DNA methyltransferases and stress-related genes expression in zebrafish larvae after exposure to heat and copper during reprogramming of DNA methylation.

    Science.gov (United States)

    Dorts, Jennifer; Falisse, Elodie; Schoofs, Emilie; Flamion, Enora; Kestemont, Patrick; Silvestre, Frédéric

    2016-10-12

    DNA methylation, a well-studied epigenetic mark, is important for gene regulation in adulthood and for development. Using genetic and epigenetic approaches, the present study aimed at evaluating the effects of heat stress and copper exposure during zebrafish early embryogenesis when patterns of DNA methylation are being established, a process called reprogramming. Embryos were exposed to 325 μg Cu/L from fertilization (<1 h post fertilization - hpf) to 4 hpf at either 26.5 °C or 34 °C, followed by incubation in clean water at 26.5 °C till 96 hpf. Significant increased mortality rates and delayed hatching were observed following exposure to combined high temperature and Cu. Secondly, both stressors, alone or in combination, significantly upregulated the expression of de novo DNA methyltransferase genes (dnmt3) along with no differences in global cytosine methylation level. Finally, Cu exposure significantly increased the expression of metallothionein (mt2) and heat shock protein (hsp70), the latter being also increased following exposure to high temperature. These results highlighted the sensitivity of early embryogenesis and more precisely of the reprogramming period to environmental challenges, in a realistic situation of combined stressors.

  9. The efficacy of SMS text messages to compensate for the effects of cognitive impairments in schizophrenia

    NARCIS (Netherlands)

    Pijnenborg, G. H. M.; Withaar, F. K.; Brouwer, W. H.; Timmerman, M. E.; van den Bosch, R. J.; Evans, J. J.

    Background and aims. Many people with schizophrenia have severe cognitive impairments that hamper their activities. The effect of pharmacological and behavioural interventions on cognitive functioning has been demonstrated, but even after successful intervention considerable impairments can remain.

  10. Successful and unsuccessful psychopaths: a neurobiological model.

    Science.gov (United States)

    Gao, Yu; Raine, Adrian

    2010-01-01

    Despite increasing interest in psychopathy research, surprisingly little is known about the etiology of non-incarcerated, successful psychopaths. This review provides an analysis of current knowledge on the similarities and differences between successful and unsuccessful psychopaths derived from five population sources: community samples, individuals from employment agencies, college students, industrial psychopaths, and serial killers. An initial neurobiological model of successful and unsuccessful psychopathy is outlined. It is hypothesized that successful psychopaths have intact or enhanced neurobiological functioning that underlies their normal or even superior cognitive functioning, which in turn helps them to achieve their goals using more covert and nonviolent methods. In contrast, in unsuccessful, caught psychopaths, brain structural and functional impairments together with autonomic nervous system dysfunction are hypothesized to underlie cognitive and emotional deficits and more overt violent offending.

  11. C/EBPα creates elite cells for iPSC reprogramming by upregulating Klf4 and increasing the levels of Lsd1 and Brd4

    DEFF Research Database (Denmark)

    Di Stefano, Bruno; Collombet, Samuel; Jakobsen, Janus Schou

    2016-01-01

    reprogrammed into iPSCs by the Yamanaka factors OSKM. Here we show that C/EBPα post-transcriptionally increases the abundance of several hundred proteins, including Lsd1, Hdac1, Brd4, Med1 and Cdk9, components of chromatin-modifying complexes present at super-enhancers. Lsd1 was found to be required for B cell...

  12. Nuclear and nuclear reprogramming during the first cell cycle in bovine nuclear transfer embryos

    DEFF Research Database (Denmark)

    Østrup, Olga; Petrovicova, Ida; Strejcek, Frantisek

    2009-01-01

    Abstract The immediate events of genomic reprogramming at somatic cell nuclear transfer (SCNT) are to high degree unknown. This study was designed to evaluate the nuclear and nucleolar changes during the first cell cycle. Bovine SCNT embryos were produced from starved bovine fibroblasts and fixed......, somatic cell nuclei introduced into enucleated oocytes displayed chromatin condensation, partial nuclear envelope breakdown, nucleolar desegregation and transcriptional quiescence already at 0.5 hpa. Somatic cell cytoplasm remained temporally attached to introduced nucleus and nucleolus was partially...... restored indicating somatic influence in the early SCNT phases. At 1-3 hpa, chromatin gradually decondensed toward the nucleus periphery and nuclear envelope reformed. From 4 hpa, the somatic cell nucleus gained a PN-like appearance and displayed NPBs suggesting ooplasmic control of development....

  13. Quantitative 1H NMR metabolomics reveals extensive metabolic reprogramming of primary and secondary metabolism in elicitor-treated opium poppy cell cultures

    Directory of Open Access Journals (Sweden)

    Vogel Hans J

    2008-01-01

    Full Text Available Abstract Background Opium poppy (Papaver somniferum produces a diverse array of bioactive benzylisoquinoline alkaloids and has emerged as a model system to study plant alkaloid metabolism. The plant is cultivated as the only commercial source of the narcotic analgesics morphine and codeine, but also produces many other alkaloids including the antimicrobial agent sanguinarine. Modulations in plant secondary metabolism as a result of environmental perturbations are often associated with the altered regulation of other metabolic pathways. As a key component of our functional genomics platform for opium poppy we have used proton nuclear magnetic resonance (1H NMR metabolomics to investigate the interplay between primary and secondary metabolism in cultured opium poppy cells treated with a fungal elicitor. Results Metabolite fingerprinting and compound-specific profiling showed the extensive reprogramming of primary metabolic pathways in association with the induction of alkaloid biosynthesis in response to elicitor treatment. Using Chenomx NMR Suite v. 4.6, a software package capable of identifying and quantifying individual compounds based on their respective signature spectra, the levels of 42 diverse metabolites were monitored over a 100-hour time course in control and elicitor-treated opium poppy cell cultures. Overall, detectable and dynamic changes in the metabolome of elicitor-treated cells, especially in cellular pools of carbohydrates, organic acids and non-protein amino acids were detected within 5 hours after elicitor treatment. The metabolome of control cultures also showed substantial modulations 80 hours after the start of the time course, particularly in the levels of amino acids and phospholipid pathway intermediates. Specific flux modulations were detected throughout primary metabolism, including glycolysis, the tricarboxylic acid cycle, nitrogen assimilation, phospholipid/fatty acid synthesis and the shikimate pathway, all of which

  14. Metabolic reprogramming of the urea cycle pathway in experimental pulmonary arterial hypertension rats induced by monocrotaline.

    Science.gov (United States)

    Zheng, Hai-Kuo; Zhao, Jun-Han; Yan, Yi; Lian, Tian-Yu; Ye, Jue; Wang, Xiao-Jian; Wang, Zhe; Jing, Zhi-Cheng; He, Yang-Yang; Yang, Ping

    2018-05-11

    Pulmonary arterial hypertension (PAH) is a rare systemic disorder associated with considerable metabolic dysfunction. Although enormous metabolomic studies on PAH have been emerging, research remains lacking on metabolic reprogramming in experimental PAH models. We aim to evaluate the metabolic changes in PAH and provide new insight into endogenous metabolic disorders of PAH. A single subcutaneous injection of monocrotaline (MCT) (60 mg kg - 1 ) was used for rats to establish PAH model. Hemodynamics and right ventricular hypertrophy were adopted to evaluate the successful establishment of PAH model. Plasma samples were assessed through targeted metabolomic profiling platform to quantify 126 endogenous metabolites. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to discriminate between MCT-treated model and control groups. Metabolite Set Enrichment Analysis was adapted to exploit the most disturbed metabolic pathways. Endogenous metabolites of MCT treated PAH model and control group were well profiled using this platform. A total of 13 plasma metabolites were significantly altered between the two groups. Metabolite Set Enrichment Analysis highlighted that a disruption in the urea cycle pathway may contribute to PAH onset. Moreover, five novel potential biomarkers in the urea cycle, adenosine monophosphate, urea, 4-hydroxy-proline, ornithine, N-acetylornithine, and two candidate biomarkers, namely, O-acetylcarnitine and betaine, were found to be highly correlated with PAH. The present study suggests a new role of urea cycle disruption in the pathogenesis of PAH. We also found five urea cycle related biomarkers and another two candidate biomarkers to facilitate early diagnosis of PAH in metabolomic profile.

  15. Pancreatic Cancer Cell Exosome-Mediated Macrophage Reprogramming and the Role of MicroRNAs 155 and 125b2 Transfection using Nanoparticle Delivery Systems

    Science.gov (United States)

    Su, Mei-Ju; Aldawsari, Hibah; Amiji, Mansoor

    2016-01-01

    Exosomes are nano-sized endosome-derived small intraluminal vesicles, which are important facilitators of intercellular communication by transporting contents, such as protein, mRNA, and microRNAs, between neighboring cells, such as in the tumor microenvironment. The purpose of this study was to understand the mechanisms of exosomes-mediated cellular communication between human pancreatic cancer (Panc-1) cells and macrophages (J771.A1) using a Transwell co-culture system. Following characterization of exosome-mediated cellular communication and pro-tumoral baseline M2 macrophage polarization, the Panc-1 cells were transfected with microRNA-155 (miR-155) and microRNA-125b-2 (miR-125b2) expressing plasmid DNA using hyaluronic acid-poly(ethylene imine)/hyaluronic acid-poly(ethylene glycol) (HA-PEI/HA-PEG) self-assembling nanoparticle-based non-viral vectors. Our results show that upon successful transfection of Panc-1 cells, the exosome content was altered leading to differential communication and reprogramming of the J774.A1 cells to an M1 phenotype. Based on these results, genetic therapies targeted towards selective manipulation of tumor cell-derived exosome content may be very promising for cancer therapy. PMID:27443190

  16. Mice with an Oncogenic HRAS Mutation are Resistant to High-Fat Diet-Induced Obesity and Exhibit Impaired Hepatic Energy Homeostasis

    Directory of Open Access Journals (Sweden)

    Daiju Oba

    2018-01-01

    Full Text Available Costello syndrome is a “RASopathy” that is characterized by growth retardation, dysmorphic facial appearance, hypertrophic cardiomyopathy and tumor predisposition. >80% of patients with Costello syndrome harbor a heterozygous germline G12S mutation in HRAS. Altered metabolic regulation has been suspected because patients with Costello syndrome exhibit hypoketotic hypoglycemia and increased resting energy expenditure, and their growth is severely retarded. To examine the mechanisms of energy reprogramming by HRAS activation in vivo, we generated knock-in mice expressing a heterozygous Hras G12S mutation (HrasG12S/+ mice as a mouse model of Costello syndrome. On a high-fat diet, HrasG12S/+ mice developed a lean phenotype with microvesicular hepatic steatosis, resulting in early death compared with wild-type mice. Under starvation conditions, hypoketosis and elevated blood levels of long-chain fatty acylcarnitines were observed, suggesting impaired mitochondrial fatty acid oxidation. Our findings suggest that the oncogenic Hras mutation modulates energy homeostasis in vivo.

  17. Myeloid-Cell-Derived VEGF Maintains Brain Glucose Uptake and Limits Cognitive Impairment in Obesity.

    Science.gov (United States)

    Jais, Alexander; Solas, Maite; Backes, Heiko; Chaurasia, Bhagirath; Kleinridders, André; Theurich, Sebastian; Mauer, Jan; Steculorum, Sophie M; Hampel, Brigitte; Goldau, Julia; Alber, Jens; Förster, Carola Y; Eming, Sabine A; Schwaninger, Markus; Ferrara, Napoleone; Karsenty, Gerard; Brüning, Jens C

    2016-05-05

    High-fat diet (HFD) feeding induces rapid reprogramming of systemic metabolism. Here, we demonstrate that HFD feeding of mice downregulates glucose transporter (GLUT)-1 expression in blood-brain barrier (BBB) vascular endothelial cells (BECs) and reduces brain glucose uptake. Upon prolonged HFD feeding, GLUT1 expression is restored, which is paralleled by increased expression of vascular endothelial growth factor (VEGF) in macrophages at the BBB. In turn, inducible reduction of GLUT1 expression specifically in BECs reduces brain glucose uptake and increases VEGF serum concentrations in lean mice. Conversely, myeloid-cell-specific deletion of VEGF in VEGF(Δmyel) mice impairs BBB-GLUT1 expression, brain glucose uptake, and memory formation in obese, but not in lean mice. Moreover, obese VEGF(Δmyel) mice exhibit exaggerated progression of cognitive decline and neuroinflammation on an Alzheimer's disease background. These experiments reveal that transient, HFD-elicited reduction of brain glucose uptake initiates a compensatory increase of VEGF production and assign obesity-associated macrophage activation a homeostatic role to restore cerebral glucose metabolism, preserve cognitive function, and limit neurodegeneration in obesity. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies

    DEFF Research Database (Denmark)

    Damgaard Jensen, Emil; Ferreira, Raphael; Jakociunas, Tadas

    2017-01-01

    on developing synthetic biology tools for orthogonal control of transcription. Most recently, the nuclease-deficient Cas9 (dCas9) has emerged as a flexible tool for controlling activation and repression of target genes, by the simple RNA-guided positioning of dCas9 in the vicinity of the target gene...... transcription start site. In this study we compared two different systems of dCas9-mediated transcriptional reprogramming, and applied them to genes controlling two biosynthetic pathways for biobased production of isoprenoids and triacylglycerols (TAGs) in baker's yeast Saccharomyces cerevisiae. By testing 101...... production and increases in TAG. Taken together, we show similar performance for a constitutive and an inducible dCas9 approach, and identify multiplex gRNA designs that can significantly perturb isoprenoid production and TAG profiles in yeast without editing the genomic context of the target genes. We also...

  19. Genetic and Epigenetic Regulation of Human Cardiac Reprogramming and Differentiation in Regenerative Medicine.

    Science.gov (United States)

    Burridge, Paul W; Sharma, Arun; Wu, Joseph C

    2015-01-01

    Regeneration or replacement of lost cardiomyocytes within the heart has the potential to revolutionize cardiovascular medicine. Numerous methodologies have been used to achieve this aim, including the engraftment of bone marrow- and heart-derived cells as well as the identification of modulators of adult cardiomyocyte proliferation. Recently, the conversion of human somatic cells into induced pluripotent stem cells and induced cardiomyocyte-like cells has transformed potential approaches toward this goal, and the engraftment of cardiac progenitors derived from human embryonic stem cells into patients is now feasible. Here we review recent advances in our understanding of the genetic and epigenetic control of human cardiogenesis, cardiac differentiation, and the induced reprogramming of somatic cells to cardiomyocytes. We also cover genetic programs for inducing the proliferation of endogenous cardiomyocytes and discuss the genetic state of cells used in cardiac regenerative medicine.

  20. Role of the GRAS transcription factor ATA/RAM1 in the transcriptional reprogramming of arbuscular mycorrhiza in Petunia hybrida

    OpenAIRE

    Rich, Melanie K.; Courty, Pierre-Emmanuel; Roux, Christophe; Reinhardt, Didier

    2017-01-01

    Background Development of arbuscular mycorrhiza (AM) requires a fundamental reprogramming of root cells for symbiosis. This involves the induction of hundreds of genes in the host. A recently identified GRAS-type transcription factor in Petunia hybrida, ATA/RAM1, is required for the induction of host genes during AM, and for morphogenesis of the fungal endosymbiont. To better understand the role of RAM1 in symbiosis, we set out to identify all genes that depend on activation by RAM1 in mycorr...

  1. Identification of Reprogrammed Myeloid Cell Transcriptomes in NSCLC.

    Directory of Open Access Journals (Sweden)

    Anna Durrans

    Full Text Available Lung cancer is the leading cause of cancer related mortality worldwide, with non-small cell lung cancer (NSCLC as the most prevalent form. Despite advances in treatment options including minimally invasive surgery, CT-guided radiation, novel chemotherapeutic regimens, and targeted therapeutics, prognosis remains dismal. Therefore, further molecular analysis of NSCLC is necessary to identify novel molecular targets that impact prognosis and the design of new-targeted therapies. In recent years, tumor "activated/reprogrammed" stromal cells that promote carcinogenesis have emerged as potential therapeutic targets. However, the contribution of stromal cells to NSCLC is poorly understood. Here, we show increased numbers of bone marrow (BM-derived hematopoietic cells in the tumor parenchyma of NSCLC patients compared with matched adjacent non-neoplastic lung tissue. By sorting specific cellular fractions from lung cancer patients, we compared the transcriptomes of intratumoral myeloid compartments within the tumor bed with their counterparts within adjacent non-neoplastic tissue from NSCLC patients. The RNA sequencing of specific myeloid compartments (immature monocytic myeloid cells and polymorphonuclear neutrophils identified differentially regulated genes and mRNA isoforms, which were inconspicuous in whole tumor analysis. Genes encoding secreted factors, including osteopontin (OPN, chemokine (C-C motif ligand 7 (CCL7 and thrombospondin 1 (TSP1 were identified, which enhanced tumorigenic properties of lung cancer cells indicative of their potential as targets for therapy. This study demonstrates that analysis of homogeneous stromal populations isolated directly from fresh clinical specimens can detect important stromal genes of therapeutic value.

  2. Metabolic reprogramming during neuronal differentiation from aerobic glycolysis to neuronal oxidative phosphorylation.

    Science.gov (United States)

    Zheng, Xinde; Boyer, Leah; Jin, Mingji; Mertens, Jerome; Kim, Yongsung; Ma, Li; Ma, Li; Hamm, Michael; Gage, Fred H; Hunter, Tony

    2016-06-10

    How metabolism is reprogrammed during neuronal differentiation is unknown. We found that the loss of hexokinase (HK2) and lactate dehydrogenase (LDHA) expression, together with a switch in pyruvate kinase gene splicing from PKM2 to PKM1, marks the transition from aerobic glycolysis in neural progenitor cells (NPC) to neuronal oxidative phosphorylation. The protein levels of c-MYC and N-MYC, transcriptional activators of the HK2 and LDHA genes, decrease dramatically. Constitutive expression of HK2 and LDHA during differentiation leads to neuronal cell death, indicating that the shut-off aerobic glycolysis is essential for neuronal survival. The metabolic regulators PGC-1α and ERRγ increase significantly upon neuronal differentiation to sustain the transcription of metabolic and mitochondrial genes, whose levels are unchanged compared to NPCs, revealing distinct transcriptional regulation of metabolic genes in the proliferation and post-mitotic differentiation states. Mitochondrial mass increases proportionally with neuronal mass growth, indicating an unknown mechanism linking mitochondrial biogenesis to cell size.

  3. Cytokeratin 19 (KRT19) has a Role in the Reprogramming of Cancer Stem Cell-Like Cells to Less Aggressive and More Drug-Sensitive Cells

    Science.gov (United States)

    Kim, Kyeongseok; Yang, Gwang-Mo; Choi, Hye Yeon

    2018-01-01

    Cytokeratin 19 (KRT19) is a cytoplasmic intermediate filament protein, which is responsible for structural rigidity and multipurpose scaffolds. In several cancers, KRT19 is overexpressed and may play a crucial role in tumorigenic transformation. In our previous study, we revealed the role of KRT19 as signaling component which mediated Wnt/NOTCH crosstalk through NUMB transcription in breast cancer. Here, we investigated the function of KRT19 in cancer reprogramming and drug resistance in breast cancer cells. We found that expression of KRT19 was attenuated in several patients-derived breast cancer tissues and patients with a low expression of KRT19 were significantly correlated with poor prognosis in breast cancer patients. Consistently, highly aggressive and drug-resistant breast cancer patient-derived cancer stem cell-like cells (konkuk university-cancer stem cell-like cell (KU-CSLCs)) displayed higher expression of cancer stem cell (CSC) markers, including ALDH1, CXCR4, and CD133, but a much lower expression of KRT19 than that is seen in highly aggressive triple negative breast cancer MDA-MB231 cells. Moreover, we revealed that the knockdown of KRT19 in MDA-MB231 cells led to an enhancement of cancer properties, such as cell proliferation, sphere formation, migration, and drug resistance, while the overexpression of KRT19 in KU-CSLCs resulted in the significant attenuation of cancer properties. KRT19 regulated cancer stem cell reprogramming by modulating the expression of cancer stem cell markers (ALDH1, CXCR4, and CD133), as well as the phosphorylation of Src and GSK3β (Tyr216). Therefore, our data may imply that the modulation of KRT19 expression could be involved in cancer stem cell reprogramming and drug sensitivity, which might have clinical implications for cancer or cancer stem cell treatment. PMID:29747452

  4. Cytokeratin 19 (KRT19) has a Role in the Reprogramming of Cancer Stem Cell-Like Cells to Less Aggressive and More Drug-Sensitive Cells.

    Science.gov (United States)

    Saha, Subbroto Kumar; Kim, Kyeongseok; Yang, Gwang-Mo; Choi, Hye Yeon; Cho, Ssang-Goo

    2018-05-09

    Cytokeratin 19 ( KRT19 ) is a cytoplasmic intermediate filament protein, which is responsible for structural rigidity and multipurpose scaffolds. In several cancers, KRT19 is overexpressed and may play a crucial role in tumorigenic transformation. In our previous study, we revealed the role of KRT19 as signaling component which mediated Wnt/NOTCH crosstalk through NUMB transcription in breast cancer. Here, we investigated the function of KRT19 in cancer reprogramming and drug resistance in breast cancer cells. We found that expression of KRT19 was attenuated in several patients-derived breast cancer tissues and patients with a low expression of KRT19 were significantly correlated with poor prognosis in breast cancer patients. Consistently, highly aggressive and drug-resistant breast cancer patient-derived cancer stem cell-like cells (konkuk university-cancer stem cell-like cell (KU-CSLCs)) displayed higher expression of cancer stem cell (CSC) markers, including ALDH1 , CXCR4 , and CD133 , but a much lower expression of KRT19 than that is seen in highly aggressive triple negative breast cancer MDA-MB231 cells. Moreover, we revealed that the knockdown of KRT19 in MDA-MB231 cells led to an enhancement of cancer properties, such as cell proliferation, sphere formation, migration, and drug resistance, while the overexpression of KRT19 in KU-CSLCs resulted in the significant attenuation of cancer properties. KRT19 regulated cancer stem cell reprogramming by modulating the expression of cancer stem cell markers ( ALDH1 , CXCR4 , and CD133 ), as well as the phosphorylation of Src and GSK3β (Tyr216). Therefore, our data may imply that the modulation of KRT19 expression could be involved in cancer stem cell reprogramming and drug sensitivity, which might have clinical implications for cancer or cancer stem cell treatment.

  5. Reaching Out to Develop the Creative Reading Potential of the Hearing Impaired in Community Colleges.

    Science.gov (United States)

    Norton, Audree L.

    Hearing impaired students need the opportunity to develop their creative reading ability (the ability to acquire ideas and information through printed words, to apply them to personal problems, and to attempt to resolve those problems). Although many hearing impaired students have the intelligence to be successful readers, they have been…

  6. Age-Related Sensory Impairments and Risk of Cognitive Impairment

    Science.gov (United States)

    Fischer, Mary E; Cruickshanks, Karen J.; Schubert, Carla R; Pinto, Alex A; Carlsson, Cynthia M; Klein, Barbara EK; Klein, Ronald; Tweed, Ted S.

    2016-01-01

    Background/Objectives To evaluate the associations of sensory impairments with the 10-year risk of cognitive impairment. Previous work has primarily focused on the relationship between a single sensory system and cognition. Design The Epidemiology of Hearing Loss Study (EHLS) is a longitudinal, population-based study of aging in the Beaver Dam, WI community. Baseline examinations were conducted in 1993 and follow-up exams have been conducted every 5 years. Setting General community Participants EHLS members without cognitive impairment at EHLS-2 (1998–2000). There were 1,884 participants (mean age = 66.7 years) with complete EHLS-2 sensory data and follow-up information. Measurements Cognitive impairment was a Mini-Mental State Examination score of impairment was a pure-tone average of hearing thresholds (0.5, 1, 2 and 4 kHz) of > 25 decibel Hearing Level in either ear. Visual impairment was Pelli-Robson contrast sensitivity of impairment was a San Diego Odor Identification Test score of impairment were independently associated with cognitive impairment risk [Hearing: Hazard Ratio (HR) = 1.90, 95% Confidence Interval (C.I.) = 1.11, 3.26; Vision: HR = 2.05, 95% C.I. = 1.24, 3.38; Olfaction: HR = 3.92, 95% C.I. = 2.45, 6.26]. However, 85% with hearing impairment, 81% with visual impairment, and 76% with olfactory impairment did not develop cognitive impairment during follow-up. Conclusion The relationship between sensory impairment and cognitive impairment was not unique to one sensory system suggesting sensorineural health may be a marker of brain aging. The development of a combined sensorineurocognitive measure may be useful in uncovering mechanisms of healthy brain aging. PMID:27611845

  7. High-fat diet reprograms the epigenome of rat spermatozoa and transgenerationally affects metabolism of the offspring

    DEFF Research Database (Denmark)

    de Castro Barbosa, Thais; Ingerslev, Lars R; Alm, Petter S

    2016-01-01

    OBJECTIVES: Chronic and high consumption of fat constitutes an environmental stress that leads to metabolic diseases. We hypothesized that high-fat diet (HFD) transgenerationally remodels the epigenome of spermatozoa and metabolism of the offspring. METHODS: F0-male rats fed either HFD or chow diet......1 male offspring showed common DNA methylation and small non-coding RNA expression signatures. Altered expression of sperm miRNA let-7c was passed down to metabolic tissues of the offspring, inducing a transcriptomic shift of the let-7c predicted targets. CONCLUSION: Our results provide insight...... into mechanisms by which HFD transgenerationally reprograms the epigenome of sperm cells, thereby affecting metabolic tissues of offspring throughout two generations....

  8. Inhibition of miRNA-212/132 improves the reprogramming of fibroblasts into induced pluripotent stem cells by de-repressing important epigenetic remodelling factors

    Directory of Open Access Journals (Sweden)

    Nils Pfaff

    2017-04-01

    Thus, conducting a full library miRNA screen we here describe a miRNA family, which markedly reduces generation of iPSC and upon inhibition in turn enhances reprogramming. These miRNAs, at least in part, exert their functions through repression of the epigenetic modulators p300 and Jarid1a, highlighting these two molecules as an endogenous epigenetic roadblock during iPSC generation.

  9. Ability to solve riddles in patients with speech and language impairments after stroke.

    Science.gov (United States)

    Savić, Goran

    2016-01-01

    Successful riddle solving requires recognition of the meaning of words, attention, concentration, memory, connectivity and analysis of riddle content, and sufficiently developed associative thinking. The aim of the study was to determine the ability to solve riddles in stroke patients who do or do not have speech and language disorders (SLDs), to determine the presence of SLDs in relation to the lesion localization, as well as to define the relationship between riddle-solving and functional impairment of a body side. The sample consisted of 88 patients. The data used included age, sex, educational level, time of stroke onset, presence of an SLD, lesion localization, and functional damage of the body side. The patients were presented with a task of solving 10 riddles. A significant SLD was present in 38.60% of the patients. Brain lesions were found distributed at 46 different brain sites. Patients with different lesion localization had different success in solving riddles. Patients with perisylvian cortex brain lesions, or patients with Wernicke and global aphasia, had the poorest results. The group with SLDs had an average success of solved riddles of 26.76% (p = 0.000). The group with right-sided functional impairments had average success of 37.14%, and the group with functional impairments of the left side of the body 56.88% (p = 0.002). Most patients with SLDs had a low ability of solving riddles. Most of the patients with left brain lesions and perisylvian cortex damage demonstrated lower ability in solving riddles in relation to patients with right hemisphere lesions.

  10. Age-Related Eye Diseases and Visual Impairment Among U.S. Adults

    Science.gov (United States)

    Chou, Chiu-Fang; Cotch, Mary Frances; Vitale, Susan; Zhang, Xinzhi; Klein, Ronald; Friedman, David S.; Klein, Barbara E.K.; Saaddine, Jinan B.

    2014-01-01

    Background Visual impairment is a common health-related disability in the U.S. The association between clinical measurements of age-related eye diseases and visual impairment in data from a national survey has not been reported. Purpose To examine common eye conditions and other correlates associated with visual impairment in the U.S. Methods Data from the 2005–2008 National Health and Nutrition Examination Survey of 5222 Americans aged ≥40 years were analyzed in 2012 for visual impairment (presenting distance visual acuity worse than 20/40 in the better-seeing eye), and visual impairment not due to refractive error (distance visual acuity worse than 20/40 after refraction). Diabetic retinopathy (DR) and age-related macular degeneration (AMD) were assessed from retinal fundus images; glaucoma was assessed from two successive frequency-doubling tests and a cup-to-disc ratio measurement. Results Prevalence of visual impairment and of visual impairment not due to refractive error was 7.5% (95% CI=6.9%, 8.1%) and 2.0% (1.7%, 2.3%), respectively. The prevalence of visual impairment not due to refractive error was significantly higher among people with AMD (2.2%) compared to those without AMD (0.8%), or with DR (3.5%) compared to those without DR (1.2%). Independent predictive factors of visual impairment not due to refractive error were AMD (OR=4.52, 95% CI=2.50, 8.17); increasing age (OR=1.09 per year, 95% CI=1.06, 1.13); and less than a high school education (OR=2.99, 95% CI=1.18, 7.55). Conclusions Visual impairment is a public health problem in the U.S. Visual impairment in two thirds of adults could be eliminated with refractive correction. Screening of the older population may identify adults at increased risk of visual impairment due to eye diseases. PMID:23790986

  11. Macrophage dysfunction impairs resolution of inflammation in the wounds of diabetic mice.

    Directory of Open Access Journals (Sweden)

    Savita Khanna

    2010-03-01

    Full Text Available Chronic inflammation is a characteristic feature of diabetic cutaneous wounds. We sought to delineate novel mechanisms involved in the impairment of resolution of inflammation in diabetic cutaneous wounds. At the wound-site, efficient dead cell clearance (efferocytosis is a pre-requisite for the timely resolution of inflammation and successful healing.Macrophages isolated from wounds of diabetic mice showed significant impairment in efferocytosis. Impaired efferocytosis was associated with significantly higher burden of apoptotic cells in wound tissue as well as higher expression of pro-inflammatory and lower expression of anti-inflammatory cytokines. Observations related to apoptotic cell load at the wound site in mice were validated in the wound tissue of diabetic and non-diabetic patients. Forced Fas ligand driven elevation of apoptotic cell burden at the wound site augmented pro-inflammatory and attenuated anti-inflammatory cytokine response. Furthermore, successful efferocytosis switched wound macrophages from pro-inflammatory to an anti-inflammatory mode.Taken together, this study presents first evidence demonstrating that diabetic wounds suffer from dysfunctional macrophage efferocytosis resulting in increased apoptotic cell burden at the wound site. This burden, in turn, prolongs the inflammatory phase and complicates wound healing.

  12. Genetic testing for hearing impairment.

    Science.gov (United States)

    Topsakal, V; Van Camp, G; Van de Heyning, P

    2005-01-01

    For some patients, genetic testing can reveal the etiology of their hearing impairment, and can provide evidence for a medical diagnosis. However, a gap between fundamental genetic research on hereditary deafness and clinical otology emerges because of the steadily increasing number of discovered genes for hereditary hearing impairment (HHI) and the comparably low clinical differentiation of the HHIs. In an attempt to keep up with the scientific progress, this article enumerates the indications of genetic testing for HHI from a clinical point of view and describes the most frequently encountered HHIs in Belgium. Domains of recent scientific interest, molecular biological aspects, and some pitfalls with HHIs are highlighted. The overview comprises bilateral congenital hearing loss, late-onset progressive high frequency hearing loss, progressive bilateral cochleo-vestibular deficit, and progressive low frequency hearing loss. Also, several syndromal forms of HHI are summarized, and the availability of genetic tests mentioned. Finally, the requirements for successful linkage analysis, an important genetic research tool for localizing the potential genes of a trait on a chromosome, are briefly described.

  13. Real-Time Detection of Important Sounds with a Wearable Vibration Based Device for Hearing-Impaired People

    Directory of Open Access Journals (Sweden)

    Mete Yağanoğlu

    2018-04-01

    Full Text Available Hearing-impaired people do not hear indoor and outdoor environment sounds, which are important for them both at home and outside. By means of a wearable device that we have developed, a hearing-impaired person will be informed of important sounds through vibrations, thereby understanding what kind of sound it is. Our system, which operates in real time, can achieve a success rate of 98% when estimating a door bell ringing sound, 99% success identifying an alarm sound, 99% success identifying a phone ringing, 91% success identifying honking, 93% success identifying brake sounds, 96% success identifying dog sounds, 97% success identifying human voice, and 96% success identifying other sounds using the audio fingerprint method. Audio fingerprint is a brief summary of an audio file, perceptively summarizing a piece of audio content. In this study, our wearable device is tested 100 times a day for 100 days on five deaf persons and 50 persons with normal hearing whose ears were covered by earphones that provided wind sounds. This study aims to improve the quality of life of deaf persons, and provide them a more prosperous life. In the questionnaire performed, deaf people rate the clarity of the system at 90%, usefulness at 97%, and the likelihood of using this device again at 100%.

  14. Combining Patient-Reprogrammed Neural Cells and Proteomics as a Model to Study Psychiatric Disorders.

    Science.gov (United States)

    Zuccoli, Giuliana S; Martins-de-Souza, Daniel; Guest, Paul C; Rehen, Stevens K; Nascimento, Juliana Minardi

    2017-01-01

    The mechanisms underlying the pathophysiology of psychiatric disorders are still poorly known. Most of the studies about these disorders have been conducted on postmortem tissue or in limited preclinical models. The development of human induced pluripotent stem cells (iPSCs) has helped to increase the translational capacity of molecular profiling studies of psychiatric disorders through provision of human neuronal-like tissue. This approach consists of generation of pluripotent cells by genetically reprogramming somatic cells to produce the multiple neural cell types as observed within the nervous tissue. The finding that iPSCs can recapitulate the phenotype of the donor also affords the possibility of using this approach to study both the disease and control states in a given medical area. Here, we present a protocol for differentiation of human pluripotent stem cells to neural progenitor cells followed by subcellular fractionation which allows the study of specific cellular organelles and proteomic analysis.

  15. Detection of infarct size safety threshold for left ventricular ejection fraction impairment in acute myocardial infarction successfully treated with primary percutaneous coronary intervention.

    Science.gov (United States)

    Sciagrà, Roberto; Cipollini, Fabrizio; Berti, Valentina; Migliorini, Angela; Antoniucci, David; Pupi, Alberto

    2013-04-01

    In acute myocardial infarction (AMI) treated by primary percutaneous coronary intervention (PCI), there is a direct relationship between myocardial damage and consequent left ventricular (LV) functional impairment. It is however unclear whether there is a safety threshold below which infarct size does not significantly affect LV ejection fraction (EF). The aim of this study was to evaluate the relationship between infarct size and LVEF in AMI patients treated by successful PCI using a specific statistical approach to identify a possible safety threshold. Among patients with recent AMI submitted to perfusion gated single photon emission computed tomography (SPECT) to define the infarct size, the data of 427 subjects with sizable infarct size were considered. The relationship between infarct size and LVEF was analysed using a simple segmented regression (SSR) model and an iterative algorithm based on robust least squares (RLS) for parameter estimation. The RLS algorithm detected two break points in the SSR model, set at infarct size values of 11.0 and 51.5 %. Because the slope coefficients of the two extreme segments of the regression line were not significant, by constraining such segments to zero slope in the SSR model, the lower break point was identified at infarct size = 8 % and the upper one at 45 %. Using a rigorous statistical approach, it is possible to demonstrate that below a threshold of 8 % the infarct size apparently does not affect the LVEF and therefore a safety threshold could be set at this value. Furthermore, the same analysis suggests that the relationship between infarct size and LVEF impairment is lost for an infarct size > 45 %.

  16. A framework for communication between visually impaired, hearing impaired and speech impaired using arduino

    Science.gov (United States)

    Sujatha, R.; Khandelwa, Prakhar; Gupta, Anusha; Anand, Nayan

    2017-11-01

    A long time ago our society accepted the notion of treating people with disabilities not as unviable and disabled but as differently-abled, recognizing their skills beyond their disabilities. The next step has to be taken by our scientific community, that is, to normalize lives of the people with disabilities and make it so as if they are no different to us. The primary step in this direction would be to normalize communication between people. People with an impaired speech or impaired vision or impaired hearing face difficulties while having a casual conversation with others. Any form of communication feels so strenuous that the impaired end up communicating just the important information and avoid a casual conversation. To normalize conversation between the impaired we need a simple and compact device which facilitates the conversation by providing the information in the desired form.

  17. 20 CFR 416.998 - If you become disabled by another impairment(s).

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false If you become disabled by another impairment... Disability Or Blindness § 416.998 If you become disabled by another impairment(s). If a new severe impairment(s) begins in or before the month in which your last impairment(s) ends, we will find that your...

  18. 20 CFR 404.1598 - If you become disabled by another impairment(s).

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false If you become disabled by another impairment... Disability § 404.1598 If you become disabled by another impairment(s). If a new severe impairment(s) begins in or before the month in which your last impairment(s) ends, we will find that your disability is...

  19. Looking at the Social Activity for Adolescents with Orthopedic Impairments

    Science.gov (United States)

    Biastro, Leslie; Frank, Heather; Larwin, Karen H.

    2015-01-01

    Adolescents with identified orthopedic impairments are often less likely to participate in social activities outside of the school setting. However, the adolescents who are able to participate in activities have higher social skills, more academic successes, and show more satisfaction in their roles as family member or friend. The aim of this…

  20. A Computer Supported Teamwork Project for People with a Visual Impairment.

    Science.gov (United States)

    Hale, Greg

    2000-01-01

    Discussion of the use of computer supported teamwork (CSTW) in team-based organizations focuses on problems that visually impaired people have reading graphical user interface software via screen reader software. Describes a project that successfully used email for CSTW, and suggests issues needing further research. (LRW)

  1. Rhyme and Syllable Recognition in Severe to Profound Hearing-Impaired Children

    Directory of Open Access Journals (Sweden)

    MohammadReza Keihani

    2003-09-01

    Full Text Available Objective: A research was conducted to assess the phonological awareness in hearing-impaired children in comparison to normal children. In this context, we discussed about the ability of these children in identification of rhyme and word segmentation to syllables. Method and Material: The sample of this study is composed of 320 children, 160 normal & 160 hearing-impaired with a hearing-loss. Of over 70 dB, studying in the 1st t 4th grade of the primary schools in Tehran. They are divided into two groups at each level (20 girls & 20 boys. Results: 1 Hearing-impaired children's scores on test related to rhyme and syllable is lower as compared with normal children. 2 Hearing-impaired children are more potent on test of word segmentation to syllables in comparison to that related to rhyme, while normal children are more successful on test of word segmentation to syllables as compared with that related to rhyme. Discussion: Hearing is an important factor in phonological awareness. Formal education at special schools doesn't compensate for the hearing impairment as to development of the phonological awareness.

  2. A wide reprogramming of histone H3 modifications during male meiosis I in rice is dependent on the Argonaute protein MEL1.

    Science.gov (United States)

    Liu, Hua; Nonomura, Ken-Ichi

    2016-10-01

    The roles of epigenetic mechanisms, including small-RNA-mediated silencing, in plant meiosis largely remain unclear, despite their importance in plant reproduction. This study unveiled that rice chromosomes are reprogrammed during the premeiosis-to-meiosis transition in pollen mother cells (PMCs). This large-scale meiotic chromosome reprogramming (LMR) continued throughout meiosis I, during which time H3K9 dimethylation (H3K9me2) was increased, and H3K9 acetylation and H3S10 phosphorylation were broadly decreased, with an accompanying immunostaining pattern shift of RNA polymerase II. LMR was dependent on the rice Argonaute protein, MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1), which is specifically expressed in germ cells prior to meiosis, because LMR was severely diminished in mel1 mutant anthers. Pivotal meiotic events, such as pre-synaptic centromere association, DNA double-strand break initiation and synapsis of homologous chromosomes, were also disrupted in this mutant. Interestingly, and as opposed to the LMR loss in most chromosomal regions, aberrant meiotic protein loading and hypermethylation of H3K9 emerged on the nucleolar organizing region in the mel1 PMCs. These results suggest that MEL1 plays important roles in epigenetic LMR to promote faithful homologous recombination and synapsis during rice meiosis. © 2016. Published by The Company of Biologists Ltd.

  3. 20 CFR 220.184 - If the annuitant becomes disabled by another impairment(s).

    Science.gov (United States)

    2010-04-01

    ... impairment(s). 220.184 Section 220.184 Employees' Benefits RAILROAD RETIREMENT BOARD REGULATIONS UNDER THE... Activity or Medical Improvement § 220.184 If the annuitant becomes disabled by another impairment(s). If a new severe impairment(s) begins in or before the month in which the last impairment(s) ends, the Board...

  4. Ability to solve riddles in patients with speech and language impairments after stroke

    Directory of Open Access Journals (Sweden)

    Savić Goran

    2016-01-01

    Full Text Available Introduction. Successful riddle solving requires recognition of the meaning of words, attention, concentration, memory, connectivity and analysis of riddle content, and sufficiently developed associative thinking. Objective. The aim of the study was to determine the ability to solve riddles in stroke patients who do or do not have speech and language disorders (SLDs, to determine the presence of SLDs in relation to the lesion localization, as well as to define the relationship between riddle-solving and functional impairment of a body side. Methods. The sample consisted of 88 patients. The data used included age, sex, educational level, time of stroke onset, presence of an SLD, lesion localization, and functional damage of the body side. The patients were presented with a task of solving 10 riddles. Results. A significant SLD was present in 38.60% of the patients. Brain lesions were found distributed at 46 different brain sites. Patients with different lesion localization had different success in solving riddles. Patients with perisylvian cortex brain lesions, or patients with Wernicke and global aphasia, had the poorest results. The group with SLDs had an average success of solved riddles of 26.76% (p = 0.000. The group with right-sided functional impairments had average success of 37.14%, and the group with functional impairments of the left side of the body 56.88% (p = 0.002. Conclusion. Most patients with SLDs had a low ability of solving riddles. Most of the patients with left brain lesions and perisylvian cortex damage demonstrated lower ability in solving riddles in relation to patients with right hemisphere lesions.

  5. Metabolic Reprogramming Regulates the Proliferative and Inflammatory Phenotype of Adventitial Fibroblasts in Pulmonary Hypertension Through the Transcriptional Corepressor C-Terminal Binding Protein-1.

    Science.gov (United States)

    Li, Min; Riddle, Suzette; Zhang, Hui; D'Alessandro, Angelo; Flockton, Amanda; Serkova, Natalie J; Hansen, Kirk C; Moldvan, Radu; McKeon, B Alexandre; Frid, Maria; Kumar, Sushil; Li, Hong; Liu, Hongbing; Caánovas, Angela; Medrano, Juan F; Thomas, Milton G; Iloska, Dijana; Plecitá-Hlavatá, Lydie; Ježek, Petr; Pullamsetti, Soni; Fini, Mehdi A; El Kasmi, Karim C; Zhang, QingHong; Stenmark, Kurt R

    2016-10-11

    Changes in metabolism have been suggested to contribute to the aberrant phenotype of vascular wall cells, including fibroblasts, in pulmonary hypertension (PH). Here, we test the hypothesis that metabolic reprogramming to aerobic glycolysis is a critical adaptation of fibroblasts in the hypertensive vessel wall that drives proliferative and proinflammatory activation through a mechanism involving increased activity of the NADH-sensitive transcriptional corepressor C-terminal binding protein 1 (CtBP1). RNA sequencing, quantitative polymerase chain reaction, 13 C-nuclear magnetic resonance, fluorescence-lifetime imaging, mass spectrometry-based metabolomics, and tracing experiments with U- 13 C-glucose were used to assess glycolytic reprogramming and to measure the NADH/NAD + ratio in bovine and human adventitial fibroblasts and mouse lung tissues. Immunohistochemistry was used to assess CtBP1 expression in the whole-lung tissues. CtBP1 siRNA and the pharmacological inhibitor 4-methylthio-2-oxobutyric acid (MTOB) were used to abrogate CtBP1 activity in cells and hypoxic mice. We found that adventitial fibroblasts from calves with severe hypoxia-induced PH and humans with idiopathic pulmonary arterial hypertension (PH-Fibs) displayed aerobic glycolysis when cultured under normoxia, accompanied by increased free NADH and NADH/NAD + ratios. Expression of the NADH sensor CtBP1 was increased in vivo and in vitro in fibroblasts within the pulmonary adventitia of humans with idiopathic pulmonary arterial hypertension and animals with PH and cultured PH-Fibs, respectively. Decreasing NADH pharmacologically with MTOB or genetically blocking CtBP1 with siRNA upregulated the cyclin-dependent genes (p15 and p21) and proapoptotic regulators (NOXA and PERP), attenuated proliferation, corrected the glycolytic reprogramming phenotype of PH-Fibs, and augmented transcription of the anti-inflammatory gene HMOX1. Chromatin immunoprecipitation analysis demonstrated that CtBP1 directly

  6. Erasure and reestablishment of random allelic expression imbalance after epigenetic reprogramming.

    Science.gov (United States)

    Jeffries, Aaron Richard; Uwanogho, Dafe Aghogho; Cocks, Graham; Perfect, Leo William; Dempster, Emma; Mill, Jonathan; Price, Jack

    2016-10-01

    Clonal level random allelic expression imbalance and random monoallelic expression provides cellular heterogeneity within tissues by modulating allelic dosage. Although such expression patterns have been observed in multiple cell types, little is known about when in development these stochastic allelic choices are made. We examine allelic expression patterns in human neural progenitor cells before and after epigenetic reprogramming to induced pluripotency, observing that loci previously characterized by random allelic expression imbalance (0.63% of expressed genes) are generally reset to a biallelic state in induced pluripotent stem cells (iPSCs). We subsequently neuralized the iPSCs and profiled isolated clonal neural stem cells, observing that significant random allelic expression imbalance is reestablished at 0.65% of expressed genes, including novel loci not found to show allelic expression imbalance in the original parental neural progenitor cells. Allelic expression imbalance was associated with altered DNA methylation across promoter regulatory regions, with clones characterized by skewed allelic expression being hypermethylated compared to their biallelic sister clones. Our results suggest that random allelic expression imbalance is established during lineage commitment and is associated with increased DNA methylation at the gene promoter. © 2016 Jeffries et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  7. The histone H3.3K36M mutation reprograms the epigenome of chondroblastomas

    Science.gov (United States)

    Fang, Dong; Gan, Haiyun; Lee, Jeong-Heon; Han, Jing; Wang, Zhiquan; Riester, Scott M.; Jin, Long; Chen, Jianji; Zhou, Hui; Wang, Jinglong; Zhang, Honglian; Yang, Na; Bradley, Elizabeth W.; Ho, Thai H.; Rubin, Brian P.; Bridge, Julia A.; Thibodeau, Stephen N; Ordog, Tamas; Chen, Yue; van Wijnen, Andre J.; Oliveira, Andre M.; Xu, Rui-Ming; Westendorf, Jennifer J.; Zhang, Zhiguo

    2016-01-01

    Over 90% of chondroblastomas contain a heterozygous mutation replacing lysine 36 with methionine (K36M) in the histone H3 variant H3.3. Here, we show that H3K36 methylation is reduced globally in chondroblastomas and in chondrocytes harboring the same genetic mutation due to inhibition of at least two H3K36 methyltransferases, MMSET and SETD2, by the H3.3K36M mutant proteins. Genes with altered expression as well as H3K36 di- and trimethylation in H3.3K36M cells are enriched in cancer pathways. In addition, H3.3K36M chondrocytes exhibit several hallmarks of cancer cells including increased ability to form colonies, resistance to apoptosis and defects in differentiation. Thus, H3.3K36M proteins reprogram H3K36 methylation landscape and contribute to tumorigenesis in part through altering the expression of cancer-associated genes. PMID:27229140

  8. Efficient non-viral reprogramming of myoblasts to stemness with a single small molecule to generate cardiac progenitor cells.

    Directory of Open Access Journals (Sweden)

    Zeeshan Pasha

    Full Text Available The current protocols for generation of induced pluripotent stem (iPS cells involve genome integrating viral vectors which may induce tumorgenesis. The aim of this study was to develop and optimize a non-viral method without genetic manipulation for reprogramming of skeletal myoblasts (SMs using small molecules.SMs from young male Oct3/4-GFP(+ transgenic mouse were treated with DNA methyltransferase (DNMT inhibitor, RG108. Two weeks later, GFP(+ colonies of SM derived iPS cells (SiPS expressing GFP and with morphological similarity of mouse embryonic stem (ESCs were formed and propagated in vitro. SiPS were positive for alkaline phosphatase activity, expressed SSEA1, displayed ES cell specific pluripotency markers and formed teratoma in nude mice. Optimization of culture conditions for embryoid body (EBs formation yielded spontaneously contracting EBs having morphological, molecular, and ultra-structural similarities with cardiomyocytes and expressed early and late cardiac markers. miR profiling showed abrogation of let-7 family and upregulation of ESCs specific miR-290-295 cluster thus indicating that SiPS were similar to ESCs in miR profile. Four weeks after transplantation into the immunocompetent mice model of acute myocardial infarction (n = 12 per group, extensive myogenesis was observed in SiPS transplanted hearts as compared to DMEM controls (n = 6 per group. A significant reduction in fibrosis and improvement in global heart function in the hearts transplanted with SiPS derived cardiac progenitor cells were observed.Reprogramming of SMs by DNMT inhibitor is a simple, reproducible and efficient technique more likely to generate transgene integration-free iPS cells. Cardiac progenitors derived from iPS cells propagated extensively in the infarcted myocardium without tumorgenesis and improved cardiac function.

  9. MUC1-C Oncoprotein Integrates a Program of EMT, Epigenetic Reprogramming and Immune Evasion in Human Carcinomas.

    Science.gov (United States)

    Rajabi, Hasan; Kufe, Donald

    2017-08-01

    The MUC1 gene evolved in mammalian species to provide protection of epithelia. The transmembrane MUC1 C-terminal subunit (MUC1-C) signals stress to the interior of the epithelial cell and, when overexpressed as in most carcinomas, functions as an oncoprotein. MUC1-C induces the epithelial-mesenchymal transition (EMT) by activating the inflammatory NF-κB p65 pathway and, in turn, the EMT-transcriptional repressor ZEB1. Emerging evidence has indicated that MUC1-C drives a program integrating the induction of EMT with activation of stem cell traits, epigenetic reprogramming and immune evasion. This mini-review focuses on the potential importance of this MUC1-C program in cancer progression. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Reprogramming towards totipotency is greatly facilitated by synergistic effects of small molecules

    Directory of Open Access Journals (Sweden)

    Kei Miyamoto

    2017-04-01

    Full Text Available Animal cloning has been achieved in many species by transplanting differentiated cell nuclei to unfertilized oocytes. However, the low efficiencies of cloning have remained an unresolved issue. Here we find that the combination of two small molecules, trichostatin A (TSA and vitamin C (VC, under culture condition with bovine serum albumin deionized by ion-exchange resins, dramatically improves the cloning efficiency in mice and 15% of cloned embryos develop to term by means of somatic cell nuclear transfer (SCNT. The improvement was not observed by adding the non-treated, rather than deionized, bovine serum. RNA-seq analyses of SCNT embryos at the two-cell stage revealed that the treatment with TSA and VC resulted in the upregulated expression of previously identified reprogramming-resistant genes. Moreover, the expression of early-embryo-specific retroelements was upregulated by the TSA and VC treatment. The enhanced gene expression was relevant to the VC-mediated reduction of histone H3 lysine 9 methylation in SCNT embryos. Our study thus shows a simply applicable method to greatly improve mouse cloning efficiency, and furthers our understanding of how somatic nuclei acquire totipotency.

  11. Chemical Cocktails Enable Hepatic Reprogramming of Mouse Fibroblasts with a Single Transcription Factor

    Directory of Open Access Journals (Sweden)

    Ren Guo

    2017-08-01

    Full Text Available Liver or hepatocytes transplantation is limited by the availability of donor organs. Functional hepatocytes independent of the donor sources may have wide applications in regenerative medicine and the drug industry. Recent studies have demonstrated that chemical cocktails may induce reprogramming of fibroblasts into a range of functional somatic cells. Here, we show that mouse fibroblasts can be transdifferentiated into the hepatocyte-like cells (iHeps using only one transcription factor (TF (Foxa1, Foxa2, or Foxa3 plus a chemical cocktail. These iHeps show typical epithelial morphology, express multiple hepatocyte-specific genes, and acquire hepatocyte functions. Genetic lineage tracing confirms the fibroblast origin of these iHeps. More interestingly, these iHeps are expandable in vitro and can reconstitute the damaged hepatic tissues of the fumarylacetoacetate hydrolase-deficient (Fah−/− mice. Our study provides a strategy to generate functional hepatocyte-like cells by using a single TF plus a chemical cocktail and is one step closer to generate the full-chemical iHeps.

  12. Cortical visual impairment

    OpenAIRE

    Koželj, Urša

    2013-01-01

    In this thesis we discuss cortical visual impairment, diagnosis that is in the developed world in first place, since 20 percent of children with blindness or low vision are diagnosed with it. The objectives of the thesis are to define cortical visual impairment and the definition of characters suggestive of the cortical visual impairment as well as to search for causes that affect the growing diagnosis of cortical visual impairment. There are a lot of signs of cortical visual impairment. ...

  13. Mycobacterium tuberculosis induces the miR-33 locus to reprogram autophagy and host lipid metabolism.

    Science.gov (United States)

    Ouimet, Mireille; Koster, Stefan; Sakowski, Erik; Ramkhelawon, Bhama; van Solingen, Coen; Oldebeken, Scott; Karunakaran, Denuja; Portal-Celhay, Cynthia; Sheedy, Frederick J; Ray, Tathagat Dutta; Cecchini, Katharine; Zamore, Philip D; Rayner, Katey J; Marcel, Yves L; Philips, Jennifer A; Moore, Kathryn J

    2016-06-01

    Mycobacterium tuberculosis (Mtb) survives in macrophages by evading delivery to the lysosome and promoting the accumulation of lipid bodies, which serve as a bacterial source of nutrients. We found that by inducing the microRNA (miRNA) miR-33 and its passenger strand miR-33*, Mtb inhibited integrated pathways involved in autophagy, lysosomal function and fatty acid oxidation to support bacterial replication. Silencing of miR-33 and miR-33* by genetic or pharmacological means promoted autophagy flux through derepression of key autophagy effectors (such as ATG5, ATG12, LC3B and LAMP1) and AMPK-dependent activation of the transcription factors FOXO3 and TFEB, which enhanced lipid catabolism and Mtb xenophagy. These data define a mammalian miRNA circuit used by Mtb to coordinately inhibit autophagy and reprogram host lipid metabolism to enable intracellular survival and persistence in the host.

  14. Application of TALE-Based Approach for Dissecting Functional MicroRNA-302/367 in Cellular Reprogramming.

    Science.gov (United States)

    Zhang, Zhonghui; Wu, Wen-Shu

    2018-01-01

    MicroRNAs are small 18-24 nt single-stranded noncoding RNA molecules involved in many biological processes, including stemness maintenance and cellular reprogramming. Current methods used in loss-of-function studies of microRNAs have several limitations. Here, we describe a new approach for dissecting miR-302/367 functions by transcription activator-like effectors (TALEs), which are natural effector proteins secreted by Xanthomonas and Ralstonia bacteria. Knockdown of the miR-302/367 cluster uses the Kruppel-associated box repressor domain fused with specific TALEs designed to bind the miR-302/367 cluster promoter. Knockout of the miR-302/367 cluster uses two pairs of TALE nucleases (TALENs) to delete the miR-302/367 cluster in human primary cells. Together, both TALE-based transcriptional repressor and TALENs are two promising approaches for loss-of-function studies of microRNA cluster in human primary cells.

  15. Issues to be resolved for the successful implementation of the Nuclear Waste Policy Act of 1982: Utilities' viewpoint

    International Nuclear Information System (INIS)

    Kauffman, J.T.; Kraft, S.P.

    1987-01-01

    This article describes issues that utility companies perceive as important for successful implementation of the NWPA. Electric utility companies with nuclear energy programs are fulfilling their commitments under the NWPA by paying over $400 million a year into the Nuclear Waste Fund as well as preparing for on-site storage of spent fuel until 1998. The current impasse in Congress over DOE's recommend second repository reprogramming is giving the industry pause to consider whether or not DOE will be allowed by Congress to live up to its 1998 obligation to the utilities. The industry is asking Congress to allow DOE to proceed with characterization of the three potential first sites, to authorize and fund the MRS, and to provide equitable payments for defense waste disposal. Also, Congress and DOE must work cooperatively to find a solution to the current impasse over the second repository program

  16. Surgical treatment for obesity: ensuring success.

    Science.gov (United States)

    Andris, Deborah A

    2005-01-01

    In the United States, obesity has reached epidemic proportions. Serious medical complications, impaired quality of life, and premature mortality are all associated with obesity. Medical conditions such as type 2 diabetes mellitus, hypertension, hyperlipidemia, or sleep apnea can improve or be cured with weight loss. Medical treatment programs focused on diet, behavior modification, and/or pharmacologic intervention have met with limited long-term success. Although surgical treatments for obesity have become popular in recent years, they should only be used as a last resort for weight loss. Not all patients can be considered appropriate candidates for surgery; therefore, guidelines based on criteria from the National Institutes of Health should be used preoperatively to help identify suitable persons. Most individuals who opt for weight-loss surgery have usually struggled for many years with losing weight and keeping it off, but surgery alone will not ensure successful weight loss. Patient education is imperative for long-term success. Moreover, any such educational regimen should include information on diet, vitamin and mineral supplementation, and lifestyle changes, as well as expected weight-loss results and improvements in comorbid conditions. Patients must be willing to commit to a long-term follow-up program intended to promote successful weight loss and weight maintenance and to prevent metabolic and nutritional complications.

  17. Putting theory of mind in its place: psychological explanations of the socio-emotional-communicative impairments in autistic spectrum disorder.

    Science.gov (United States)

    Boucher, Jill

    2012-05-01

    In this review, the history of the theory of mind (ToM) theory of autistic spectrum disorder (ASD) is outlined (in which ToM is indexed by success on false belief tasks), and the explanatory power and psychological causes of impaired ToM in ASD are critically discussed. It is concluded that impaired ToM by itself has only limited explanatory power, but that explorations of the psychological precursors of impaired ToM have been fruitful in increasing understanding of mindreading impairments in ASD (where 'mindreading' refers those abilities that underlie triadic interaction as well as ToM). It is argued that early explanations of impaired mindreading are untenable for various reasons, but that impairments of dyadic interaction in ASD that could lead to impaired ability to represent others' mental states may be the critical psychological cause, or causes, of impaired ToM. The complexity of causal routes to impaired ToM is emphasized.

  18. Metabolic Reprogramming Regulates the Proliferative and Inflammatory Phenotype of Adventitial Fibroblasts in Pulmonary Hypertension Through the Transcriptional Co-Repressor C-terminal Binding Protein-1

    Science.gov (United States)

    Li, Min; Riddle, Suzette; Zhang, Hui; D’Alessandro, Angelo; Flockton, Amanda; Serkova, Natalie J.; Hansen, Kirk C.; Moldvan, Radu; McKeon, B. Alexandre; Frid, Maria; Kumar, Sushil; Li, Hong; Liu, Hongbing; Cánovas, Angela; Medrano, Juan F.; Thomas, Milton G.; Iloska, Dijana; Plecita-Hlavata, Lydie; Ježek, Petr; Pullamsetti, Soni; Fini, Mehdi A.; El Kasmi, Karim C.; Zhang, Qinghong; Stenmark, Kurt R.

    2016-01-01

    Background Changes in metabolism have been suggested to contribute to the aberrant phenotype of vascular wall cells including fibroblasts in pulmonary hypertension (PH). Herein, we test the hypothesis that metabolic reprogramming to aerobic glycolysis is a critical adaptation of fibroblasts in the hypertensive vessel wall that drives proliferative and pro-inflammatory activation through a mechanism involving increased activity of the NADH-sensitive transcriptional co-repressor C-terminal binding protein 1 (CtBP1). Methods RNA-Sequencing, qPCR, 13C-NMR, fluorescence-lifetime imaging, mass spectrometry-based metabolomics and tracing experiments with U-13C-glucose were used to assess glycolytic reprogramming and to measure NADH/NAD+ ratio in bovine and human adventitial fibroblasts, and mouse lung tissues. Immunohistochemistry was utilized to assess CtBP1 expression in the whole lung tissues. CtBP1 siRNA and the pharmacologic inhibitor 4-methylthio-2-oxobutyric acid (MTOB) were utilized to abrogate CtBP1 activity in cells and hypoxic mice. Results We found adventitial fibroblasts from calves with severe hypoxia-induced PH and humans with IPAH (PH-Fibs) displayed aerobic glycolysis when cultured under normoxia, accompanied by increased free NADH and NADH/NAD+ ratios. Expression of the NADH sensor CtBP1 was increased in vivo and in vitro in fibroblasts within the pulmonary adventitia of humans with IPAH and animals with PH and cultured PH-Fibs, respectively. Decreasing NADH pharmacologically with MTOB, or genetically blocking CtBP1 using siRNA, upregulated the cyclin-dependent genes (p15 and p21) and pro-apoptotic regulators (NOXA and PERP), attenuated proliferation, corrected the glycolytic reprogramming phenotype of PH-Fibs, and augmented transcription of the anti-inflammatory gene HMOX1. ChIP analysis demonstrated that CtBP1 directly binds the HMOX1 promoter. Treatment of hypoxic mice with MTOB decreased glycolysis and expression of inflammatory genes, attenuated

  19. Irreversible barrier to the reprogramming of donor cells in cloning with mouse embryos and embryonic stem cells.

    Science.gov (United States)

    Ono, Yukiko; Kono, Tomohiro

    2006-08-01

    Somatic cloning does not always result in ontogeny in mammals, and development is often associated with various abnormalities and embryo loss with a high frequency. This is considered to be due to aberrant gene expression resulting from epigenetic reprogramming errors. However, a fundamental question in this context is whether the developmental abnormalities reported to date are specific to somatic cloning. The aim of this study was to determine the stage of nuclear differentiation during development that leads to developmental abnormalities associated with embryo cloning. In order to address this issue, we reconstructed cloned embryos using four- and eight-cell embryos, morula embryos, inner cell mass (ICM) cells, and embryonic stem cells as donor nuclei and determined the occurrence of abnormalities such as developmental arrest and placentomegaly, which are common characteristics of all mouse somatic cell clones. The present analysis revealed that an acute decline in the full-term developmental competence of cloned embryos occurred with the use of four- and eight-cell donor nuclei (22.7% vs. 1.8%) in cases of standard embryo cloning and with morula and ICM donor nuclei (11.4% vs. 6.6%) in serial nuclear transfer. Histological observation showed abnormal differentiation and proliferation of trophoblastic giant cells in the placentae of cloned concepti derived from four-cell to ICM cell donor nuclei. Enlargement of placenta along with excessive proliferation of the spongiotrophoblast layer and glycogen cells was observed in the clones derived from morula embryos and ICM cells. These results revealed that irreversible epigenetic events had already started to occur at the four-cell stage. In addition, the expression of genes involved in placentomegaly is regulated at the blastocyst stage by irreversible epigenetic events, and it could not be reprogrammed by the fusion of nuclei with unfertilized oocytes. Hence, developmental abnormalities such as placentomegaly as

  20. Visual impairment and blindness in Hungary.

    Science.gov (United States)

    Szabó, Dorottya; Sándor, Gábor László; Tóth, Gábor; Pék, Anita; Lukács, Regina; Szalai, Irén; Tóth, Georgina Zsófia; Papp, András; Nagy, Zoltán Zsolt; Limburg, Hans; Németh, János

    2018-03-01

    The aim of this study was to estimate the prevalence and causes of blindness, severe visual impairment (SVI), moderate visual impairment (MVI), and early visual impairment (EVI) and its causes in an established market economy of Europe. A cross-sectional population-based survey. A sample size of 3675 was calculated using the standard Rapid Assessment of Avoidable Blindness (RAAB) software in Hungary. A total of 105 clusters of 35 people aged 50 years or older were randomly selected with probability proportionate to size by the Hungarian Central Statistical Office. Households within the clusters were selected using compact segment sampling. Visual acuity (VA) was assessed with a Snellen tumbling E-chart with or without a pinhole in the households. The adjusted prevalences of bilateral blindness, SVI, MVI and EVI were 0.9% (95% CI: 0.6-1.2), 0.5% (95% CI: 0.2-0.7), 5.1% (95% CI: 4.3-5.9) and 6.9% (95% CI: 5.9-7.9), respectively. The major causes of blindness in Hungary were age-related macular degeneration (AMD; 27.3%) and other posterior segment diseases (27.3%), cataract (21.2%) and glaucoma (12.1%). Cataract was the main cause of SVI, MVI and EVI. Cataract surgical coverage (CSC) was 90.7%. Of all bilateral blindness in Hungary, 45.5% was considered avoidable. This study proved that RAAB methodology can be successfully conducted in industrialized countries, which often lack reliable epidemiologic data. The prevalence of blindness was relatively low, with AMD and other posterior segment diseases being the leading causes, and cataract is still a significant cause of visual impairment. © 2017 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

  1. Regulators of pluripotency and their implications in regenerative medicine

    Directory of Open Access Journals (Sweden)

    El-Badawy A

    2015-04-01

    Full Text Available Ahmed El-Badawy, Nagwa El-Badri Center of Excellence for Stem Cells and Regenerative Medicine, Zewail City of Science and Technology, Giza, Egypt Abstract: The ultimate goal of regenerative medicine is to replace damaged tissues with new functioning ones. This can potentially be accomplished by stem cell transplantation. While stem cell transplantation for blood diseases has been increasingly successful, widespread application of stem cell therapy in the clinic has shown limited results. Despite successful efforts to refine existing methodologies and to develop better ones for reprogramming, clinical application of stem cell therapy suffers from issues related to the safety of the transplanted cells, as well as the low efficiency of reprogramming technology. Better understanding of the underlying mechanism(s involved in pluripotency should accelerate the clinical application of stem cell transplantation for regenerative purposes. This review outlines the main decision-making factors involved in pluripotency, focusing on the role of microRNAs, epigenetic modification, signaling pathways, and toll-like receptors. Of special interest is the role of toll-like receptors in pluripotency, where emerging data indicate that the innate immune system plays a vital role in reprogramming. Based on these data, we propose that nongenetic mechanisms for reprogramming provide a novel and perhaps an essential strategy to accelerate application of regenerative medicine in the clinic. Keywords: dedifferentiation, transdifferentiation, reprogramming, pluripotency, microRNAs, epigenetic modifications, signaling pathways, toll-like receptors

  2. Virtue ethics and the selection of children with impairments: a reply to Rosalind McDougall.

    Science.gov (United States)

    Saenz, Carla

    2010-11-01

    In 'Parental Virtues: A New Way of Thinking about the Morality of Reproductive Actions' Rosalind McDougall proposes a virtue-based framework to assess the morality of child selection. Applying the virtue-based account to the selection of children with impairments does not lead, according to McDougall, to an unequivocal answer to the morality of selecting impaired children. In 'Impairment, Flourishing, and the Moral Nature of Parenthood,' she also applies the virtue-based account to the discussion of child selection, and claims that couples with an impairment are morally justified in selecting a child with the same impairment. This claim, she maintains, reveals that the flourishing of a child should be understood as requiring environment-specific characteristics. I argue that McDougall's argument begs the question. More importantly, it does not do justice to virtue ethics. I also question to what extent a virtue ethics framework can be successfully applied to discussions about the moral permissibility of reproductive actions. © 2009 Blackwell Publishing Ltd.

  3. Virtue Ethics and the Selection of Children with Impairments A Reply to Rosalind McDougall

    Science.gov (United States)

    Saenz, Carla

    2009-01-01

    In “Parental Virtues: A New Way of Thinking about the Morality of Reproductive Actions” Rosalind McDougall proposes a virtue-based framework to assess the morality of child selection. Applying the virtue-based account to the selection of children with impairments does not lead, according to McDougall, to an unequivocal answer to the morality of selecting impaired children. In “Impairment, Flourishing, and the Moral Nature of Parenthood,” she also applies the virtue-based account to the discussion of child selection, and claims that couples with an impairment are morally justified in selecting a child with the same impairment. This claim, she maintains, reveals that the flourishing of a child should be understood as requiring environment-specific characteristics. I argue that McDougall’s argument begs the question. More importantly, it does not do justice to virtue ethics. I also question to what extent a virtue ethics framework can be successfully applied to discussions about the moral permissibility of reproductive actions. PMID:19508307

  4. Glucocorticoids mediate stress-induced impairment of retrieval of stimulus-response memory.

    Science.gov (United States)

    Atsak, Piray; Guenzel, Friederike M; Kantar-Gok, Deniz; Zalachoras, Ioannis; Yargicoglu, Piraye; Meijer, Onno C; Quirarte, Gina L; Wolf, Oliver T; Schwabe, Lars; Roozendaal, Benno

    2016-05-01

    Acute stress and elevated glucocorticoid hormone levels are well known to impair the retrieval of hippocampus-dependent 'declarative' memory. Recent findings suggest that stress might also impair the retrieval of non-hippocampal memories. In particular, stress shortly before retention testing was shown to impair the retrieval of striatal stimulus-response associations in humans. However, the mechanism underlying this stress-induced retrieval impairment of non-hippocampal stimulus-response memory remains elusive. In the present study, we investigated whether an acute elevation in glucocorticoid levels mediates the impairing effects of stress on retrieval of stimulus-response memory. Male Sprague-Dawley rats were trained on a stimulus-response task in an eight-arm radial maze until they learned to associate a stimulus, i.e., cue, with a food reward in one of the arms. Twenty-four hours after successful acquisition, they received a systemic injection of vehicle, corticosterone (1mg/kg), the corticosterone-synthesis inhibitor metyrapone (35mg/kg) or were left untreated 1h before retention testing. We found that the corticosterone injection impaired the retrieval of stimulus-response memory. We further found that the systemic injection procedure per se was stressful as the vehicle administration also increased plasma corticosterone levels and impaired the retrieval of stimulus-response memory. However, memory retrieval was not impaired when rats were tested 2min after the systemic vehicle injection, before any stress-induced elevation in corticosterone levels had occurred. Moreover, metyrapone treatment blocked the effect of injection stress on both plasma corticosterone levels and memory retrieval impairment, indicating that the endogenous corticosterone response mediates the stress-induced memory retrieval impairment. None of the treatments affected rats' locomotor activity or motivation to search for the food reward within the maze. These findings show that stress

  5. DNA Methylation in Embryo Development: Epigenetic Impact of ART (Assisted Reproductive Technologies).

    Science.gov (United States)

    Canovas, Sebastian; Ross, Pablo J; Kelsey, Gavin; Coy, Pilar

    2017-11-01

    DNA methylation can be considered a component of epigenetic memory with a critical role during embryo development, and which undergoes dramatic reprogramming after fertilization. Though it has been a focus of research for many years, the reprogramming mechanism is still not fully understood. Recent results suggest that absence of maintenance at DNA replication is a major factor, and that there is an unexpected role for TET3-mediated oxidation of 5mC to 5hmC in guarding against de novo methylation. Base-resolution and genome-wide profiling methods are enabling more comprehensive assessments of the extent to which ART might impair DNA methylation reprogramming, and which sequence elements are most vulnerable. Indeed, as we also review here, studies showing the effect of culture media, ovarian stimulation or embryo transfer on the methylation pattern of embryos emphasize the need to face ART-associated defects and search for strategies to mitigate adverse effects on the health of ART-derived children. © 2017 WILEY Periodicals, Inc.

  6. Effective intervention for expressive grammar in children with specific language impairment.

    Science.gov (United States)

    Smith-Lock, Karen M; Leitao, Suze; Lambert, Lara; Nickels, Lyndsey

    2013-01-01

    Children with specific language impairment are known to struggle with expressive grammar. While some studies have shown successful intervention under laboratory conditions, there is a paucity of evidence for the effectiveness of grammar treatment in young children in community settings. To evaluate the effectiveness of a school-based intervention programme for expressive grammar in 5-year-olds with specific language impairment. Thirty-four 5-year-old children attending a specialized school for children with language impairment participated in the study. Nineteen children received treatment for expressive grammar (experimental group) and 15 children received a control treatment. Treatment consisted of weekly 1-h sessions of small group activities in a classroom setting for 8 weeks. Techniques included direct instruction, focused stimulation, recasting and imitation. Results were analysed at the group level and as a case series with each child as their own control in a single-subject design. There was a significant difference in grammatical performance pre- and post-treatment for children who received grammar treatment (Cohen's d = 1.24), but not for a group of children who received a control treatment. Further, no difference in performance was found in the equivalent time period prior to treatment, nor for an untreated target. Treatment success was more pronounced in children without articulation difficulties which interfered with their ability to produce the grammatical targets (Cohen's d = 1.66). Individual analyses indicated the treatment effect was significant for the majority of children. Individually targeted intervention delivered in small groups in a classroom setting was effective in improving production of expressive grammatical targets in 5-year-old children with specific language impairment. © 2013 Royal College of Speech and Language Therapists.

  7. Human monocytes undergo functional re-programming during sepsis mediated by hypoxia-inducible factor-1α.

    Science.gov (United States)

    Shalova, Irina N; Lim, Jyue Yuan; Chittezhath, Manesh; Zinkernagel, Annelies S; Beasley, Federico; Hernández-Jiménez, Enrique; Toledano, Victor; Cubillos-Zapata, Carolina; Rapisarda, Annamaria; Chen, Jinmiao; Duan, Kaibo; Yang, Henry; Poidinger, Michael; Melillo, Giovanni; Nizet, Victor; Arnalich, Francisco; López-Collazo, Eduardo; Biswas, Subhra K

    2015-03-17

    Sepsis is characterized by a dysregulated inflammatory response to infection. Despite studies in mice, the cellular and molecular basis of human sepsis remains unclear and effective therapies are lacking. Blood monocytes serve as the first line of host defense and are equipped to recognize and respond to infection by triggering an immune-inflammatory response. However, the response of these cells in human sepsis and their contribution to sepsis pathogenesis is poorly understood. To investigate this, we performed a transcriptomic, functional, and mechanistic analysis of blood monocytes from patients during sepsis and after recovery. Our results revealed the functional plasticity of monocytes during human sepsis, wherein they transited from a pro-inflammatory to an immunosuppressive phenotype, while enhancing protective functions like phagocytosis, anti-microbial activity, and tissue remodeling. Mechanistically, hypoxia inducible factor-1α (HIF1α) mediated this functional re-programming of monocytes, revealing a potential mechanism for their therapeutic targeting to regulate human sepsis. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Emotional arousal impairs association-memory: Roles of amygdala and hippocampus.

    Science.gov (United States)

    Madan, Christopher R; Fujiwara, Esther; Caplan, Jeremy B; Sommer, Tobias

    2017-08-01

    Emotional arousal is well-known to enhance memory for individual items or events, whereas it can impair association memory. The neural mechanism of this association memory impairment by emotion is not known: In response to emotionally arousing information, amygdala activity may interfere with hippocampal associative encoding (e.g., via prefrontal cortex). Alternatively, emotional information may be harder to unitize, resulting in reduced availability of extra-hippocampal medial temporal lobe support for emotional than neutral associations. To test these opposing hypotheses, we compared neural processes underlying successful and unsuccessful encoding of emotional and neutral associations. Participants intentionally studied pairs of neutral and negative pictures (Experiments 1-3). We found reduced association-memory for negative pictures in all experiments, accompanied by item-memory increases in Experiment 2. High-resolution fMRI (Experiment 3) indicated that reductions in associative encoding of emotional information are localizable to an area in ventral-lateral amygdala, driven by attentional/salience effects in the central amygdala. Hippocampal activity was similar during both pair types, but a left hippocampal cluster related to successful encoding was observed only for negative pairs. Extra-hippocampal associative memory processes (e.g., unitization) were more effective for neutral than emotional materials. Our findings suggest that reduced emotional association memory is accompanied by increases in activity and functional coupling within the amygdala. This did not disrupt hippocampal association-memory processes, which indeed were critical for successful emotional association memory formation. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. The usefulness of science knowledge for parents of hearing-impaired children.

    Science.gov (United States)

    Shauli, Sophie; Baram-Tsabari, Ayelet

    2018-04-01

    Hearing-impaired children's chances of integrating into hearing society largely depend on their parents, who need to learn vast amounts of science knowledge in the field of hearing. This study characterized the role played by science knowledge in the lives of nonscientists faced with science-related decisions by examining the interactions between general science knowledge, contextual science knowledge in the field of hearing, and parents' advocacy knowledge and attitudes. Based on six semi-structured interviews and 115 questionnaires completed by parents of hearing-impaired children, contextual science knowledge emerged as the only predictor for having slightly better advocacy attitudes and knowledge (5.5% explained variance). Although general science knowledge was the best predictor of contextual knowledge (14% of explained variance), it was not a direct predictor of advocacy knowledge and attitudes. Science knowledge plays some role in the lives of hearing-impaired families, even if they do not list it as a resource for successful rehabilitation.

  10. Heart Development, Diseases, and Regeneration - New Approaches From Innervation, Fibroblasts, and Reprogramming.

    Science.gov (United States)

    Ieda, Masaki

    2016-09-23

    It is well known that cardiac function is tightly controlled by neural activity; however, the molecular mechanism of cardiac innervation during development and the relationship with heart disease remain undetermined. My work has revealed the molecular networks that govern cardiac innervation and its critical roles in heart diseases such as silent myocardial ischemia and arrhythmias. Cardiomyocytes proliferate during embryonic development, but lose their proliferative capacity after birth. Cardiac fibroblasts are a major source of cells during fibrosis and induce cardiac hypertrophy after myocardial injury in the adult heart. Despite the importance of fibroblasts in the adult heart, the role of fibroblasts in embryonic heart development was previously not determined. I demonstrated that cardiac fibroblasts play important roles in myocardial growth and cardiomyocyte proliferation during embryonic development, and I identified key paracrine factors and signaling pathways. In contrast to embryonic cardiomyocytes, adult cardiomyocytes have little regenerative capacity, leading to heart failure and high mortality rates after myocardial infarction. Leveraging the knowledge of developmental biology, I identified cardiac reprogramming factors that can directly convert resident cardiac fibroblasts into cardiomyocytes for heart regeneration. These findings greatly improved our understanding of heart development and diseases, and provide a new strategy for heart regenerative therapy. (Circ J 2016; 80: 2081-2088).

  11. Mesenchymal Cell Reprogramming in Experimental MPLW515L Mouse Model of Myelofibrosis.

    Directory of Open Access Journals (Sweden)

    Ying Han

    Full Text Available Myelofibrosis is an indicator of poor prognosis in myeloproliferative neoplasms (MPNs, but the precise mechanism(s contributing to extracellular matrix remodeling and collagen deposition in the bone marrow (BM niche remains unanswered. In this study, we isolated mesenchymal stromal cells (MSCs from mice transplanted with wild-type thrombopoietin receptor (MPLWT and MPLW515L retroviral-transduced bone marrow. Using MSCs derived from MPLW515-transplant recipients, excessive collagen deposition was maintained in the absence of the virus and neoplastic hematopoietic cells suggested that the MSCs were reprogrammed in vivo. TGFβ production by malignant megakaryocytes plays a definitive role promoting myelofibrosis in MPNs. However, TGFβ was equally expressed by MSCs derived from MPLWT and MPLW515L expressing mice and the addition of neutralizing anti-TGFβ antibody only partially reduced collagen secretion in vitro. Interestingly, profibrotic MSCs displayed increased levels of pSmad3 and pSTAT3 suggesting that inflammatory mediators cooperating with the TGFβ-receptor signaling may maintain the aberrant phenotype ex vivo. FGFb is a known suppressor of TGFβ signaling. Reduced collagen deposition by FGFb-treated MSCs derived from MPLW515L mice suggests that the activating pathway is vulnerable to this suppressive mediator. Therefore, our findings have implications for the future investigation of therapies to reverse fibrosis in MPNs.

  12. Mesenchymal Cell Reprogramming in Experimental MPLW515L Mouse Model of Myelofibrosis.

    Science.gov (United States)

    Han, Ying; Yue, Lanzhu; Wei, Max; Ren, Xiubao; Shao, Zonghong; Zhang, Ling; Levine, Ross L; Epling-Burnette, Pearlie K

    2017-01-01

    Myelofibrosis is an indicator of poor prognosis in myeloproliferative neoplasms (MPNs), but the precise mechanism(s) contributing to extracellular matrix remodeling and collagen deposition in the bone marrow (BM) niche remains unanswered. In this study, we isolated mesenchymal stromal cells (MSCs) from mice transplanted with wild-type thrombopoietin receptor (MPLWT) and MPLW515L retroviral-transduced bone marrow. Using MSCs derived from MPLW515-transplant recipients, excessive collagen deposition was maintained in the absence of the virus and neoplastic hematopoietic cells suggested that the MSCs were reprogrammed in vivo. TGFβ production by malignant megakaryocytes plays a definitive role promoting myelofibrosis in MPNs. However, TGFβ was equally expressed by MSCs derived from MPLWT and MPLW515L expressing mice and the addition of neutralizing anti-TGFβ antibody only partially reduced collagen secretion in vitro. Interestingly, profibrotic MSCs displayed increased levels of pSmad3 and pSTAT3 suggesting that inflammatory mediators cooperating with the TGFβ-receptor signaling may maintain the aberrant phenotype ex vivo. FGFb is a known suppressor of TGFβ signaling. Reduced collagen deposition by FGFb-treated MSCs derived from MPLW515L mice suggests that the activating pathway is vulnerable to this suppressive mediator. Therefore, our findings have implications for the future investigation of therapies to reverse fibrosis in MPNs.

  13. Memory Impairment in Children with Language Impairment

    Science.gov (United States)

    Baird, Gillian; Dworzynski, Katharina; Slonims, Vicky; Simonoff, Emily

    2010-01-01

    Aim: The aim of this study was to assess whether any memory impairment co-occurring with language impairment is global, affecting both verbal and visual domains, or domain specific. Method: Visual and verbal memory, learning, and processing speed were assessed in children aged 6 years to 16 years 11 months (mean 9y 9m, SD 2y 6mo) with current,…

  14. A Role for the Krebs Cycle Intermediate Citrate in Metabolic Reprogramming in Innate Immunity and Inflammation

    Directory of Open Access Journals (Sweden)

    Niamh C. Williams

    2018-02-01

    Full Text Available Metabolism in immune cells is no longer thought of as merely a process for adenosine triphosphate (ATP production, biosynthesis, and catabolism. The reprogramming of metabolic pathways upon activation is also for the production of metabolites that can act as immune signaling molecules. Activated dendritic cells (DCs and macrophages have an altered Krebs cycle, one consequence of which is the accumulation of both citrate and succinate. Citrate is exported from the mitochondria via the mitochondrial citrate- carrier. Cytosolic metabolism of citrate to acetyl-coenzyme A (acetyl-CoA is important for both fatty-acid synthesis and protein acetylation, both of which have been linked to macrophage and DC activation. Citrate-derived itaconate has a direct antibacterial effect and also has been shown to act as an anti-inflammatory agent, inhibiting succinate dehydrogenase. These findings identify citrate as an important metabolite for macrophage and DC effector function.

  15. DNA methylation at a bovine alpha satellite I repeat CpG site during development following fertilization and somatic cell nuclear transfer.

    Directory of Open Access Journals (Sweden)

    Christine Couldrey

    Full Text Available Incomplete epigenetic reprogramming is postulated to contribute to the low developmental success following somatic cell nuclear transfer (SCNT. Here, we describe the epigenetic reprogramming of DNA methylation at an alpha satellite I CpG site (αsatI-5 during development of cattle generated either by artificial insemination (AI or in vitro fertilization (IVF and SCNT. Quantitative methylation analysis identified that SCNT donor cells were highly methylated at αsatI-5 and resulting SCNT blastocysts showed significantly more methylation than IVF blastocysts. At implantation, no difference in methylation was observed between SCNT and AI in trophoblast tissue at αsatI-5, however, SCNT embryos were significantly hyper-methylated compared to AI controls at this time point. Following implantation, DNA methylation at αsatI-5 decreased in AI but not SCNT placental tissues. In contrast to placenta, the proportion of methylation at αsatI-5 remained high in adrenal, kidney and muscle tissues during development. Differences in the average proportion of methylation were smaller in somatic tissues than placental tissues but, on average, SCNT somatic tissues were hyper-methylated at αsatI-5. Although sperm from all bulls was less methylated than somatic tissues at αsatI-5, on average this site remained hyper-methylated in sperm from cloned bulls compared with control bulls. This developmental time course confirms that epigenetic reprogramming does occur, at least to some extent, following SCNT. However, the elevated methylation levels observed in SCNT blastocysts and cellular derivatives implies that there is either insufficient time or abundance of appropriate reprogramming factors in oocytes to ensure complete reprogramming. Incomplete reprogramming at this CpG site may be a contributing factor to low SCNT success rates, but more likely represents the tip of the iceberg in terms of incompletely reprogramming. Until protocols ensure the epigenetic

  16. Putting Theory of Mind in Its Place: Psychological Explanations of the Socio-Emotional-Communicative Impairments in Autistic Spectrum Disorder

    Science.gov (United States)

    Boucher, Jill

    2012-01-01

    In this review, the history of the theory of mind (ToM) theory of autistic spectrum disorder (ASD) is outlined (in which ToM is indexed by success on false belief tasks), and the explanatory power and psychological causes of impaired ToM in ASD are critically discussed. It is concluded that impaired ToM by itself has only limited explanatory…

  17. Cognitive impairment and stroke in elderly patients

    Directory of Open Access Journals (Sweden)

    Lo Coco D

    2016-03-01

    Full Text Available Daniele Lo Coco,1 Gianluca Lopez,1 Salvatore Corrao,2,31Neurology and Stroke Unit, 2Department of Internal Medicine, National Relevance and High Specialization Hospital Trust ARNAS Civico, Di Cristina, Benfratelli, Palermo, 3Centre of Research for Effectiveness and Appropriateness in Medicine (C.R.E.A.M., Di.Bi.M.I.S., University of Palermo, Palermo, Italy Abstract: We reviewed current knowledge about the interaction between stroke and vascular risk factors and the development of cognitive impairment and dementia. Stroke is increasingly recognized as an important cause of cognitive problems and has been implicated in the development of both Alzheimer's disease and vascular dementia. The prevalence of cognitive impairment after stroke is high, and their combined effects significantly increase the cost of care and health resource utilization, with reflections on hospital readmissions and increased mortality rates. There is also substantial evidence that vascular risk factors (such as hypertension, diabetes, obesity, dyslipidemia, and tobacco smoking are independently associated with an increased risk of cognitive decline and dementia. Thus, a successful management of these factors, as well as optimal acute stroke management, might have a great impact on the development of cognitive impairment. Notwithstanding, the pathological link between cognitive impairment, stroke, and vascular risk factors is complex and still partially unclear so that further studies are needed to better elucidate the boundaries of this relationship. Many specific pharmacological treatments, including anticholinergic drugs and antihypertensive medications, and nonpharmacological approaches, such as diet, cognitive rehabilitation, and physical activity, have been studied for patients with vascular cognitive impairment, but the optimal care is still far away. Meanwhile, according to the most recent knowledge, optimal stroke care should also include cognitive assessment in the

  18. Theory of Mind in Bipolar Disorder, with Comparison to the Impairments Observed in Schizophrenia.

    Science.gov (United States)

    Mitchell, Rachel L C; Young, Allan H

    2015-01-01

    Our ability to make sense of information on the potential intentions and dispositions of others is of paramount importance for understanding their communicative intent, and for judging what an appropriate reaction might be. Thus, anything that impinges on this ability has the potential to cause significant social impairment, and compromise an individual's level of functioning. Both bipolar disorder and schizophrenia are known to feature theory of mind impairment. We conducted a theoretical review to determine the extent and types of theory of mind impairment in bipolar disorder, and evaluate their relationship to medication and symptoms. We also considered possible mediatory mechanisms, and set out to discover what else could be learnt about the impairment in bipolar disorder by comparison to the profile of impairment in schizophrenia. The literature established that in bipolar disorder (i) some form of theory of mind impairment has been observed in all mood states, including euthymia, (ii) the form of theory of mind assessed and task used to make the assessment influence the impairment observed, and (iii) there might be some relationship to cognitive impairment, although a relationship to standard clinical variables was harder to establish. What also became clear in the literature on bipolar disorder itself was the possible relationship of theory of mind impairment to history of psychotic symptoms. Direct comparative studies, including patients with schizophrenia, were thus examined, and provided several important directions for future research on the bases of impairment in bipolar disorder. Particularly prominent was the issue of whether theory of mind impairment could be considered a candidate endophenotype for the psychoses, although current evidence suggests that this may be premature. The differences in impairment across schizophrenia and bipolar disorder may, however, have genuine differential effects on social functioning and the likely success of

  19. Theory of Mind in Bipolar Disorder, with Comparison to the Impairments Observed in Schizophrenia

    Science.gov (United States)

    Mitchell, Rachel L. C.; Young, Allan H.

    2016-01-01

    Our ability to make sense of information on the potential intentions and dispositions of others is of paramount importance for understanding their communicative intent, and for judging what an appropriate reaction might be. Thus, anything that impinges on this ability has the potential to cause significant social impairment, and compromise an individual’s level of functioning. Both bipolar disorder and schizophrenia are known to feature theory of mind impairment. We conducted a theoretical review to determine the extent and types of theory of mind impairment in bipolar disorder, and evaluate their relationship to medication and symptoms. We also considered possible mediatory mechanisms, and set out to discover what else could be learnt about the impairment in bipolar disorder by comparison to the profile of impairment in schizophrenia. The literature established that in bipolar disorder (i) some form of theory of mind impairment has been observed in all mood states, including euthymia, (ii) the form of theory of mind assessed and task used to make the assessment influence the impairment observed, and (iii) there might be some relationship to cognitive impairment, although a relationship to standard clinical variables was harder to establish. What also became clear in the literature on bipolar disorder itself was the possible relationship of theory of mind impairment to history of psychotic symptoms. Direct comparative studies, including patients with schizophrenia, were thus examined, and provided several important directions for future research on the bases of impairment in bipolar disorder. Particularly prominent was the issue of whether theory of mind impairment could be considered a candidate endophenotype for the psychoses, although current evidence suggests that this may be premature. The differences in impairment across schizophrenia and bipolar disorder may, however, have genuine differential effects on social functioning and the likely success of

  20. Psychopathological traits in college students from top-ranking french schools: Do autistic features impair success in science when associated with schizotypal traits?

    Science.gov (United States)

    Choteau, Laura; Raynal, Patrick; Goutaudier, Nelly; Chabrol, Henri

    2016-03-30

    The link between personality and the interest of individuals for science has not been thoroughly explored. In this report, we studied psychopathological traits in students studying science in French top-ranking institutions. Three hundred and forty seven individuals answered questionnaires assessing autistic and schizotypal dimensions, as well as anxiety, depression symptomatology and attachment quality. A cluster analysis based on autistic and schizotypal traits led to the identification of 4 distinct profiles: a "low trait cluster", a "moderate autistic trait cluster", a "moderate schizotypal trait cluster" and a "high trait cluster" (HTC) composed of individuals with high scores on both autistic and schizotypal scales. Each cluster represented 20.1-27.1% of participants and was clearly different from the three others, both on autistic and on schizotypal dimensions. These groups could be also typified by their level of anxiety, depression or degraded attachment, which are proportional to the extent of psychopathological traits. Moreover, students from the HTC cluster displayed lower academic results, thus implying that autistic traits might impair success in science when they are associated with moderate schizotypal personality features. This study also suggests that depression and anxiety might mediate performance inhibition in the HTC group. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  1. Derivation and characterization of sleeping beauty transposon-mediated porcine induced pluripotent stem cells

    DEFF Research Database (Denmark)

    Kues, Wilfried A.; Herrmann, Doris; Barg-Kues, Brigitte

    2013-01-01

    the nonviral Sleeping Beauty transposon system to deliver the reprogramming factors Oct4, Sox2, Klf4, and cMyc. Successful reprogramming to a pluripotent state was indicated by changes in cell morphology and reactivation of the Oct4-EGFP reporter. The transposon-reprogrammed induced pluripotent stem (i......PS) cells showed long-term proliferation in vitro over >40 passages, expressed transcription factors typical of embryonic stem cells, including OCT4, NANOG, SOX2, REX1, ESRRB, DPPA5, and UTF1 and surface markers of pluripotency, including SSEA-1 and TRA-1-60. In vitro differentiation resulted in derivatives......The domestic pig is an important large animal model for preclinical testing of novel cell therapies. Recently, we produced pluripotency reporter pigs in which the Oct4 promoter drives expression of the enhanced green fluorescent protein (EGFP). Here, we reprogrammed Oct4-EGFP fibroblasts employing...

  2. Impaired face recognition is associated with social inhibition.

    Science.gov (United States)

    Avery, Suzanne N; VanDerKlok, Ross M; Heckers, Stephan; Blackford, Jennifer U

    2016-02-28

    Face recognition is fundamental to successful social interaction. Individuals with deficits in face recognition are likely to have social functioning impairments that may lead to heightened risk for social anxiety. A critical component of social interaction is how quickly a face is learned during initial exposure to a new individual. Here, we used a novel Repeated Faces task to assess how quickly memory for faces is established. Face recognition was measured over multiple exposures in 52 young adults ranging from low to high in social inhibition, a core dimension of social anxiety. High social inhibition was associated with a smaller slope of change in recognition memory over repeated face exposure, indicating participants with higher social inhibition showed smaller improvements in recognition memory after seeing faces multiple times. We propose that impaired face learning is an important mechanism underlying social inhibition and may contribute to, or maintain, social anxiety. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  3. Transcription reprogramming during root nodule development in Medicago truncatula.

    Directory of Open Access Journals (Sweden)

    Sandra Moreau

    Full Text Available Many genes which are associated with root nodule development and activity in the model legume Medicago truncatula have been described. However information on precise stages of activation of these genes and their corresponding transcriptional regulators is often lacking. Whether these regulators are shared with other plant developmental programs also remains an open question. Here detailed microarray analyses have been used to study the transcriptome of root nodules induced by either wild type or mutant strains of Sinorhizobium meliloti. In this way we have defined eight major activation patterns in nodules and identified associated potential regulatory genes. We have shown that transcription reprogramming during consecutive stages of nodule differentiation occurs in four major phases, respectively associated with (i early signalling events and/or bacterial infection; plant cell differentiation that is either (ii independent or (iii dependent on bacteroid differentiation; (iv nitrogen fixation. Differential expression of several genes involved in cytokinin biosynthesis was observed in early symbiotic nodule zones, suggesting that cytokinin levels are actively controlled in this region. Taking advantage of databases recently developed for M. truncatula, we identified a small subset of gene expression regulators that were exclusively or predominantly expressed in nodules, whereas most other regulators were also activated under other conditions, and notably in response to abiotic or biotic stresses. We found evidence suggesting the activation of the jasmonate pathway in both wild type and mutant nodules, thus raising questions about the role of jasmonate during nodule development. Finally, quantitative RT-PCR was used to analyse the expression of a series of nodule regulator and marker genes at early symbiotic stages in roots and allowed us to distinguish several early stages of gene expression activation or repression.

  4. Reading interest and information needs of persons with visual impairment in Nigeria

    Directory of Open Access Journals (Sweden)

    ’Niran Adetoro

    2010-01-01

    Full Text Available Information materials can only become usable to persons with visual impairment when they are transcribed into alternative formats. Over time, the transcription and provision of alternative formats in Nigeria by libraries has not been based on users’ reading interest and information needs. This study delves into the reading interests and information needs of persons with visual impairment in Nigeria. Survey research design was adopted and the study purposively focused on southwestern Nigeria. Using stratified proportionate random sampling techniques, data was gathered by questionnaires namely the Visually Impaired Adult Questionnaire VIAQ (= 0.75 and Visually Impaired Student Questionnaire VISQ (= 0.78 from fourteen selected libraries stratified into non-governmental, public, tertiary institutions and secondary schools. Of the 563 copies of the questionnaire that were administered, 401 (71.3% were successfully completed and used for the study. The study found that adults with visual impairment had high reading interests in religious, business, and entertainment materials, among others. Secondary school respondents had high reading interest in art subjects, reference materials, manuals and animal story materials. Both respondents showed high information needs in expected and relevant areas. Braille materials (58.3% are the most preferred source of information generally. Adult respondents preferred Braille (72.4%, while the secondary school respondents preferred Talking books/audio recordings (55%. Transcription and provision of information materials for the visually impaired through libraries should be based on knowledge of their reading interest and information needs.

  5. Susceptibility of functional impairment scales to noncredible responses in the clinical evaluation of adult ADHD.

    Science.gov (United States)

    Fuermaier, Anselm B M; Tucha, Oliver; Koerts, Janneke; Butzbach, Marah; Weisbrod, Matthias; Aschenbrenner, Steffen; Tucha, Lara

    2018-05-01

    A growing body of research questions the reliance of symptom self-reports in the clinical evaluation of attention-deficit/hyperactivity disorder (ADHD) in adulthood. A recent study suggested that also impairment reports are vulnerable to noncredible responses, as derived from a simulation design using a global functional impairment scale. The present study aims to add evidence to this issue, by using an ADHD specific impairment scale in a simulation design on large samples. Impairment ratings on the Weiss Functional Impairment Rating Scale (WFIRS) of 62 patients with ADHD were compared to 142 healthy individuals who were instructed to show normal behavior. Furthermore, impairment ratings of patients with ADHD were compared to ratings of 330 healthy individuals who were randomly assigned to one of four simulation conditions that were instructed to complete the scale as if they had ADHD. Patients with ADHD reported higher levels of impairment than the healthy control group in all domains of life. Furthermore, individuals instructed to feign ADHD indicated higher levels of impairments in most domains of life compared to control participants and genuine patients with ADHD. The group differences between individuals feigning ADHD and individuals with genuine ADHD, however, were only small to moderate. Further analyses revealed that the WFRIS was not useful to successfully differentiate genuine from feigned ADHD. The present study confirms the conclusion that self-reported impairments are susceptible to noncredible responses and should be used with caution in the clinical evaluation of adult ADHD.

  6. Visual Impairment

    Science.gov (United States)

    ... site Sitio para adolescentes Body Mind Sexual Health Food & Fitness Diseases & Conditions Infections Drugs & Alcohol School & Jobs Sports Expert Answers (Q&A) Staying Safe Videos for Educators Search English Español Visual Impairment KidsHealth / For Teens / Visual Impairment What's in ...

  7. Different Patterns of Theory of Mind Impairment in Mild Cognitive Impairment.

    Science.gov (United States)

    Moreau, Noémie; Rauzy, Stéphane; Bonnefoi, Bernadette; Renié, Laurent; Martinez-Almoyna, Laurent; Viallet, François; Champagne-Lavau, Maud

    2015-01-01

    Theory of Mind refers to the ability to infer other’s mental states, their beliefs, intentions, or knowledge. To date, only two studies have reported the presence of Theory of Mind impairment in mild cognitive impairment (MCI). In the present study,we evaluated 20 MCI patients and compared them with 25 healthy control participants using two Theory of Mind tasks. The first task was a false belief paradigm as frequently used in the literature, and the second one was a referential communication task,assessing Theory of Mind in a real situation of interaction and which had never been used before in this population. The results showed that MCI patients presented difficulties inferring another person’s beliefs about reality and attributing knowledge to them in a situation of real-life interaction. Two different patterns of Theory of Mind emerged among the patients. In comparison with the control group, some MCI patients demonstrated impairment only in the interaction task and presented isolated episodicmemory impairment, while others were impaired in both Theory of Mind tasks and presented cognitive impairment impacting both episodic memory and executive functioning. Theory of Mind is thus altered in the very early stages of cognitive impairment even in real social interaction, which could impact precociously relationships in daily life.

  8. Neural correlates of spatial navigation changes in mild cognitive impairment and Alzheimer’s disease

    Directory of Open Access Journals (Sweden)

    Kamil eVlček

    2014-03-01

    Full Text Available Although the memory impairment is a hallmark of Alzheimer’s disease (AD, AD has also been characterized by spatial disorientation, which is present from its early stages. Spatial disorientation in AD manifests itself in getting lost in familiar and unfamiliar places and have been characterized more specifically using spatial navigation tests in both real space and virtual environments as an impairment in multiple spatial abilities, including allocentric and egocentric navigation strategies, visuospatial perception or selection of relevant information for successful navigation. Patients suffering mild cognitive impairment (MCI, who are at a high risk of development of dementia, show impairment in a subset of these abilities, mainly connected with allocentric and egocentric processing. While spatial disorientation in typical AD patients probably reflects neurodegenerative changes in medial and posterior temporal, parietal and frontal lobes and retrosplenial cortex, the impairment of spatial navigation in MCI seem to be connected mainly with the medial temporal and also parietal brain changes. In this review we will summarize the signs of brain disease in most MCI and AD patients showing in various tasks of spatial memory and navigation.

  9. Social impairment in conversation: disfluency and compensatory mechanisms

    DEFF Research Database (Denmark)

    Fusaroli, Riccardo; Lambrechts, Anna; Weed, Ethan

    of interactions with adults with ASD will help us understand how social impairment affects the life of people with ASD and which compensatory mechanisms can be used to minimize its effects. Objectives: We want to develop automated quantitative methods to assess dysfluency and compensatory dynamics in conversation......-taking measures, we observe clear compensatory dynamics at work in the interviews. The more disfluency is displayed in the participant, the more the interviewer provides scaffolding. Future work will investigate whether these effects are modulated by practice and context, and how they affect the success...

  10. Expression Profiling of Differentiating Emerin-Null Myogenic Progenitor Identifies Molecular Pathways Implicated in Their Impaired Differentiation

    Directory of Open Access Journals (Sweden)

    Ashvin Iyer

    2017-10-01

    Full Text Available Mutations in the gene encoding emerin cause Emery-Dreifuss muscular dystrophy (EDMD, a disorder causing progressive skeletal muscle wasting, irregular heart rhythms and contractures of major tendons. RNA sequencing was performed on differentiating wildtype and emerin-null myogenic progenitors to identify molecular pathways implicated in EDMD, 340 genes were uniquely differentially expressed during the transition from day 0 to day 1 in wildtype cells. 1605 genes were uniquely expressed in emerin-null cells; 1706 genes were shared among both wildtype and emerin-null cells. One thousand and forty-seven transcripts showed differential expression during the transition from day 1 to day 2. Four hundred and thirty-one transcripts showed altered expression in both wildtype and emerin-null cells. Two hundred and ninety-five transcripts were differentially expressed only in emerin-null cells and 321 transcripts were differentially expressed only in wildtype cells. DAVID, STRING and Ingenuity Pathway Analysis identified pathways implicated in impaired emerin-null differentiation, including cell signaling, cell cycle checkpoints, integrin signaling, YAP/TAZ signaling, stem cell differentiation, and multiple muscle development and myogenic differentiation pathways. Functional enrichment analysis showed biological functions associated with the growth of muscle tissue and myogenesis of skeletal muscle were inhibited. The large number of differentially expressed transcripts upon differentiation induction suggests emerin functions during transcriptional reprograming of progenitors to committed myoblasts.

  11. 20 CFR 220.102 - Non-severe impairment(s), defined.

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false Non-severe impairment(s), defined. 220.102 Section 220.102 Employees' Benefits RAILROAD RETIREMENT BOARD REGULATIONS UNDER THE RAILROAD RETIREMENT... these include— (1) Physical functions such as walking, standing, sitting, lifting, pushing, pulling...

  12. DNA Methylation at a Bovine Alpha Satellite I Repeat CpG Site during Development following Fertilization and Somatic Cell Nuclear Transfer

    OpenAIRE

    Couldrey, Christine; Wells, David N.

    2013-01-01

    Incomplete epigenetic reprogramming is postulated to contribute to the low developmental success following somatic cell nuclear transfer (SCNT). Here, we describe the epigenetic reprogramming of DNA methylation at an alpha satellite I CpG site (αsatI-5) during development of cattle generated either by artificial insemination (AI) or in vitro fertilization (IVF) and SCNT. Quantitative methylation analysis identified that SCNT donor cells were highly methylated at αsatI-5 and resulting SCNT bla...

  13. Dystypia: isolated typing impairment without aphasia, apraxia or visuospatial impairment.

    Science.gov (United States)

    Otsuki, Mika; Soma, Yoshiaki; Arihiro, Shoji; Watanabe, Yoshimasa; Moriwaki, Hiroshi; Naritomi, Hiroaki

    2002-01-01

    We report a 60-year-old right-handed Japanese man who showed an isolated persistent typing impairment without aphasia, agraphia, apraxia or any other neuropsychological deficit. We coined the term 'dystypia' for this peculiar neuropsychological manifestation. The symptom was caused by an infarction in the left frontal lobe involving the foot of the second frontal convolution and the frontal operculum. The patient's typing impairment was not attributable to a disturbance of the linguistic process, since he had no aphasia or agraphia. The impairment was not attributable to the impairment of the motor execution process either, since he had no apraxia. Thus, his typing impairment was deduced to be based on a disturbance of the intermediate process where the linguistic phonological information is converted into the corresponding performance. We hypothesized that there is a specific process for typing which branches from the motor programming process presented in neurolinguistic models. The foot of the left second frontal convolution and the operculum may play an important role in the manifestation of 'dystypia'. Copyright 2002 S. Karger AG, Basel

  14. Theory of mind in bipolar disorder and its comparison to the impairments observed in schizophrenia.

    Directory of Open Access Journals (Sweden)

    Rachel L. C. Mitchell

    2016-01-01

    Full Text Available Our ability to make sense of information on the potential intentions and dispositions of others is of paramount importance for understanding their communicative intent, and for judging what an appropriate reaction might be. Thus anything that impinges on this ability has the potential to cause significant social impairment, and compromise an individual’s level of functioning. Both bipolar disorder and schizophrenia are known to feature theory of mind impairment. We conducted a theoretical review to determine the extent and types of theory of mind impairment in bipolar disorder, and evaluate their relationship to medication and symptoms. We also considered possible mediatory mechanisms, and set out to discover what else could be learnt about the impairment in bipolar disorder by comparison to the profile of impairment in schizophrenia. The literature established that in bipolar disorder (i some form of theory of mind impairment has been observed in all mood states, including euthymia, (ii the form of theory of mind assessed and task used to make the assessment influence the impairment observed, and (iii there might be some relationship to cognitive impairment, although a relationship to standard clinical variables was harder to establish. What also became clear in the literature on bipolar disorder itself was the possible relationship of theory of mind impairment to history of psychotic symptoms. Direct comparative studies including patients with schizophrenia were thus examined, and provided several important directions for future research on the bases of impairment in bipolar disorder. Particularly prominent was the issue of whether theory of mind impairment could be considered a candidate endophenotype for the psychoses, although current evidence suggests this may be premature. The differences in impairment across schizophrenia and bipolar disorder may, however, have genuine differential effects on social functioning and the likely success

  15. Impact of visual impairment on the lives of young adults in the Netherlands: a concept-mapping approach.

    Science.gov (United States)

    Elsman, Ellen Bernadette Maria; van Rens, Gerardus Hermanus Maria Bartholomeus; van Nispen, Ruth Marie Antoinette

    2017-12-01

    While the impact of visual impairments on specific aspects of young adults' lives is well recognised, a systematic understanding of its impact on all life aspects is lacking. This study aims to provide an overview of life aspects affected by visual impairment in young adults (aged 18-25 years) using a concept-mapping approach. Visually impaired young adults (n = 22) and rehabilitation professionals (n = 16) participated in online concept-mapping workshops (brainstorm procedure), to explore how having a visual impairment influences the lives of young adults. Statements were categorised based on similarity and importance. Using multidimensional scaling, concept maps were produced and interpreted. A total of 59 and 260 statements were generated by young adults and professionals, respectively, resulting in 99 individual statements after checking and deduplication. The combined concept map revealed 11 clusters: work, study, information and regulations, social skills, living independently, computer, social relationships, sport and activities, mobility, leisure time, and hobby. The concept maps provided useful insight into activities influenced by visual impairments in young adults, which can be used by rehabilitation centres to improve their services. This might help in goal setting, rehabilitation referral and successful transition to adult life, ultimately increasing participation and quality of life. Implications for rehabilitation Having a visual impairment affects various life-aspects related to participation, including activities related to work, study, social skills and relationships, activities of daily living, leisure time and mobility. Concept-mapping helped to identify the life aspects affected by low vision, and quantify these aspects in terms of importance according to young adults and low vision rehabilitation professionals. Low vision rehabilitation centres should focus on all life aspects found in this study when identifying the needs of young

  16. SATB1 tethers multiple gene loci to reprogram expression profiledriving breast cancer metastasis

    Energy Technology Data Exchange (ETDEWEB)

    Han, Hye-Jung; Kohwi, Yoshinori; Kohwi-Shigematsu, Terumi

    2006-07-13

    Global changes in gene expression occur during tumor progression, as indicated by expression profiling of metastatic tumors. How this occurs is poorly understood. SATB1 functions as a genome organizer by folding chromatin via tethering multiple genomic loci and recruiting chromatin remodeling enzymes to regulate chromatin structure and expression of a large number of genes. Here we show that SATB1 is expressed at high levels in aggressive breast cancer cells, and is undetectable in non-malignant breast epithelial cells. Importantly, RNAi-mediated removal of SATB1 from highly-aggressive MDA-MB-231 cells altered the expression levels of over 1200 genes, restored breast-like acinar polarity in three-dimensional cultures, and prevented the metastastic phenotype in vivo. Conversely, overexpression of SATB1 in the less-aggressive breast cancer cell line Hs578T altered the gene expression profile and increased metastasis dramatically in vivo. Thus, SATB1 is a global regulator of gene expression in breast cancer cells, directly regulating crucial metastasis-associated genes, including ERRB2 (HER2/NEU), TGF-{beta}1, matrix metalloproteinase 3, and metastasin. The identification of SATB1 as a protein that re-programs chromatin organization and transcription profiles to promote breast cancer metastasis suggests a new model for metastasis and may provide means of therapeutic intervention.

  17. Impaired cognitive plasticity and goal-directed control in adolescent obsessive-compulsive disorder.

    Science.gov (United States)

    Gottwald, Julia; de Wit, Sanne; Apergis-Schoute, Annemieke M; Morein-Zamir, Sharon; Kaser, Muzaffer; Cormack, Francesca; Sule, Akeem; Limmer, Winifred; Morris, Anna Conway; Robbins, Trevor W; Sahakian, Barbara J

    2018-01-22

    Youths with obsessive-compulsive disorder (OCD) experience severe distress and impaired functioning at school and at home. Critical cognitive domains for daily functioning and academic success are learning, memory, cognitive flexibility and goal-directed behavioural control. Performance in these important domains among teenagers with OCD was therefore investigated in this study. A total of 36 youths with OCD and 36 healthy comparison subjects completed two memory tasks: Pattern Recognition Memory (PRM) and Paired Associates Learning (PAL); as well as the Intra-Extra Dimensional Set Shift (IED) task to quantitatively gauge learning as well as cognitive flexibility. A subset of 30 participants of each group also completed a Differential-Outcome Effect (DOE) task followed by a Slips-of-Action Task, designed to assess the balance of goal-directed and habitual behavioural control. Adolescent OCD patients showed a significant learning and memory impairment. Compared with healthy comparison subjects, they made more errors on PRM and PAL and in the first stages of IED involving discrimination and reversal learning. Patients were also slower to learn about contingencies in the DOE task and were less sensitive to outcome devaluation, suggesting an impairment in goal-directed control. This study advances the characterization of juvenile OCD. Patients demonstrated impairments in all learning and memory tasks. We also provide the first experimental evidence of impaired goal-directed control and lack of cognitive plasticity early in the development of OCD. The extent to which the impairments in these cognitive domains impact academic performance and symptom development warrants further investigation.

  18. Deletion or Inhibition of the Oxygen Sensor PHD1 Protects against Ischemic Stroke via Reprogramming of Neuronal Metabolism.

    Science.gov (United States)

    Quaegebeur, Annelies; Segura, Inmaculada; Schmieder, Roberta; Verdegem, Dries; Decimo, Ilaria; Bifari, Francesco; Dresselaers, Tom; Eelen, Guy; Ghosh, Debapriva; Davidson, Shawn M; Schoors, Sandra; Broekaert, Dorien; Cruys, Bert; Govaerts, Kristof; De Legher, Carla; Bouché, Ann; Schoonjans, Luc; Ramer, Matt S; Hung, Gene; Bossaert, Goele; Cleveland, Don W; Himmelreich, Uwe; Voets, Thomas; Lemmens, Robin; Bennett, C Frank; Robberecht, Wim; De Bock, Katrien; Dewerchin, Mieke; Ghesquière, Bart; Fendt, Sarah-Maria; Carmeliet, Peter

    2016-02-09

    The oxygen-sensing prolyl hydroxylase domain proteins (PHDs) regulate cellular metabolism, but their role in neuronal metabolism during stroke is unknown. Here we report that PHD1 deficiency provides neuroprotection in a murine model of permanent brain ischemia. This was not due to an increased collateral vessel network. Instead, PHD1(-/-) neurons were protected against oxygen-nutrient deprivation by reprogramming glucose metabolism. Indeed, PHD1(-/-) neurons enhanced glucose flux through the oxidative pentose phosphate pathway by diverting glucose away from glycolysis. As a result, PHD1(-/-) neurons increased their redox buffering capacity to scavenge oxygen radicals in ischemia. Intracerebroventricular injection of PHD1-antisense oligonucleotides reduced the cerebral infarct size and neurological deficits following stroke. These data identify PHD1 as a regulator of neuronal metabolism and a potential therapeutic target in ischemic stroke. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Sezary syndrome after successful treatment of Hodgkin's Disease

    International Nuclear Information System (INIS)

    Buechner, S.A.

    1981-01-01

    A patient had a cutaneous T-cell lymphoma, appearing clinically as Sezary syndrome, that developed two years after successful treatment of Hodgkin's disease with combined radiotherapy and chemotherapy. Clinical, histologic, and electron microscopic observations were made during the course of the Sezary syndrome. The malignant cells in the cell infiltrates and in the peripheral blood were characterized as T cells. There is a possible relationship of the cutaneous T-cell lymphoma to impaired immune surveillance in this patient and to the potential carcinogenicity of combined radiotherapy and chemotherapy

  20. The relationship between impaired driving crashes and beliefs about impaired driving: do residents in high crash rate counties have greater concerns about impaired driving?

    Science.gov (United States)

    Beck, Kenneth H; Yan, Alice F; Wang, Min Qi; Kerns, Timothy J; Burch, Cynthia A

    2009-04-01

    The purpose of this investigation was to examine the relationship between impaired driving crashes and public beliefs and concerns about impaired driving across each of Maryland's twenty-four counties (including Baltimore City). It was hypothesized that residents of counties that experience higher impaired driving crashes would express more concerns about impaired driving and perceive more risks about driving impaired than residents of counties that have lower rates of impaired driving. Data for alcohol impaired driving crashes were obtained for the years 2004-2006. These data were compared to public opinion data that was obtained annually by random-digit-dial telephone surveys from 2004 to 2007. Concerns about drunk driving as well as perceptions of the likelihood of being stopped by the police if one were to drive after having too much to drink were related to counties with higher serious impaired driving crash rates, as were perceptions that the police and the legal system were too lenient. Perceptions about the likelihood of being stopped by the police were higher in those counties with more impaired driving enforcement activity. Perceptions of concern appear to be shaped more by crash exposure than enforcement activity. Campaigns that address impaired driving prevention should substantially increase enforcement, strengthen the adjudication process of impaired drivers, and emphasize the potential seriousness of drinking-driving crashes in their promotional activities.

  1. Direct evidence of impaired neuronal Na/K-ATPase pump function in alternating hemiplegia of childhood.

    Science.gov (United States)

    Simmons, Christine Q; Thompson, Christopher H; Cawthon, Bryan E; Westlake, Grant; Swoboda, Kathryn J; Kiskinis, Evangelos; Ess, Kevin C; George, Alfred L

    2018-03-19

    Mutations in ATP1A3 encoding the catalytic subunit of the Na/K-ATPase expressed in mammalian neurons cause alternating hemiplegia of childhood (AHC) as well as an expanding spectrum of other neurodevelopmental syndromes and neurological phenotypes. Most AHC cases are explained by de novo heterozygous ATP1A3 mutations, but the fundamental molecular and cellular consequences of these mutations in human neurons are not known. In this study, we investigated the electrophysiological properties of neurons generated from AHC patient-specific induced pluripotent stem cells (iPSCs) to ascertain functional disturbances underlying this neurological disease. Fibroblasts derived from two subjects with AHC, a male and a female, both heterozygous for the common ATP1A3 mutation G947R, were reprogrammed to iPSCs. Neuronal differentiation of iPSCs was initiated by neurogenin-2 (NGN2) induction followed by co-culture with mouse glial cells to promote maturation of cortical excitatory neurons. Whole-cell current clamp recording demonstrated that, compared with control iPSC-derived neurons, neurons differentiated from AHC iPSCs exhibited a significantly lower level of ouabain-sensitive outward current ('pump current'). This finding correlated with significantly depolarized potassium equilibrium potential and depolarized resting membrane potential in AHC neurons compared with control neurons. In this cellular model, we also observed a lower evoked action potential firing frequency when neurons were held at their resting potential. However, evoked action potential firing frequencies were not different between AHC and control neurons when the membrane potential was clamped to -80 mV. Impaired neuronal excitability could be explained by lower voltage-gated sodium channel availability at the depolarized membrane potential observed in AHC neurons. Our findings provide direct evidence of impaired neuronal Na/K-ATPase ion transport activity in human AHC neurons and demonstrate the potential

  2. A behavioral rehabilitation intervention for amnestic Mild Cognitive Impairment

    Science.gov (United States)

    Greenaway, Melanie C.; Hanna, Sherrie M.; Lepore, Susan W.; Smith, Glenn E.

    2010-01-01

    Individuals with amnestic Mild Cognitive Impairment (MCI) currently have few treatment options for combating their memory loss. The Memory Support System (MSS) is a calendar and organization system with accompanying 6-week curriculum designed for individuals with progressive memory impairment. Ability to learn the MSS and its utility were assessed in 20 participants. Participants were significantly more likely to successfully use the calendar system after training. Ninety-five percent were compliant with the MSS at training completion, and 89% continued to be compliant at follow-up. Outcome measures revealed a medium effect size for improvement in functional ability. Subjects further reported improved independence, self-confidence, and mood. This initial examination of the MSS suggests that with appropriate training, individuals with amnestic MCI can and will use a memory notebook system to help compensate for memory loss. These results are encouraging that the MSS may help with the symptoms of memory decline in MCI. PMID:18955724

  3. 20 CFR 416.921 - What we mean by a not severe impairment(s) in an adult.

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false What we mean by a not severe impairment(s) in an adult. 416.921 Section 416.921 Employees' Benefits SOCIAL SECURITY ADMINISTRATION SUPPLEMENTAL... Disability § 416.921 What we mean by a not severe impairment(s) in an adult. (a) Non-severe impairment(s). An...

  4. Attitude of Regular and Itinerant Teachers Towards the Inclusion of Hearing Impairment Children

    Directory of Open Access Journals (Sweden)

    Kamal Parhoon

    2014-12-01

    Full Text Available Objectives: Inclusive education is a process of enabling all children to learn and participate effectively within mainstream school systems. It does not segregate children who have different abilities or needs. This article explores the attitudes of regular and itinerant teachers about inclusion of hearing impairment children in their schools in general education. Methods: In a descriptive Survey research design, the sample included 100 teachers (50 regular and 50 itinerant who were selected randomly, according to a multistage sampling method. Data was collected by using questionnaire with 32 questions regarding their attitudes. One-way Analysis of Variance and t-test were performed to obtain between- group comparisons. Results: The results indicated that the teacher's positive attitudes towards inclusive educational system of students with hearing impairment. Significant difference in attitudes was observed, based on the teaching experience, gender, level of teaching. The results also indicate that most teachers are agreeable to the inclusion of students with hearing impairment in their classrooms. Discussion: successful inclusion for hearing impairment children in regular classrooms entails the positive attitudes of Regular and itinerant teachers through a systematic programming within the classroom.

  5. The combined effect of visual impairment and cognitive impairment on disability in older people.

    Science.gov (United States)

    Whitson, Heather E; Cousins, Scott W; Burchett, Bruce M; Hybels, Celia F; Pieper, Carl F; Cohen, Harvey J

    2007-06-01

    To determine the risk of disability in individuals with coexisting visual and cognitive impairment and to compare the magnitude of risk associated with visual impairment, cognitive impairment, or the multimorbidity. Prospective cohort. North Carolina. Three thousand eight hundred seventy-eight participants in the North Carolina Established Populations for the Epidemiologic Studies of the Elderly with nonmissing visual status, cognitive status, and disability status data at baseline Short Portable Mental Status Questionnaire (cognitive impairment defined as > or =4 errors), self reported visual acuity (visual impairment defined as inability to see well enough to recognize a friend across the street or to read newspaper print), demographic and health-related variables, disability status (activities of daily living (ADLs), instrumental activities of daily living (IADLs), mobility), death, and time to nursing home placement. Participants with coexisting visual and cognitive impairment were at greater risk of IADL disability (odds ratio (OR)=6.50, 95% confidence interval (CI)=4.34-9.75), mobility disability (OR=4.04, 95% CI=2.49-6.54), ADL disability (OR=2.84, 95% CI=1.87-4.32), and incident ADL disability (OR=3.66, 95%, CI=2.36-5.65). In each case, the estimated OR associated with the multimorbidity was greater than the estimated OR associated with visual or cognitive impairment alone, a pattern that was not observed for other adverse outcomes assessed. No significant interactions were observed between cognitive impairment and visual impairment as predictors of disability status. Individuals with coexisting visual impairment and cognitive impairment are at high risk of disability, with each condition contributing additively to disability risk. Further study is needed to improve functional trajectories in patients with this prevalent multimorbidity. When visual or cognitive impairment is present, efforts to maximize the other function may be beneficial.

  6. Diet-induced obesity impairs endometrial stromal cell decidualization: a potential role for impaired autophagy.

    Science.gov (United States)

    Rhee, Julie S; Saben, Jessica L; Mayer, Allyson L; Schulte, Maureen B; Asghar, Zeenat; Stephens, Claire; Chi, Maggie M-Y; Moley, Kelle H

    2016-06-01

    What effect does diet-induced obesity have on endometrial stromal cell (ESC) decidualization? Diet-induced obesity impairs ESC decidualization. Decidualization is important for successful implantation and subsequent health of the pregnancy. Compared with normal-weight women, obese women have lower pregnancy rates (both spontaneous and by assisted reproductive technology), higher rates of early pregnancy loss and poorer oocyte quality. Beginning at 6 weeks of age, female C57Bl/6J mice were fed either a high-fat/high-sugar diet (HF/HS; 58% Fat Energy/Sucrose) or a diet of standard mouse chow (CON; 13% Fat) for 12 weeks. At this point, metabolic parameters were measured. Some of the mice (n = 9 HF/HS and 9 CON) were mated with reproductively competent males, and implantation sites were assessed. Other mice (n = 11 HF/HS and 10 CON) were mated with vasectomized males, and artificial decidualization was induced. For in vitro human studies of primary ESCs, endometrial tissue was obtained via biopsy from normo-ovulatory patients without history of infertility (obese = BMI > 30 kg/m(2), n = 11 and lean = BMI treatment with cAMP and medroxyprogesterone. The level of expression of decidualization markers was assessed by RT-qPCR (mRNA) and western blotting (protein). ATP content of ESCs was measured, and levels of autophagy were assessed by western blotting of the autophagy regulators acetyl coa carboxylase (ACC) and ULK1 (Ser 317). Autophagic flux was measured by western blot of the marker LC3b-II. Mice exposed to an HF/HS diet became obese and metabolically impaired. HF/HS-exposed mice mated to reproductively competent males had smaller implantation sites in early pregnancy (P obese women than in those of normal-weight women (Ptreatment abrogated this increase. Many aspects of obesity and metabolic impairment could contribute to the decidualization defects observed in the HF/HS-exposed mice. Although our findings suggest that both autophagy and decidualization are impaired

  7. Extent and neural basis of semantic memory impairment in mild cognitive impairment.

    Science.gov (United States)

    Barbeau, Emmanuel J; Didic, Mira; Joubert, Sven; Guedj, Eric; Koric, Lejla; Felician, Olivier; Ranjeva, Jean-Philippe; Cozzone, Patrick; Ceccaldi, Mathieu

    2012-01-01

    An increasing number of studies indicate that semantic memory is impaired in mild cognitive impairment (MCI). However, the extent and the neural basis of this impairment remain unknown. The aim of the present study was: 1) to evaluate whether all or only a subset of semantic domains are impaired in MCI patients; and 2) to assess the neural substrate of the semantic impairment in MCI patients using voxel-based analysis of MR grey matter density and SPECT perfusion. 29 predominantly amnestic MCI patients and 29 matched control subjects participated in this study. All subjects underwent a full neuropsychological assessment, along with a battery of five tests evaluating different domains of semantic memory. A semantic memory composite Z-score was established on the basis of this battery and was correlated with MRI grey matter density and SPECT perfusion measures. MCI patients were found to have significantly impaired performance across all semantic tasks, in addition to their anterograde memory deficit. Moreover, no temporal gradient was found for famous faces or famous public events and knowledge for the most remote decades was also impaired. Neuroimaging analyses revealed correlations between semantic knowledge and perirhinal/entorhinal areas as well as the anterior hippocampus. Therefore, the deficits in the realm of semantic memory in patients with MCI is more widespread than previously thought and related to dysfunction of brain areas beyond the limbic-diencephalic system involved in episodic memory. The severity of the semantic impairment may indicate a decline of semantic memory that began many years before the patients first consulted.

  8. Congenital hearing impairment

    Energy Technology Data Exchange (ETDEWEB)

    Robson, Caroline D. [Children' s Hospital and Harvard Medical School, Division of Neuroradiology, Department of Radiology, Boston, MA (United States)

    2006-04-15

    Establishing the etiology of congenital hearing impairment can significantly improve treatment for certain causes of hearing loss and facilitates genetic counseling. High-resolution CT and MRI have contributed to the evaluation and management of hearing impairment. In addition, with the identification of innumerable genetic loci and genetic defects involved in hearing loss, genetic testing has emerged as an invaluable tool in the assessment of hearing impairment. Some of the common forms of congenital hearing loss are reviewed and their imaging features illustrated. (orig.)

  9. Congenital hearing impairment

    International Nuclear Information System (INIS)

    Robson, Caroline D.

    2006-01-01

    Establishing the etiology of congenital hearing impairment can significantly improve treatment for certain causes of hearing loss and facilitates genetic counseling. High-resolution CT and MRI have contributed to the evaluation and management of hearing impairment. In addition, with the identification of innumerable genetic loci and genetic defects involved in hearing loss, genetic testing has emerged as an invaluable tool in the assessment of hearing impairment. Some of the common forms of congenital hearing loss are reviewed and their imaging features illustrated. (orig.)

  10. Physical Impairment

    Science.gov (United States)

    Trewin, Shari

    Many health conditions can lead to physical impairments that impact computer and Web access. Musculoskeletal conditions such as arthritis and cumulative trauma disorders can make movement stiff and painful. Movement disorders such as tremor, Parkinsonism and dystonia affect the ability to control movement, or to prevent unwanted movements. Often, the same underlying health condition also has sensory or cognitive effects. People with dexterity impairments may use a standard keyboard and mouse, or any of a wide range of alternative input mechanisms. Examples are given of the diverse ways that specific dexterity impairments and input mechanisms affect the fundamental actions of Web browsing. As the Web becomes increasingly sophisticated, and physically demanding, new access features at the Web browser and page level will be necessary.

  11. Rapid Generation of Human Genetic Loss-of-Function iPSC Lines by Simultaneous Reprogramming and Gene Editing

    Directory of Open Access Journals (Sweden)

    Andrew M. Tidball

    2017-09-01

    Full Text Available Specifically ablating genes in human induced pluripotent stem cells (iPSCs allows for studies of gene function as well as disease mechanisms in disorders caused by loss-of-function (LOF mutations. While techniques exist for engineering such lines, we have developed and rigorously validated a method of simultaneous iPSC reprogramming while generating CRISPR/Cas9-dependent insertions/deletions (indels. This approach allows for the efficient and rapid formation of genetic LOF human disease cell models with isogenic controls. The rate of mutagenized lines was strikingly consistent across experiments targeting four different human epileptic encephalopathy genes and a metabolic enzyme-encoding gene, and was more efficient and consistent than using CRISPR gene editing of established iPSC lines. The ability of our streamlined method to reproducibly generate heterozygous and homozygous LOF iPSC lines with passage-matched isogenic controls in a single step provides for the rapid development of LOF disease models with ideal control lines, even in the absence of patient tissue.

  12. High Current Anxiety Symptoms, But Not a Past Anxiety Disorder Diagnosis, are Associated with Impaired Fear Extinction

    Science.gov (United States)

    Duits, Puck; Cath, Danielle C.; Heitland, Ivo; Baas, Johanna M. P.

    2016-01-01

    Although impaired fear extinction has repeatedly been demonstrated in patients with anxiety disorders, little is known about whether these impairments persist after treatment. The current comparative exploratory study investigated fear extinction in 26 patients treated for their anxiety disorder in the years preceding the study as compared to 17 healthy control subjects. Fear-potentiated startle and subjective fear were measured in a cue and context fear conditioning paradigm within a virtual reality environment. Results indicated no differences in fear extinction between treated anxiety patients and control subjects. However, scores on the Beck Anxiety Inventory across all participants revealed impaired extinction of fear potentiated startle in subjects with high compared to low anxiety symptoms over the past week. Taken together, this exploratory study found no support for impaired fear extinction in treated anxiety patients, and implies that current anxiety symptoms rather than previous patient status determine the success of extinction. PMID:26955364

  13. Adapting for Impaired Patrons.

    Science.gov (United States)

    Schuyler, Michael

    1999-01-01

    Describes how a library, with an MCI Corporation grant, approached the process of setting up computers for the visually impaired. Discusses preparations, which included hiring a visually-impaired user as a consultant and contacting the VIP (Visually Impaired Persons) group; equipment; problems with the graphical user interface; and training.…

  14. Reprogramming of H3K27me3 is critical for acquisition of pluripotency from cultured Arabidopsis tissues.

    Directory of Open Access Journals (Sweden)

    Chongsheng He

    2012-08-01

    Full Text Available In plants, multiple detached tissues are capable of forming a pluripotent cell mass, termed callus, when cultured on media containing appropriate plant hormones. Recent studies demonstrated that callus resembles the root-tip meristem, even if it is derived from aerial organs. This finding improves our understanding of the regeneration process of plant cells; however, the molecular mechanism that guides cells of different tissue types to form a callus still remains elusive. Here, we show that genome-wide reprogramming of histone H3 lysine 27 trimethylation (H3K27me3 is a critical step in the leaf-to-callus transition. The Polycomb Repressive Complex 2 (PRC2 is known to function in establishing H3K27me3. By analyzing callus formation of mutants corresponding to different histone modification pathways, we found that leaf blades and/or cotyledons of the PRC2 mutants curly leaf swinger (clf swn and embryonic flower2 (emf2 were defective in callus formation. We identified the H3K27me3-covered loci in leaves and calli by a ChIP-chip assay, and we found that in the callus H3K27me3 levels decreased first at certain auxin-pathway genes. The levels were then increased at specific leaf genes but decreased at a number of root-regulatory genes. Changes in H3K27me3 levels were negatively correlated with expression levels of the corresponding genes. One possible role of PRC2-mediated H3K27me3 in the leaf-to-callus transition might relate to elimination of leaf features by silencing leaf-regulatory genes, as most leaf-preferentially expressed regulatory genes could not be silenced in the leaf explants of clf swn. In contrast to the leaf explants, the root explants of both clf swn and emf2 formed calli normally, possibly because the root-to-callus transition bypasses the leaf gene silencing process. Furthermore, our data show that PRC2-mediated H3K27me3 and H3K27 demethylation act in parallel in the reprogramming of H3K27me3 during the leaf-to-callus transition

  15. Control of amino acid transport coordinates metabolic reprogramming in T-cell malignancy.

    Science.gov (United States)

    Grzes, K M; Swamy, M; Hukelmann, J L; Emslie, E; Sinclair, L V; Cantrell, D A

    2017-12-01

    This study explores the regulation and importance of System L amino acid transport in a murine model of T-cell acute lymphoblastic leukemia (T-ALL) caused by deletion of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). There has been a strong focus on glucose transport in leukemias but the present data show that primary T-ALL cells have increased transport of multiple nutrients. Specifically, increased leucine transport in T-ALL fuels mammalian target of rapamycin complex 1 (mTORC1) activity which then sustains expression of hypoxia inducible factor-1α (HIF1α) and c-Myc; drivers of glucose metabolism in T cells. A key finding is that PTEN deletion and phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P 3 ) accumulation is insufficient to initiate leucine uptake, mTORC1 activity, HIF1α or c-Myc expression in T cells and hence cannot drive T-ALL metabolic reprogramming. Instead, a key regulator for leucine transport in T-ALL is identified as NOTCH. Mass spectrometry based proteomics identifies SLC7A5 as the predominant amino acid transporter in primary PTEN -/- T-ALL cells. Importantly, expression of SLC7A5 is critical for the malignant transformation induced by PTEN deletion. These data reveal the importance of regulated amino acid transport for T-cell malignancies, highlighting how a single amino acid transporter can have a key role.

  16. DELETION OR INHIBITION OF THE OXYGEN SENSOR PHD1 PROTECTS AGAINST ISCHEMIC STROKE VIA REPROGRAMMING OF NEURONAL METABOLISM

    Science.gov (United States)

    Quaegebeur, Annelies; Segura, Inmaculada; Schmieder, Roberta; Verdegem, Dries; Decimo, Ilaria; Bifari, Francesco; Dresselaers, Tom; Eelen, Guy; Ghosh, Debapriva; Schoors, Sandra; Janaki Raman, Sudha Rani; Cruys, Bert; Govaerts, Kristof; De Legher, Carla; Bouché, Ann; Schoonjans, Luc; Ramer, Matt S.; Hung, Gene; Bossaert, Goele; Cleveland, Don W.; Himmelreich, Uwe; Voets, Thomas; Lemmens, Robin; Bennett, C. Frank; Robberecht, Wim; De Bock, Katrien; Dewerchin, Mieke; Fendt, Sarah-Maria; Ghesquière, Bart; Carmeliet, Peter

    2016-01-01

    Summary The oxygen-sensing prolyl hydroxylase domain proteins (PHDs) regulate cellular metabolism, but their role in neuronal metabolism during stroke is unknown. Here we report that PHD1 deficiency provides neuroprotection in a murine model of permanent brain ischemia. This was not due to an increased collateral vessel network, nor to enhanced neurotrophin expression. Instead, PHD1−/− neurons were protected against oxygen-nutrient deprivation by reprogramming glucose metabolism. Indeed, PHD1−/− neurons enhanced glucose flux through the oxidative pentose phosphate pathway by diverting glucose from glycolysis. As a result, PHD1−/− neurons increased their redox buffering capacity to scavenge oxygen radicals in ischemia. Intracerebroventricular injection of PHD1-antisense oligonucleotides reduced the cerebral infarct size and neurological deficits following stroke. These data identify PHD1 as a novel regulator of neuronal metabolism and a potential therapeutic target in ischemic stroke. PMID:26774962

  17. CD147 reprograms fatty acid metabolism in hepatocellular carcinoma cells through Akt/mTOR/SREBP1c and P38/PPARα pathways.

    Science.gov (United States)

    Li, Jibin; Huang, Qichao; Long, Xiaoyu; Zhang, Jing; Huang, Xiaojun; Aa, Jiye; Yang, Hushan; Chen, Zhinan; Xing, Jinliang

    2015-12-01

    CD147 is a transmembrane glycoprotein which is highly expressed in various human cancers including hepatocellular carcinoma (HCC). A drug Licartin developed with (131)Iodine-labeled antibody against CD147 has been approved by the Chinese Food and Drug Administration (FDA) and enters into clinical use for HCC treatment. Increasing lines of evidence indicate that CD147 is implicated in the metabolism of cancer cells, especially glycolysis. However, the molecular mechanism underlying the relationship between CD147 and aberrant tumor lipid metabolism remains elusive. We systematically investigated the role of CD147 in the regulation of lipid metabolism, including de novo lipogenesis and fatty acid β-oxidation, in HCC cells and explored the underlying molecular mechanisms. Bioinformatic analysis and experimental evidence demonstrated that CD147 significantly contributed to the reprogramming of fatty acid metabolism in HCC cells mainly through two mechanisms. On one hand, CD147 upregulated the expression of sterol regulatory element binding protein 1c (SREBP1c) by activating the Akt/mTOR signaling pathway, which in turn directly activated the transcription of major lipogenic genes FASN and ACC1 to promote de novo lipogenesis. On the other hand, CD147 downregulated peroxisome proliferator-activated receptor alpha (PPARα) and its transcriptional target genes CPT1A and ACOX1 by activating the p38 MAPK signaling pathway to inhibit fatty acid β-oxidation. Moreover, in vitro and in vivo assays indicated that the CD147-mediated reprogramming of fatty acid metabolism played a critical role in the proliferation and metastasis of HCC cells. Our findings demonstrate that CD147 is a critical regulator of fatty acid metabolism, which provides a strong line of evidence for this molecule to be used as a drug target in cancer treatment. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  18. Generation of integration-free induced pluripotent stem cells (GZHMUi001-A by reprogramming peripheral blood mononuclear cells from a 47, XXX syndrome patient

    Directory of Open Access Journals (Sweden)

    Yuchang Chen

    2017-08-01

    Full Text Available 47, XXX syndrome is one of several sex-chromosomal aneuploidies, and it has an incidence of approximately 1/1000 in newborn females. Because of heterogeneity in X-inactivation, these patients may exhibit a variety of clinical symptoms. Here, we report the generation of an integration-free human induced pluripotent stem cell line (GZHMUi001-A by using Sendai virus to reprogram peripheral blood mononuclear cells from a 47, XXX syndrome patient with premature ovarian failure. This 47, XXX iPS cell line has characteristics of pluripotent stem cells and is a useful tool for the investigation of this X chromosome aneuploid disease.

  19. Generation of integration-free induced pluripotent stem cells (GZHMUi001-A) by reprogramming peripheral blood mononuclear cells from a 47, XXX syndrome patient.

    Science.gov (United States)

    Chen, Yuchang; Ou, Zhanhui; Song, Bing; Xian, Yexing; Ouyang, Shuming; Xie, Yuhuan; Xue, Yanting; Sun, Xiaofang

    2017-08-01

    47, XXX syndrome is one of several sex-chromosomal aneuploidies, and it has an incidence of approximately 1/1000 in newborn females. Because of heterogeneity in X-inactivation, these patients may exhibit a variety of clinical symptoms. Here, we report the generation of an integration-free human induced pluripotent stem cell line (GZHMUi001-A) by using Sendai virus to reprogram peripheral blood mononuclear cells from a 47, XXX syndrome patient with premature ovarian failure. This 47, XXX iPS cell line has characteristics of pluripotent stem cells and is a useful tool for the investigation of this X chromosome aneuploid disease. Copyright © 2017. Published by Elsevier B.V.

  20. Initiation of epigenetic reprogramming of the X chromosome in somatic nuclei transplanted to a mouse oocyte.

    Science.gov (United States)

    Bao, Siqin; Miyoshi, Naoki; Okamoto, Ikuhiro; Jenuwein, Thomas; Heard, Edith; Azim Surani, M

    2005-08-01

    The active and inactive X chromosomes have distinct epigenetic marks in somatic nuclei, which undergo reprogramming after transplantation into oocytes. We show that, despite the disappearance of Xist RNA coating in 30 min, the epigenetic memory of the inactive X persists with the precocious appearance of histone H3 trimethylation of lysine 27 (H3-3meK27), without the expected colocalization with Eed/Ezh2. Subsequently, Xist re-appears on the original inactive X, and the silent Xist on the active X undergoes re-activation, resulting in unusual biallelic Xist RNA domains. Despite this abnormal Xist expression pattern, colocalization of H3-3meK27 and Eed is thereafter confined to a single Xist domain, which is presumably on the original inactive X. These epigenetic events differ markedly from the kinetics of preferential paternal X inactivation in normal embryos. All the epigenetic marks on the X are apparently erased in the epiblast, suggesting that the oocyte and epiblast may have distinct properties for stepwise programming of the genome.

  1. Naming of objects, faces and buildings in mild cognitive impairment.

    Science.gov (United States)

    Ahmed, Samrah; Arnold, Robert; Thompson, Sian A; Graham, Kim S; Hodges, John R

    2008-06-01

    Accruing evidence suggests that the cognitive deficits in very early Alzheimer's Disease (AD) are not confined to episodic memory, with a number of studies documenting semantic memory deficits, especially for knowledge of people. To investigate whether this difficulty in naming famous people extends to other proper names based information, three naming tasks - the Graded Naming Test (GNT), which uses objects and animals, the Graded Faces Test (GFT) and the newly designed Graded Buildings Test (GBT) - were administered to 69 participants (32 patients in the early prodromal stage of AD, so-called Mild Cognitive Impairment (MCI), and 37 normal control participants). Patients were found to be impaired on all three tests compared to controls, although naming of objects was significantly better than naming of faces and buildings. Discriminant analysis successfully predicted group membership for 100% controls and 78.1% of patients. The results suggest that even in cases that do not yet fulfil criteria for AD naming of famous people and buildings is impaired, and that both these semantic domains show greater vulnerability than general semantic knowledge. A semantic deficit together with the hallmark episodic deficit may be common in MCI, and that the use of graded tasks tapping semantic memory may be useful for the early identification of patients with MCI.

  2. Insights into the Feelings, Thoughts, and Behaviors of Children with Visual Impairments: A Focus Group Study Prior to Adapting a Cognitive Behavior Therapy-Based Anxiety Intervention

    Science.gov (United States)

    Visagie, Lisa; Loxton, Helene; Stallard, Paul; Silverman, Wendy K.

    2017-01-01

    Introduction: Anxiety is the most common psychological problem reported among children with visual impairments. Although cognitive behavior therapy interventions have proven successful in treating childhood anxiety, it is unclear whether they are suitable and accessible for children who have visual impairments. This study aimed to determine if and…

  3. Improving Access to Accommodations: Reducing Political and Institutional Barriers for Canadian Postsecondary Students with Visual Impairments

    Science.gov (United States)

    Ostrowski, Christopher P.

    2016-01-01

    Challenges to obtaining quality academic accommodations for students with visual impairments in postsecondary education hinder accessibility and the success of such students. The limitations of current policies and practices intended to address the needs of students in Canada are examined and potential solutions are discussed. Further systemic…

  4. Consolidation and reconsolidation are impaired by oral propranolol administered before but not after memory (re)activation in humans.

    Science.gov (United States)

    Thomas, Émilie; Saumier, Daniel; Pitman, Roger K; Tremblay, Jacques; Brunet, Alain

    2017-07-01

    Propranolol administered immediately after learning or after recall has been found to impair memory consolidation or reconsolidation (respectively) in animals, but less reliably so in humans. Since reconsolidation impairment has been proposed as a treatment for mental disorders that have at their core an emotional memory, it is desirable to understand how to reliably reduce the strength of pathogenic memories in humans. We postulated that since humans (unlike experimental animals) typically receive propranolol orally, this introduces a delay before this drug can exert its memory impairment effects, which may render it less effective. As a means to test this, in two double-blind placebo-controlled experiments, we examined the capacity of propranolol to impair consolidation and reconsolidation as a function of timing of ingestion in healthy subjects. In Experiment 1, (n=36), propranolol administered immediately after learning or recall failed to impair the consolidation or reconsolidation of the memory of a standardized slideshow with an accompanying emotional story. In Experiment 2 (n=50), propranolol given 60-75min before learning or recall successfully impaired memory consolidation and reconsolidation. These results suggest that it is possible to achieve reliable memory impairment in humans if propranolol is given before learning or before recall, but not after. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. High current anxiety symptoms, but not a past anxiety disorder diagnosis, are associated with impaired fear extinction.

    Directory of Open Access Journals (Sweden)

    Puck eDuits

    2016-02-01

    Full Text Available Although impaired fear extinction has repeatedly been demonstrated in patients with anxiety disorders, little is known about whether these impairments persist after treatment. The current comparative exploratory study investigated fear extinction in 26 patients treated for their anxiety disorder in the years preceding the study as compared to 17 healthy control subjects. Fear-potentiated startle and subjective fear were measured in a cue and context fear conditioning paradigm within a virtual reality environment. Results indicated no differences in fear extinction between treated anxiety patients and control subjects. However, scores on the Beck Anxiety Inventory across all participants revealed impaired extinction of fear potentiated startle in subjects with high compared to low anxiety symptoms over the past week. Taken together, this exploratory study found no support for impaired fear extinction in treated anxiety patients, and implies that current anxiety symptoms rather than previous patient status determine the success of extinction.

  6. Cloning from stem cells: different lineages, different species, same story.

    Science.gov (United States)

    Oback, Björn

    2009-01-01

    Following nuclear transfer (NT), the most stringent measure of extensive donor cell reprogramming is development into viable offspring. This is referred to as cloning efficiency and quantified as the proportion of cloned embryos transferred into surrogate mothers that survive into adulthood. Cloning efficiency depends on the ability of the enucleated recipient cell to carry out the reprogramming reactions ('reprogramming ability') and the ability of the nuclear donor cell to be reprogrammed ('reprogrammability'). It has been postulated that reprogrammability of the somatic donor cell epigenome is inversely proportional to its differentiation status. In order to test this hypothesis, reprogrammability was compared between undifferentiated stem cells and their differentiated isogenic progeny. In the mouse, cells of divergent differentiation status from the neuronal, haematopoietic and skin epithelial lineage were tested. In cattle and deer, skeletal muscle and antler cells, respectively, were used as donors. No conclusive correlation between differentiation status and cloning efficiency was found, indicating that somatic donor cell type may not be the limiting factor for cloning success. This may reflect technical limitations of the NT-induced reprogramming assay. Alternatively, differentiation status and reprogrammability may be unrelated, making all cells equally difficult to reprogramme once they have left the ground state of pluripotency.

  7. Nuclear delivery of recombinant OCT4 by chitosan nanoparticles for transgene-free generation of protein-induced pluripotent stem cells.

    Science.gov (United States)

    Tammam, Salma; Malak, Peter; Correa, Daphne; Rothfuss, Oliver; Azzazy, Hassan M E; Lamprecht, Alf; Schulze-Osthoff, Klaus

    2016-06-21

    Protein-based reprogramming of somatic cells is a non-genetic approach for the generation of induced pluripotent stem cells (iPSCs), whereby reprogramming factors, such as OCT4, SOX2, KLF4 and c-MYC, are delivered as functional proteins. The technique is considered safer than transgenic methods, but, unfortunately, most protein-based protocols provide very low reprogramming efficiencies. In this study, we developed exemplarily a nanoparticle (NP)-based delivery system for the reprogramming factor OCT4. To this end, we expressed human OCT4 in Sf9 insect cells using a baculoviral expression system. Recombinant OCT4 showed nuclear localization in Sf9 cells indicating proper protein folding. In comparison to soluble OCT4 protein, encapsulation of OCT4 in nuclear-targeted chitosan NPs strongly stabilized its DNA-binding activity even under cell culture conditions. OCT4-loaded NPs enabled cell treatment with high micromolar concentrations of OCT4 and successfully delivered active OCT4 into human fibroblasts. Chitosan NPs therefore provide a promising tool for the generation of transgene-free iPSCs.

  8. Effects of acute alcohol intoxication on automated processing: evidence from the double-step paradigm.

    Science.gov (United States)

    Vorstius, Christian; Radach, Ralph; Lang, Alan R

    2012-02-01

    Reflexive and voluntary levels of processing have been studied extensively with respect to possible impairments due to alcohol intoxication. This study examined alcohol effects at the 'automated' level of processing essential to many complex visual processing tasks (e.g., reading, visual search) that involve ongoing modifications or reprogramming of well-practiced routines. Data from 30 participants (16 male) were collected in two counterbalanced sessions (alcohol vs. no-alcohol control; mean breath alcohol concentration = 68 mg/dL vs. 0 mg/dL). Eye movements were recorded during a double-step task where 75% of trials involved two target stimuli in rapid succession (inter-stimulus interval [ISI]=40, 70, or 100 ms) so that they could elicit two distinct saccades or eye movements (double steps). On 25% of trials a single target appeared. Results indicated that saccade latencies were longer under alcohol. In addition, the proportion of single-step responses and the mean saccade amplitude (length) of primary saccades decreased significantly with increasing ISI. The key novel finding, however, was that the reprogramming time needed to cancel the first saccade and adjust saccade amplitude was extended significantly by alcohol. The additional time made available by prolonged latencies due to alcohol was not utilized by the saccade programming system to decrease the number of two-step responses. These results represent the first demonstration of specific alcohol-induced programming deficits at the automated level of oculomotor processing.

  9. Work stressors and impaired sleep: rumination as a mediator.

    Science.gov (United States)

    Berset, Martial; Elfering, Achim; Lüthy, Stefan; Lüthi, Simon; Semmer, Norbert K

    2011-04-01

    An association between stress at work and impaired sleep is theoretically plausible and supported by empirical evidence. The current study's main aim was to investigate how the influence of stressors is carried over into the evening and the night. We assume that this relationship is mediated by perseverative cognitions. We tested this assumption in two cross-sectional samples with structural equation modeling, using bootstrapped standard errors to test for significance. Effort–reward imbalance and time pressure were used as stressors, and rumination as a measure for perseverative cognitions. Results show that the stressors are related to perseverative cognitions, and these are related to impaired sleep in both samples. Indirect effects are significant in both samples. With rumination controlled, direct effects of stressors on sleep are only significant in one out of four cases. Thus, there is full mediation in three out of four cases, and partial mediation in the fourth one. Our results underscore the notion that perseverative cognitions are crucial for transferring negative effects of work stressors into private life, including sleep, thus hindering individuals to successfully recover.

  10. The role of motivation for treatment success.

    Science.gov (United States)

    Medalia, Alice; Saperstein, Alice

    2011-09-01

    Learning during skills-based psychosocial treatments for schizophrenia is influenced by the motivating properties of the treatment context and the motivational orientation of the client. Given that motivational impairment is a core feature of schizophrenia with significant functional implications, intervention strategies emphasizing extrinsic and/or intrinsic goals may be prescribed to enhance skill learning and treatment outcomes. The purpose of this article is to consider the role that motivation plays in treatment success by evaluating the relationship between motivation and learning during cognitive remediation for schizophrenia. As intrinsic motivation (IM) is most often associated with learning, we will integrate research findings which address 3 main questions: (1) is IM in schizophrenia static or dynamic, (2) is it possible to manipulate the state of being intrinsically motivated and if so do manipulations of IM affect learning? and (3) can motivation theory be translated into clinical practice? This knowledge can facilitate treatment strategies to address the low base rate of IM that is characteristic of schizophrenia and can be applied to cognitive remediation as well as other psychosocial interventions which require learning for treatment success.

  11. Dependence and caregiver burden in Alzheimer's disease and mild cognitive impairment.

    LENUS (Irish Health Repository)

    Gallagher, Damien

    2011-03-01

    The dependence scale has been designed to be sensitive to the overall care needs of the patient and is considered distinct from standard measures of functional ability in this regard. Little is known regarding the relationship between patient dependence and caregiver burden. We recruited 100 patients with Alzheimer\\'s disease or mild cognitive impairment and their caregivers through a memory clinic. Patient function, dependence, hours of care, cognition, neuropsychiatric symptoms, and caregiver burden were assessed. Dependence was significantly correlated with caregiver burden. Functional decline and dependence were most predictive of caregiver burden in patients with mild impairment while behavioral symptoms were most predictive in patients with moderate to severe disease. The dependence scale demonstrated good utility as a predictor of caregiver burden. Interventions to reduce caregiver burden should address patient dependence, functional decline, and behavioral symptoms while successful management of the latter becomes more critical with disease progression.

  12. Impaired implicit learning and feedback processing after stroke.

    Science.gov (United States)

    Lam, J M; Globas, C; Hosp, J A; Karnath, H-O; Wächter, T; Luft, A R

    2016-02-09

    The ability to learn is assumed to support successful recovery and rehabilitation therapy after stroke. Hence, learning impairments may reduce the recovery potential. Here, the hypothesis is tested that stroke survivors have deficits in feedback-driven implicit learning. Stroke survivors (n=30) and healthy age-matched control subjects (n=21) learned a probabilistic classification task with brain activation measured using functional magnetic resonance imaging in a subset of these individuals (17 stroke and 10 controls). Stroke subjects learned slower than controls to classify cues. After being rewarded with a smiley face, they were less likely to give the same response when the cue was repeated. Stroke subjects showed reduced brain activation in putamen, pallidum, thalamus, frontal and prefrontal cortices and cerebellum when compared with controls. Lesion analysis identified those stroke survivors as learning-impaired who had lesions in frontal areas, putamen, thalamus, caudate and insula. Lesion laterality had no effect on learning efficacy or brain activation. These findings suggest that stroke survivors have deficits in reinforcement learning that may be related to dysfunctional processing of feedback-based decision-making, reward signals and working memory. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  13. Post-stroke cognitive impairments

    Directory of Open Access Journals (Sweden)

    Elena Anatolyevna Katunina

    2013-01-01

    Full Text Available Post-stroke cognitive impairments are common effects of stroke. Vascular cognitive impairments are characterized by the heterogeneity of the neuropsychological profile in relation to the site and pattern of stroke. Their common trait is the presence of dysregulation secondary to frontal dysfunction. The treatment of vascular cognitive impairments should be multimodality and aimed at stimulating neuroplasticity processes, restoring neurotransmitter imbalance, and preventing recurrent vascular episodes.

  14. Characteristics of reading and understanding of hearing impaired students in classes VI-VIII

    Directory of Open Access Journals (Sweden)

    Mustaf Morina

    2015-03-01

    Full Text Available Good reading has a very important role in the development of children with hearing impaired; also reading in explicit way is one of the crucial factors which affect the oral language development of children with hearing impaired. The best form and possibility of improvement, development of oral language, development of communicating, receipt of information, knowledge, and ideas over the world, is reading. When the auditory perception is damaged reading is poor. Hearing impairment causes a lot of problems in the development of personality of children with hearing impairment in these fields: poor development of vocabulary, poor quality of lexica, poor quality of sentences, and disorder in articulation. The purpose of this research is to verify the following: 1-Speed of reading of hearing impaired children, 2-The number of errors, 3-The kind of errors, 4-To understand the text in the context of the degree of hearing impairment, age (class, success in school and gender. This theoretical-experimental study was made with students from two schools; special school “Mother Teresa” in Prizren and Primary School “Elena Gjika” in Prishtina (class attached. The research included a total of 32 students (respondent 27 students (respondent from special schools “Mother Teresa” in Prizren and 5 elementary school students “Elena Gjika” Prishtina, all these students are with hearing impairment. From 32 students involved in the research, 23 were male and 9 female. The research was done by applying a text fables “The fox and the raven” watched and analyzed in terms of three dimensions. The research results have shown that students with hearing impairments have considerable problems in many aspects; in terms of speed of reading, students with hearing impairment have stagnated compared with their peers in the ratio 8/1. In terms of reading errors have stagnated considered being incomparable. In terms of understanding the text students with hearing

  15. CD147: a small molecule transporter ancillary protein at the crossroad of multiple hallmarks of cancer and metabolic reprogramming.

    Science.gov (United States)

    Kendrick, Agnieszka A; Schafer, Johnathon; Dzieciatkowska, Monika; Nemkov, Travis; D'Alessandro, Angelo; Neelakantan, Deepika; Ford, Heide L; Pearson, Chad G; Weekes, Colin D; Hansen, Kirk C; Eisenmesser, Elan Z

    2017-01-24

    Increased expression of CD147 in pancreatic cancer has been proposed to play a critical role in cancer progression via CD147 chaperone function for lactate monocarboxylate transporters (MCTs). Here, we show for the first time that CD147 interacts with membrane transporters beyond MCTs and exhibits a protective role for several of its interacting partners. CD147 prevents its interacting partner's proteasome-dependent degradation and incorrect plasma membrane localization through the CD147 transmembrane (TM) region. The interactions with transmembrane small molecule and ion transporters identified here indicate a central role of CD147 in pancreatic cancer metabolic reprogramming, particularly with respect to amino acid anabolism and calcium signaling. Importantly, CD147 genetic ablation prevents pancreatic cancer cell proliferation and tumor growth in vitro and in vivo in conjunction with metabolic rewiring towards amino acid anabolism, thus paving the way for future combined pharmacological treatments.

  16. Centrosome Dysfunction Contributes To Chromosome Instability, Chromoanagenesis And Genome Reprograming In Cancer.

    Directory of Open Access Journals (Sweden)

    German A Pihan

    2013-11-01

    Full Text Available The unique ability of centrosomes to nucleate and organize microtubules makes them unrivaled conductors of important interphase processes, such as intracellular payload traffic, cell polarity, cell locomotion, and organization of the immunologic synapse. But it is in mitosis that centrosomes loom large, for they orchestrate, with clockmaker’s precision, the assembly and functioning of the mitotic spindle, ensuring the equal partitioning of the replicated genome into daughter cells. Centrosome dysfunction is inextricably linked to aneuploidy and chromosome instability, both hallmarks of cancer cells. Several aspects of centrosome function in normal and cancer cells have been molecularly characterized during the last two decades, greatly enhancing our mechanistic understanding of this tiny organelle. Whether centrosome defects alone can cause cancer, remains unanswered. Until recently, the aggregate of the evidence had suggested that centrosome dysfunction, by deregulating the fidelity of chromosome segregation, promotes and accelerates the characteristic Darwinian evolution of the cancer genome enabled by increased mutational load and/or decreased DNA repair. Very recent experimental work has shown that missegreated chromosomes resulting from centrosome dysfunction may experience extensive DNA damage, suggesting additional dimensions to the role of centrosomes in cancer. Centrosome dysfunction is particularly prevalent in tumors in which the genome has undergone extensive structural rearrangements and chromosome domain reshuffling. Ongoing gene reshuffling reprograms the genome for continuous growth, survival, and evasion of the immune system. Manipulation of molecular networks controlling centrosome function may soon become a viable target for specific therapeutic intervention in cancer, particularly since normal cells, which lack centrosome alterations, may be spared the toxicity of such therapies.

  17. PARIS reprograms glucose metabolism by HIF-1α induction in dopaminergic neurodegeneration.

    Science.gov (United States)

    Kang, Hojin; Jo, Areum; Kim, Hyein; Khang, Rin; Lee, Ji-Yeong; Kim, Hanna; Park, Chi-Hu; Choi, Jeong-Yun; Lee, Yunjong; Shin, Joo-Ho

    2018-01-22

    Our previous study found that PARIS (ZNF746) transcriptionally suppressed transketolase (TKT), a key enzyme in pentose phosphate pathway (PPP) in the substantia nigra (SN) of AAV-PARIS injected mice. In this study, we revealed that PARIS overexpression reprogrammed glucose metabolic pathway, leading to the increment of glycolytic proteins along with TKT reduction in the SN of AAV-PARIS injected mice. Knock-down of TKT in differentiated SH-SY5Y cells led to an increase of glycolytic enzymes and decrease of PPP-related enzymes whereas overexpression of TKT restored PARIS-mediated glucose metabolic shift, suggesting that glucose metabolic alteration by PARIS is TKT-dependent. Inhibition of PPP by either PARIS overexpression or TKT knock-down elevated the level of H 2 O 2 , and diminished NADPH and GSH levels, ultimately triggering the induction of HIF-1α, a master activator of glycolysis. In addition, TKT inhibition by stereotaxic injection of oxythiamine demonstrated slight decrement of dopaminergic neurons (DNs) in SN but not cortical neurons in the cortex, suggesting that TKT might be a survival factor of DNs. In differentiated SH-SY5Y, cell toxicity by GFP-PARIS was partially restored by introduction of Flag-TKT and siRNA-HIF-1α. We also observed the increase of HIF-1α and glycolytic hexokinase 2 in the SN of Parkinson's disease patients. Taken together, these results suggest that PARIS accumulation might distort the balance of glucose metabolism, providing clues for understanding mechanism underlying selective DNs death by PARIS. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Mild Cognitive Impairment (MCI)

    Science.gov (United States)

    Mild cognitive impairment (MCI) Overview Mild cognitive impairment (MCI) is an intermediate stage between the expected cognitive decline of normal aging and the more-serious decline of dementia. It ...

  19. An index of reservoir habitat impairment

    Science.gov (United States)

    Miranda, L.E.; Hunt, K.M.

    2011-01-01

    Fish habitat impairment resulting from natural and anthropogenic watershed and in-lake processes has in many cases reduced the ability of reservoirs to sustain native fish assemblages and fisheries quality. Rehabilitation of impaired reservoirs is hindered by the lack of a method suitable for scoring impairment status. To address this limitation, an index of reservoir habitat impairment (IRHI) was developed by merging 14 metrics descriptive of common impairment sources, with each metric scored from 0 (no impairment) to 5 (high impairment) by fisheries scientists with local knowledge. With a plausible range of 5 to 25, distribution of the IRHI scores ranged from 5 to 23 over 482 randomly selected reservoirs dispersed throughout the USA. The IRHI reflected five impairment factors including siltation, structural habitat, eutrophication, water regime, and aquatic plants. The factors were weakly related to key reservoir characteristics including reservoir area, depth, age, and usetype, suggesting that common reservoir descriptors are poor predictors of fish habitat impairment. The IRHI is rapid and inexpensive to calculate, provides an easily understood measure of the overall habitat impairment, allows comparison of reservoirs and therefore prioritization of restoration activities, and may be used to track restoration progress. The major limitation of the IRHI is its reliance on unstandardized professional judgment rather than standardized empirical measurements. ?? 2010 US Government.

  20. Overexpression of HvIcy6 in Barley Enhances Resistance against Tetranychus urticae and Entails Partial Transcriptomic Reprogramming

    Directory of Open Access Journals (Sweden)

    M. Estrella Santamaria

    2018-03-01

    Full Text Available Cystatins have been largely used for pest control against phytophagous species. However, cystatins have not been commonly overexpressed in its cognate plant species to test their pesticide capacity. Since the inhibitory role of barley HvCPI-6 cystatin against the phytophagous mite Tetranychus urticae has been previously demonstrated, the purpose of our study was to determine if barley transgenic lines overexpressing its own HvIcy6 gene were more resistant against this phytophagous infestation. Besides, a transcriptomic analysis was done to find differential expressed genes among wild-type and transformed barley plants. Barley plants overexpressing HvIcy6 cystatin gene remained less susceptible to T. urticae attack when compared to wild-type plants, with a significant lesser foliar damaged area and a lower presence of the mite. Transcriptomic analysis revealed a certain reprogramming of cellular metabolism and a lower expression of several genes related to photosynthetic activity. Therefore, although caution should be taken to discard potential deleterious pleiotropic effects, cystatins may be used as transgenes with impact on agricultural crops by conferring enhanced levels of resistance to phytophagous pests.

  1. Assisting the Blind and Visually Impaired: Guidelines for Eye Health Workers and Other Helpers

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    Sue Stevens

    2003-01-01

    Full Text Available As eye health workers, we give much attention to learning and teaching the importance of health education and the prevention and treatment of eye disease. Despite our gained knowledge, sadly, our efforts are not always successful and we are presented with the responsibility and challenge of caring for people who have to cope with visual impairment, perhaps for the rest of their lives. We have to understand their difficulties, recognise their abilities and learn how to cooperate and communicate with them in a social as well as hospital environment. It is often within the eye hospital itself that the lack in education of health workers and their understanding of the assistance needs of blind and visually impaired patients is all too evident.

  2. Ploidy-dependent changes in the epigenome of symbiotic cells correlate with specific patterns of gene expression

    KAUST Repository

    Nagymihá ly, Marianna; Veluchamy, Alaguraj; Gyö rgypá l, Zoltá n; Ariel, Federico; Jé gu, Teddy; Benhamed, Moussa; Szűcs, Attila; Kereszt, Attila; Mergaert, Peter; Kondorosi, É va

    2017-01-01

    The formation of symbiotic nodule cells in Medicago truncatula is driven by successive endoreduplication cycles and transcriptional reprogramming in different temporal waves including the activation of more than 600 cysteine-rich NCR genes expressed

  3. Generation of induced pluripotent stem cells (iPSCs from a Bernard–Soulier syndrome patient carrying a W71R mutation in the GPIX gene

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    Lourdes Lopez-Onieva

    2016-05-01

    Full Text Available We generated an induced pluripotent stem cell (iPSC line from a Bernard–Soulier Syndrome (BSS patient carrying the mutation p.Trp71Arg in the GPIX locus (BSS1-PBMC-iPS4F4. Peripheral blood mononuclear cells (PBMCs were reprogrammed using heat sensitive non-integrative Sendai viruses containing the reprogramming factors Oct3/4, SOX2, KLF4 and c-MYC. Successful silencing of the exogenous reprogramming factors was checked by RT-PCR. Characterization of BSS1-PBMC-iPS4F4 included mutation analysis of GPIX locus, Short Tandem Repeats (STR profiling, alkaline phosphatase enzymatic activity, analysis of conventional pluripotency-associated factors at mRNA and protein level and in vivo differentiation studies. BSS1-PBMC-iPS4F4 will provide a powerful tool to study BSS.

  4. Patterns of Semantic Memory Impairment in Mild Cognitive Impairment

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    Sven Joubert

    2008-01-01

    Full Text Available Although the semantic memory impairment has been largely documented in Alzheimer's disease, little is known about semantic memory in the preclinical phase of the disease (Mild Cognitive Impairment. The purpose of this study was to document the nature of semantic breakdown using a battery of tests assessing different aspects of conceptual knowledge: knowledge about common objects, famous people and famous public events. Results indicate that all domains of semantic memory were impaired in MCI individuals but knowledge about famous people and famous events was affected to a greater extent than knowledge about objects. This pattern of results suggests that conceptual entities with distinctive and unique properties may be more prone to semantic breakdown in MCI. In summary, results of this study support the view that genuine semantic deficits are present in MCI. It could be useful to investigate the etiological outcome of patients failing or succeeding at such tests.

  5. Nasal Colivelin treatment ameliorates memory impairment related to Alzheimer's disease.

    Science.gov (United States)

    Yamada, Marina; Chiba, Tomohiro; Sasabe, Jumpei; Terashita, Kenzo; Aiso, Sadakazu; Matsuoka, Masaaki

    2008-07-01

    Humanin (HN) and its derivatives, such as Colivelin (CLN), suppress neuronal death induced by insults related to Alzheimer's disease (AD) by activating STAT3 in vitro. They also ameliorate functional memory impairment of mice induced by anticholinergic drugs or soluble toxic amyloid-beta (Abeta) in vivo when either is directly administered into the cerebral ventricle or intraperitoneally injected. However, the mechanism underlying the in vivo effect remains uncharacterized. In addition, from the standpoint of clinical application, drug delivery methods that are less invasive and specific to the central nervous system (CNS) should be developed. In this study, we show that intranasally (i.n.) administered CLN can be successfully transferred to CNS via the olfactory bulb. Using several behavioral tests, we have demonstrated that i.n. administered CLN ameliorates memory impairment of AD models in a dose-responsive manner. Attenuation of AD-related memory impairment by HN derivatives such as CLN appears to be correlated with an increase in STAT3 phosphorylation levels in the septohippocampal region, suggesting that anti-AD activities of HN derivatives may be mediated by activation of STAT3 in vivo as they are in vitro. We further demonstrate that CLN treatment inhibits an Abeta induced decrease in the number of choline acetyltransferase (ChAT)-positive neurons in the medial septum. Combined with the finding that HN derivatives upregulate mRNA expression of neuronal ChAT and vesicular acetylcholine transporter (VAChT) in vitro, it is assumed that CLN may ameliorate memory impairment of AD models by supporting cholinergic neurotransmission, which is at least partly mediated by STAT3-mediated transcriptional upregulation of ChAT and VAChT.

  6. Arterial stiffness and cognitive impairment.

    Science.gov (United States)

    Li, Xiaoxuan; Lyu, Peiyuan; Ren, Yanyan; An, Jin; Dong, Yanhong

    2017-09-15

    Arterial stiffness is one of the earliest indicators of changes in vascular wall structure and function and may be assessed using various indicators, such as pulse-wave velocity (PWV), the cardio-ankle vascular index (CAVI), the ankle-brachial index (ABI), pulse pressure (PP), the augmentation index (AI), flow-mediated dilation (FMD), carotid intima media thickness (IMT) and arterial stiffness index-β. Arterial stiffness is generally considered an independent predictor of cardiovascular and cerebrovascular diseases. To date, a significant number of studies have focused on the relationship between arterial stiffness and cognitive impairment. To investigate the relationships between specific arterial stiffness parameters and cognitive impairment, elucidate the pathophysiological mechanisms underlying the relationship between arterial stiffness and cognitive impairment and determine how to interfere with arterial stiffness to prevent cognitive impairment, we searched PUBMED for studies regarding the relationship between arterial stiffness and cognitive impairment that were published from 2000 to 2017. We used the following key words in our search: "arterial stiffness and cognitive impairment" and "arterial stiffness and cognitive impairment mechanism". Studies involving human subjects older than 30years were included in the review, while irrelevant studies (i.e., studies involving subjects with comorbid kidney disease, diabetes and cardiac disease) were excluded from the review. We determined that arterial stiffness severity was positively correlated with cognitive impairment. Of the markers used to assess arterial stiffness, a higher PWV, CAVI, AI, IMT and index-β and a lower ABI and FMD were related to cognitive impairment. However, the relationship between PP and cognitive impairment remained controversial. The potential mechanisms linking arterial stiffness and cognitive impairment may be associated with arterial pulsatility, as greater arterial pulsatility

  7. Assessment of Hearing Impaired Youth.

    Science.gov (United States)

    Hicks, Doin E., Ed.; And Others

    1980-01-01

    The issue of Directions contains 11 articles on assessment of hearing impaired individuals. Entries have the following titles and authors: "Classroom Assessment Techniques for Hearing Impaired Students--A Literature Review" (B. McKee, M. Hausknecht); "Informal Assessment of Hearing Impaired Students In the Classroom" (B. Culhane, R. Hein);…

  8. Reproductive success and contaminant associations in tree swallows (Tachycineta bicolor) used to assess a Beneficial Use Impairment in U.S. and Binational Great Lakes’ Areas of Concern

    Science.gov (United States)

    Custer, Christine M.; Custer, Thomas W.; Etterson, Matthew A.; Dummer, Paul; Goldberg, Diana R.; Franson, J. Christian

    2018-01-01

    During 2010-2014, tree swallow (Tachycineta bicolor) reproductive success was monitored at 68 sites across all 5 Great Lakes, including 58 sites located within Great Lakes Areas of Concern (AOCs) and 10 non-AOCs. Sample eggs were collected from tree swallow clutches and analyzed for contaminants including polychlorinated biphenyls (PCBs), dioxins and furans, polybrominated diphenyl ethers, and 34 other organic compounds. Contaminant data were available for 360 of the clutches monitored. Markov chain multistate modeling was used to assess the importance of 5 ecological variables and 11 of the dominant contaminants in explaining the pattern of egg and nestling failure rates. Four of 5 ecological variables (Female Age, Date within season, Year, and Site) were important explanatory variables. Of the 11 contaminants, only total dioxin and furan toxic equivalents (TEQs) explained a significant amount of the egg failure probabilities. Neither total PCBs nor PCB TEQs explained the variation in egg failure rates. In a separate analysis, polycyclic aromatic hydrocarbon exposure in nestling diet, used as a proxy for female diet during egg laying, was significantly correlated with the daily probability of egg failure. The 8 sites within AOCs which had poorer reproduction when compared to 10 non-AOC sites, the measure of impaired reproduction as defined by the Great Lakes Restoration Initiative, were associated with exposure to dioxins and furan TEQs, PAHs, or depredation. Only 2 sites had poorer reproduction than the poorest performing non-AOC. Using a classic (non-modeling) approach to estimating reproductive success, 82% of nests hatched at least 1 egg, and 75% of eggs laid, excluding those collected for contaminant analyses, hatched.

  9. Metabolic Reprogramming During Purine Stress in the Protozoan Pathogen Leishmania donovani

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Jessica L.; Yates, Phillip A.; Soysa, Radika; Alfaro, Joshua F.; Yang, Feng; Burnum-Johnson, Kristin E.; Petyuk, Vladislav A.; Weitz, Karl K.; Camp, David G.; Smith, Richard D.; Wilmarth, Phillip A.; David, Larry L.; Ramasamy, Gowthaman; Myler, Peter J.; Carter, Nicola S.

    2014-02-27

    The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over 3 months, indicating that the response to purine starvation is robust and engenders parasite survival under conditions of extreme scarcity. To understand metabolic reprogramming during purine starvation we have employed global approaches. Whole proteome comparisons between purine-starved and purine-replete parasites over a 6-48 h span have revealed a temporal and coordinated response to purine starvation. Purine transporters and enzymes involved in acquisition at the cell surface are upregulated within a few hours of purine removal from the media, while other key purine salvage components are upregulated later in the time-course and more modestly. After 48 h, the proteome of purine-starved parasites is extensively remodeled and adaptations to purine stress appear tailored to deal with both purine deprivation and general stress. To probe the molecular mechanisms affecting proteome remodeling in response to purine starvation, comparative RNA-seq analyses, qRT-PCR, and luciferase reporter assays were performed on purine-starved versus purine-replete parasites. While the regulation of a minority of proteins tracked with changes at the mRNA level, for many regulated proteins it appears that proteome remodeling during purine stress occurs primarily via translational and/or post-translational mechanisms.

  10. Doxycycline Impairs Mitochondrial Function and Protects Human Glioma Cells from Hypoxia-Induced Cell Death: Implications of Using Tet-Inducible Systems.

    Science.gov (United States)

    Luger, Anna-Luisa; Sauer, Benedikt; Lorenz, Nadja I; Engel, Anna L; Braun, Yannick; Voss, Martin; Harter, Patrick N; Steinbach, Joachim P; Ronellenfitsch, Michael W

    2018-05-17

    Inducible gene expression is an important tool in molecular biology research to study protein function. Most frequently, the antibiotic doxycycline is used for regulation of so-called tetracycline (Tet)-inducible systems. In contrast to stable gene overexpression, these systems allow investigation of acute and reversible effects of cellular protein induction. Recent reports have already called for caution when using Tet-inducible systems as the employed antibiotics can disturb mitochondrial function and alter cellular metabolism by interfering with mitochondrial translation. Reprogramming of energy metabolism has lately been recognized as an important emerging hallmark of cancer and is a central focus of cancer research. Therefore, the scope of this study was to systematically analyze dose-dependent metabolic effects of doxycycline on a panel of glioma cell lines with concomitant monitoring of gene expression from Tet-inducible systems. We report that doxycycline doses commonly used with inducible expression systems (0.01⁻1 µg/mL) substantially alter cellular metabolism: Mitochondrial protein synthesis was inhibited accompanied by reduced oxygen and increased glucose consumption. Furthermore, doxycycline protected human glioma cells from hypoxia-induced cell death. An impairment of cell growth was only detectable with higher doxycycline doses (10 µg/mL). Our findings describe settings where doxycycline exerts effects on eukaryotic cellular metabolism, limiting the employment of Tet-inducible systems.

  11. Students with hearing impairment at a South African university: Self-identity and disclosure

    Science.gov (United States)

    2016-01-01

    Background A growing number of students with hearing loss are being granted access to higher education in South Africa due to the adoption of inclusive educational policies. However, available statistics indicate that participation by students with hearing impairments in higher education remains low and research suggests that support provisioning for those who do gain access is inadequate. Objectives This article aims to illustrate that the assumed self-identity of students with hearing impairment influences their choice to disclose their disability. The choice not to disclose their hearing loss prevents them from accessing the necessary reasonable accommodations and this in turn may affect their eventual educational success. Method Reported here is a qualitative descriptive case study at a South African university. Purposive sampling methods were employed. Data were gathered from in-depth interviews with seven students with hearing impairment ranging from moderate to profound, using spoken language. Constructivist grounded theory was used as an approach to the process of generating and transforming the data, as well as the construction of theory. Findings All the student participants identified as having a hearing rather than a D/deaf identity cultural paradigm and viewed themselves as ‘normal’. Linked to this was their unwillingness to disclose their hearing impairment and thus access support. Conclusion It is crucially important for academic, support and administrative staff to be aware of both the assumed ‘hearing’ identity and therefore subsequent non-disclosure practices of students with a hearing impairment using the oral method of communication. Universities need to put measures in place to encourage students to voluntarily disclose their hearing impairment in order to provide more targeted teaching and learning support. This could lead to improved educational outcomes for students. PMID:28730053

  12. Repair of dense connective tissues via biomaterial-mediated matrix reprogramming of the wound interface.

    Science.gov (United States)

    Qu, Feini; Pintauro, Michael P; Haughan, Joanne E; Henning, Elizabeth A; Esterhai, John L; Schaer, Thomas P; Mauck, Robert L; Fisher, Matthew B

    2015-01-01

    Repair of dense connective tissues in adults is limited by their intrinsic hypocellularity and is exacerbated by a dense extracellular matrix (ECM) that impedes cellular migration to and local proliferation at the wound site. Conversely, healing in fetal tissues occurs due in part to an environment conducive to cell mobility and division. Here, we investigated whether the application of a degradative enzyme, collagenase, could reprogram the adult wound margin to a more fetal-like state, and thus abrogate the biophysical impediments that hinder migration and proliferation. We tested this concept using the knee meniscus, a commonly injured structure for which few regenerative approaches exist. To focus delivery and degradation to the wound interface, we developed a system in which collagenase was stored inside poly(ethylene oxide) (PEO) electrospun nanofibers and released upon hydration. Through a series of in vitro and in vivo studies, our findings show that partial digestion of the wound interface improves repair by creating a more compliant and porous microenvironment that expedites cell migration to and/or proliferation at the wound margin. This innovative approach of targeted manipulation of the wound interface, focused on removing the naturally occurring barriers to adult tissue repair, may find widespread application in the treatment of injuries to a variety of dense connective tissues. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Human mesenchymal stromal cell-secreted lactate induces M2-macrophage differentiation by metabolic reprogramming

    Science.gov (United States)

    Civini, Sara; Pacelli, Consiglia; Dieng, Mame Massar; Lemieux, William; Jin, Ping; Bazin, Renée; Patey, Natacha; Marincola, Francesco M.; Moldovan, Florina; Zaouter, Charlotte; Trudeau, Louis-Eric; Benabdhalla, Basma; Louis, Isabelle; Beauséjour, Christian; Stroncek, David; Le Deist, Françoise; Haddad, Elie

    2016-01-01

    Human mesenchymal stromal cells (MSC) have been shown to dampen immune response and promote tissue repair, but the underlying mechanisms are still under investigation. Herein, we demonstrate that umbilical cord-derived MSC (UC-MSC) alter the phenotype and function of monocyte-derived dendritic cells (DC) through lactate-mediated metabolic reprogramming. UC-MSC can secrete large quantities of lactate and, when present during monocyte-to-DC differentiation, induce instead the acquisition of M2-macrophage features in terms of morphology, surface markers, migratory properties and antigen presentation capacity. Microarray expression profiling indicates that UC-MSC modify the expression of metabolic-related genes and induce a M2-macrophage expression signature. Importantly, monocyte-derived DC obtained in presence of UC-MSC, polarize naïve allogeneic CD4+ T-cells into Th2 cells. Treatment of UC-MSC with an inhibitor of lactate dehydrogenase strongly decreases lactate concentration in culture supernatant and abrogates the effect on monocyte-to-DC differentiation. Metabolic analysis further revealed that UC-MSC decrease oxidative phosphorylation in differentiating monocytes while strongly increasing the spare respiratory capacity proportional to the amount of secreted lactate. Because both MSC and monocytes are recruited in vivo at the site of tissue damage and inflammation, we propose the local increase of lactate concentration induced by UC-MSC and the consequent enrichment in M2-macrophage generation as a mechanism to achieve immunomodulation. PMID:27070086

  14. Isonitrosoacetophenone drives transcriptional reprogramming in Nicotiana tabacum cells in support of innate immunity and defense.

    Directory of Open Access Journals (Sweden)

    Arnaud T Djami-Tchatchou

    Full Text Available Plants respond to various stress stimuli by activating broad-spectrum defense responses both locally as well as systemically. As such, identification of expressed genes represents an important step towards understanding inducible defense responses and assists in designing appropriate intervention strategies for disease management. Genes differentially expressed in tobacco cell suspensions following elicitation with isonitrosoacetophenone (INAP were identified using mRNA differential display and pyro-sequencing. Sequencing data produced 14579 reads, which resulted in 198 contigs and 1758 singletons. Following BLAST analyses, several inducible plant defense genes of interest were identified and classified into functional categories including signal transduction, transcription activation, transcription and protein synthesis, protein degradation and ubiquitination, stress-responsive, defense-related, metabolism and energy, regulation, transportation, cytoskeleton and cell wall-related. Quantitative PCR was used to investigate the expression of 17 selected target genes within these categories. Results indicate that INAP has a sensitising or priming effect through activation of salicylic acid-, jasmonic acid- and ethylene pathways that result in an altered transcriptome, with the expression of genes involved in perception of pathogens and associated cellular re-programming in support of defense. Furthermore, infection assays with the pathogen Pseudomonas syringae pv. tabaci confirmed the establishment of a functional anti-microbial environment in planta.

  15. Latrunculin A treatment prevents abnormal chromosome segregation for successful development of cloned embryos.

    Directory of Open Access Journals (Sweden)

    Yukari Terashita

    Full Text Available Somatic cell nuclear transfer to an enucleated oocyte is used for reprogramming somatic cells with the aim of achieving totipotency, but most cloned embryos die in the uterus after transfer. While modifying epigenetic states of cloned embryos can improve their development, the production rate of cloned embryos can also be enhanced by changing other factors. It has already been shown that abnormal chromosome segregation (ACS is a major cause of the developmental failure of cloned embryos and that Latrunculin A (LatA, an actin polymerization inhibitor, improves F-actin formation and birth rate of cloned embryos. Since F-actin is important for chromosome congression in embryos, here we examined the relation between ACS and F-actin in cloned embryos. Using LatA treatment, the occurrence of ACS decreased significantly whereas cloned embryo-specific epigenetic abnormalities such as dimethylation of histone H3 at lysine 9 (H3K9me2 could not be corrected. In contrast, when H3K9me2 was normalized using the G9a histone methyltransferase inhibitor BIX-01294, the Magea2 gene-essential for normal development but never before expressed in cloned embryos-was expressed. However, this did not increase the cloning success rate. Thus, non-epigenetic factors also play an important role in determining the efficiency of mouse cloning.

  16. Latrunculin A Treatment Prevents Abnormal Chromosome Segregation for Successful Development of Cloned Embryos

    Science.gov (United States)

    Terashita, Yukari; Yamagata, Kazuo; Tokoro, Mikiko; Itoi, Fumiaki; Wakayama, Sayaka; Li, Chong; Sato, Eimei; Tanemura, Kentaro; Wakayama, Teruhiko

    2013-01-01

    Somatic cell nuclear transfer to an enucleated oocyte is used for reprogramming somatic cells with the aim of achieving totipotency, but most cloned embryos die in the uterus after transfer. While modifying epigenetic states of cloned embryos can improve their development, the production rate of cloned embryos can also be enhanced by changing other factors. It has already been shown that abnormal chromosome segregation (ACS) is a major cause of the developmental failure of cloned embryos and that Latrunculin A (LatA), an actin polymerization inhibitor, improves F-actin formation and birth rate of cloned embryos. Since F-actin is important for chromosome congression in embryos, here we examined the relation between ACS and F-actin in cloned embryos. Using LatA treatment, the occurrence of ACS decreased significantly whereas cloned embryo-specific epigenetic abnormalities such as dimethylation of histone H3 at lysine 9 (H3K9me2) could not be corrected. In contrast, when H3K9me2 was normalized using the G9a histone methyltransferase inhibitor BIX-01294, the Magea2 gene—essential for normal development but never before expressed in cloned embryos—was expressed. However, this did not increase the cloning success rate. Thus, non-epigenetic factors also play an important role in determining the efficiency of mouse cloning. PMID:24205216

  17. Impaired absorption of marked oligopeptide Glycine-I Tyrosine-Glycine after successful autologous-allotopic ileal mucosa transplantation in beagles.

    Science.gov (United States)

    Beiler, H A; Steinorth, J; Witt, A; Mier, W; Mohammed, A; Waag, K L; Zachariou, Z

    2004-10-01

    After establishing a method for ileal mucosa transplantation in an animal model, the authors investigated the absorptive capacity for oligopeptides of the transplanted mucosa. In 14 beagle dogs the authors transplanted ileal mucosa in a vascularized demucosed segment of the transverse colon. The colonic wall-ileal mucosa complex then was integrated in the ileal continuity. Six animals were lost owing to operative complications. Absorptive capacity for oligopeptides was measured in the remaining 8 animals with the iodine 131 (131I)-marked tripeptide glycine-tyrosine-glycine before and 4 weeks after transplantation. The results were compared and analyzed with the Student's t test for matched pairs. Blood concentrations of the marked tripeptide with P value less than .05 were considered as a significant reduction in the absorptive capacity of the transplanted ileal mucosa. After fixation with glutaraldehyd graft, uptake of the colonic wall-ileal mucosa complex was evaluated histologically in 8 animals. In all 8 animals, a 100% graft uptake was verified in all sections. Fifteen minutes after application of 15 MBc Glycine-131I-Tyrosine-Glycine there was no significant difference in the absorption between normal and transplanted ileal mucosa. After 30 minutes, the absorption of the transplanted ileal mucosa showed a tendency (P < .1) for an impaired uptake of the marked tripeptide. However, 60 minutes after application the difference in the absorptive capacity of the transplanted ileal mucosa was significant (P < .05). Autologous allotopic ileal mucosa transplantation is feasible; however, an impaired absorption of oligopeptides of the transplanted mucosa 4 weeks after transplantation could be observed.

  18. [Multilingualism and specific language impairment].

    Science.gov (United States)

    Arkkila, Eva; Smolander, Sini; Laasonen, Marja

    2013-01-01

    Specific language impairment is one of the most common developmental disturbances in childhood. With the increase of the foreign language population group an increasing number of children assimilating several languages and causing concern in language development attend clinical examinations. Knowledge of factors underlying the specific language impairment and the specific impairment in general, special features of language development of those learning several languages, as well as the assessment and support of the linguistic skills of a multilingual child is essential. The risk of long-term problems and marginalization is high for children having specific language impairment.

  19. DNA methylation patterns in tissues from mid-gestation bovine foetuses produced by somatic cell nuclear transfer show subtle abnormalities in nuclear reprogramming

    Directory of Open Access Journals (Sweden)

    Lee Rita SF

    2010-03-01

    Full Text Available Abstract Background Cloning of cattle by somatic cell nuclear transfer (SCNT is associated with a high incidence of pregnancy failure characterized by abnormal placental and foetal development. These abnormalities are thought to be due, in part, to incomplete re-setting of the epigenetic state of DNA in the donor somatic cell nucleus to a state that is capable of driving embryonic and foetal development to completion. Here, we tested the hypothesis that DNA methylation patterns were not appropriately established during nuclear reprogramming following SCNT. A panel of imprinted, non-imprinted genes and satellite repeat sequences was examined in tissues collected from viable and failing mid-gestation SCNT foetuses and compared with similar tissues from gestation-matched normal foetuses generated by artificial insemination (AI. Results Most of the genomic regions examined in tissues from viable and failing SCNT foetuses had DNA methylation patterns similar to those in comparable tissues from AI controls. However, statistically significant differences were found between SCNT and AI at specific CpG sites in some regions of the genome, particularly those associated with SNRPN and KCNQ1OT1, which tended to be hypomethylated in SCNT tissues. There was a high degree of variation between individuals in methylation levels at almost every CpG site in these two regions, even in AI controls. In other genomic regions, methylation levels at specific CpG sites were tightly controlled with little variation between individuals. Only one site (HAND1 showed a tissue-specific pattern of DNA methylation. Overall, DNA methylation patterns in tissues of failing foetuses were similar to apparently viable SCNT foetuses, although there were individuals showing extreme deviant patterns. Conclusion These results show that SCNT foetuses that had developed to mid-gestation had largely undergone nuclear reprogramming and that the epigenetic signature at this stage was not a

  20. Vascular cognitive impairment

    Directory of Open Access Journals (Sweden)

    N.V. Vakhnina

    2014-01-01

    Full Text Available Vascular pathology of the brain is the second most common cause of cognitive impairment after Alzheimer's disease. The article describes the modern concepts of etiology, pathogenetic mechanisms, clinical features and approaches to diagnosis and therapy of vascular cognitive impairment (VCI. Cerebrovascular accident, chronic cerebral circulatory insufficiency and their combination, sometimes in combination with a concomitant neurodegenerative process, are shown to be the major types of brain lesions leading to VCI. The clinical presentation of VCI is characterized by the neuropsychological status dominated by impairment of the executive frontal functions (planning, control, attention in combination with focal neurological symptoms. The diagnosis is based on comparing of the revealed neuropsychological and neurological features with neuroimaging data. Neurometabolic, acetylcholinergic, glutamatergic, and other vasoactive drugs and non-pharmacological methods are widely used to treat VCI.