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

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    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.

  2. Simultaneous Reprogramming and Gene Correction of Patient Fibroblasts

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    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.

  3. 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.

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

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    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.

  5. 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...

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

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    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

  7. 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.

  8. 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.

  9. Constitutive Reprogramming of Fibroblast Mitochondrial Metabolism in Pulmonary Hypertension

    Czech Academy of Sciences Publication Activity Database

    Plecitá-Hlavatá, Lydie; Tauber, Jan; Li, M.; Zhang, H.; Flockton, A. R.; Pullamsetti, S. S.; Chelladurai, P.; D'Alessandro, A.; El Kasmi, K. C.; Ježek, Petr; Stenmark, K. R.

    2016-01-01

    Roč. 55, č. 1 (2016), s. 47-57 ISSN 1044-1549 R&D Projects: GA MŠk(CZ) LH11055; GA MŠk(CZ) LH15071 Institutional support: RVO:67985823 Keywords : mitochondria * complex I * oxidative metabolism * pulmonary hypertension * adventitial fibroblasts Subject RIV: ED - Physiology Impact factor: 4.100, year: 2016

  10. 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...

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

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    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.

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

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    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

  13. Heart Development, Diseases, and Regeneration - New Approaches From Innervation, Fibroblasts, and Reprogramming.

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    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).

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

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    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.

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

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    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.

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

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    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

  17. 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

    Full Text Available Nupura S Bhise,1,* Karl J Wahlin,2,* Donald J Zack,2–4 Jordan J Green1,21Department of Biomedical Engineering, Translational Tissue Engineering Center, and Institute for Nanobiotechnology, 2Department of Ophthalmology, The Johns Hopkins University School of Medicine, Baltimore, MD, 3Solomon H Snyder Department of Neuroscience, Department of Molecular Biology and Genetics, and Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; 4Institut de la Vision, Paris, France*These authors contributed equally to this workBackground: Gene delivery can potentially be used as a therapeutic for treating genetic diseases, including neurodegenerative diseases, as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs from human fibroblasts.Methods: A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling.Results: 1-(3-aminopropyl-4-methylpiperazine end-terminated poly(1,4-butanediol diacrylate-co-4-amino-1-butanol polymer (B4S4E7 self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available

  18. Chemical Cocktails Enable Hepatic Reprogramming of Mouse Fibroblasts with a Single Transcription Factor

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    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.

  19. 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.

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

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    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.

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

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    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.

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

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

    2013-01-01

    Gene delivery can potentially be used as a therapeutic for treating genetic diseases, including neurodegenerative diseases, as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester) nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs) from human fibroblasts. A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling. 1-(3-aminopropyl)-4-methylpiperazine end-terminated poly(1,4-butanediol diacry-late-co-4-amino-1-butanol) polymer (B4S4E7) self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available 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. This

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

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    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

  4. An atomic-force-microscopy study of the structure of surface layers of intact fibroblasts

    Science.gov (United States)

    Khalisov, M. M.; Ankudinov, A. V.; Penniyaynen, V. A.; Nyapshaev, I. A.; Kipenko, A. V.; Timoshchuk, K. I.; Podzorova, S. A.; Krylov, B. V.

    2017-02-01

    Intact embryonic fibroblasts on a collagen-treated substrate have been studied by atomic-force microscopy (AFM) using probes of two types: (i) standard probes with tip curvature radii of 2-10 nm and (ii) special probes with a calibrated 325-nm SiO2 ball radius at the tip apex. It is established that, irrespective of probe type, the average maximum fibroblast height is on a level of 1.7 μm and the average stiffness of the probe-cell contact amounts to 16.5 mN/m. The obtained AFM data reveal a peculiarity of the fibroblast structure, whereby its external layers move as a rigid shell relative to the interior and can be pressed inside to a depth dependent on the load only.

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

    Directory of Open Access Journals (Sweden)

    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

  6. In vitro comparison of human fibroblasts from intact and ruptured ACL for use in tissue engineering

    Directory of Open Access Journals (Sweden)

    T Brune

    2007-12-01

    Full Text Available The present study compares fibroblasts extracted from intact and ruptured human anterior cruciate ligaments (ACL for creation of a tissue engineered ACL-construct, made of porcine small intestinal submucosal extracellular matrix (SIS-ECM seeded with these ACL cells. The comparison is based on histological, immunohistochemical and RT-PCR analyses. Differences were observed between cells in a ruptured ACL (rACL and cells in an intact ACL (iACL, particularly with regard to the expression of integrin subunits and smooth muscle actin (SMA. Despite these differences in the cell source, both cell populations behaved similarly when seeded on an SIS-ECM scaffold, with similar cell morphology, connective tissue organization and composition, SMA and integrin expression. This study shows the usefulness of naturally occurring scaffolds such as SIS-ECM for the study of cell behaviour in vitro, and illustrates the possibility to use autologous cells extracted from ruptured ACL biopsies as a source for tissue engineered ACL constructs.

  7. Plasma intact fibroblast growth factor 23 levels in women with anorexia nervosa

    Directory of Open Access Journals (Sweden)

    Otani Makoto

    2008-04-01

    Full Text Available Abstract Background Fibroblast growth factor (FGF23 is a novel phosphaturic factor associated with inorganic phosphate homeostasis. Previous human studies have shown that serum FGF23 levels increase in response to a high phosphate diet. For anorexia nervosa (AN patients, inorganic phosphate homeostasis is important in the clinical course, such as in refeeding syndrome. The purpose of this study was to determine plasma levels of intact FGF23 (iFGF23 in restricting-type AN (AN-R patients, binge-eating/purging-type AN (AN-BP patients, and healthy controls. Methods The subjects consisted of 6 female AN-R patients, 6 female AN-BP patients, and 11 healthy female controls; both inpatients and outpatients were included. Plasma iFGF23, 1,25-dihydroxyvitamin D (1,25-(OH2D, and 25-hydroxyvitamin D (25-OHD levels were measured. Data are presented as the median and the range. A two-tailed Mann-Whitney U-test with Bonferroni correction was used to assess differences among the three groups, and a value of p Results There were no differences between AN-R patients and controls in the iFGF23 and 1,25-(OH2D levels. In AN-BP patients, the iFGF23 level (41.3 pg/ml; range, 6.1–155.5 pg/ml was significantly higher than in controls (3.8 pg/ml; range, not detected-21.3 pg/ml; p = 0.001, and the 1,25-(OH2D was significantly lower in AN-BP patients (7.0 pg/ml; range, 4.2–33.7 pg/ml than in controls (39.7 pg/ml; range, 6.3–58.5 pg/ml; p = 0.015. No differences in plasma 25-OHD levels were observed among the groups. Conclusion This preliminary study is the first to show that plasma iFGF23 levels are increased in AN-BP patients, and that these elevated plasma FGF23 levels might be related to the decrease in plasma 1,25-(OH2D levels.

  8. 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

  9. 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

  10. 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.

  11. Novel GALNT3 mutations causing hyperostosis-hyperphosphatemia syndrome result in low intact fibroblast growth factor 23 concentrations.

    Science.gov (United States)

    Ichikawa, Shoji; Guigonis, Vincent; Imel, Erik A; Courouble, Mélanie; Heissat, Sophie; Henley, John D; Sorenson, Andrea H; Petit, Barbara; Lienhardt, Anne; Econs, Michael J

    2007-05-01

    Hyperostosis-hyperphosphatemia syndrome (HHS) is a rare metabolic disorder characterized by hyperphosphatemia and localized hyperostosis. HHS is caused by mutations in GALNT3, which encodes UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- acetylgalactosaminyltransferase 3. Familial tumoral calcinosis (TC), characterized by ectopic calcifications and hyperphosphatemia, is caused by mutations in the GALNT3 or fibroblast growth factor 23 (FGF23) genes. Our objective was to identify mutations in FGF23 or GALNT3 and determine serum FGF23 levels in an HHS patient. Mutation detection in FGF23 and GALNT3 was performed by DNA sequencing, and serum FGF23 concentrations were measured by ELISA. A 5-year-old French boy with HHS and his family members participated. The patient presented with painful cortical lesions in his leg. Radiographs of the affected bone showed diaphyseal hyperostosis. The lesional tissue comprised trabeculae of immature, woven bone surrounded by fibrous tissue. Biochemistry revealed elevated phosphate, tubular maximum rate for phosphate reabsorption per deciliter of glomerular filtrate, and 1,25-dihydroxyvitamin D levels. The patient was a compound heterozygote for two novel GALNT3 mutations. His parents and brother were heterozygous for one of the mutations and had no biochemical abnormalities. Intact FGF23 level in the patient was low normal, whereas C-terminal FGF23 was elevated, a pattern similar to TC. The presence of GALNT3 mutations and elevated C-terminal, but low intact serum FGF23, levels in HHS resemble those seen in TC, suggesting that HHS and TC are different manifestations of the same disorder. The absence of biochemical abnormalities in the heterozygous individuals suggests that one normal allele is sufficient for secretion of intact FGF23.

  12. A feeder- and xeno-free human induced pluripotent stem cell line obtained from primary human dermal fibroblasts with epigenetic repression of reprogramming factors expression: GPCCi001-A

    Directory of Open Access Journals (Sweden)

    Michał Stefan Lach

    2017-04-01

    Full Text Available The primary human dermal fibroblasts (PHDFs from breast cancer patient were obtained to generate the human induced pluripotent stem cell line GPCCi001-A via lentiviral transfection. Thus, a modified EF1a-hSTEMCCA-loxP with tetO operator which regulates transgene expression was used. This method takes advantage of epigenetic regulation of transcription and allows for stable silencing of the reprogramming factors in obtained hiPS cells. To increase the potential utility of hiPSCs for clinical applications, they were adapted to feeder- and xeno-free conditions. The pluripotency of GPCCi001-A cell line and ability to differentiate into three germ layers was confirmed.

  13. Ablation of the Galnt3 Gene Leads to Low-Circulating Intact Fibroblast Growth Factor 23 (Fgf23) Concentrations and Hyperphosphatemia Despite Increased Fgf23 Expression

    OpenAIRE

    Ichikawa, Shoji; Sorenson, Andrea H.; Austin, Anthony M.; Mackenzie, Donald S.; Fritz, Timothy A.; Moh, Akira; Hui, Siu L.; Econs, Michael J.

    2009-01-01

    Familial tumoral calcinosis is characterized by ectopic calcifications and hyperphosphatemia. The disease is caused by inactivating mutations in fibroblast growth factor 23 (FGF23), Klotho (KL), and uridine diphosphate-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3). In vitro studies indicate that GALNT3 O-glycosylates a phosphaturic hormone, FGF23, and prevents its proteolytic processing, thereby allowing secretion of intact FGF23. In this study we generat...

  14. Plasma intact fibroblast growth factor 23 levels in women with bulimia nervosa: A cross-sectional pilot study

    Directory of Open Access Journals (Sweden)

    Yoshiuchi Kazuhiro

    2011-06-01

    Full Text Available Abstract Fibroblast growth factor (FGF 23, a circulating 26-kDa peptide produced by osteogenic cells, is a novel phosphaturic factor. In our previous study, binge-eating/purging type anorexia nervosa (AN-BP patients had elevated plasma intact FGF23 (iFGF23 levels, while restricting type (AN-R patients had plasma iFGF23 levels similar to healthy controls. Although bulimia nervosa (BN patients as well as some patients with AN-BP regularly engage in binge eating, there have been no studies regarding plasma iFGF23 levels in BN patients. Therefore, this study was performed to determine plasma iFGF23 concentrations in BN patients and healthy controls. The study population consisted of 13 female BN patients and 11 healthy female controls. Blood samples were collected from all subjects after overnight fasting. Plasma iFGF23 was measured using an ELISA kit in a cross-sectional manner. The two-tailed Mann-Whitney U-test was used to assess differences between BN patients and healthy controls. In addition, BN patients were divided into two groups based on questionnaire-reported binge eating frequency immediately prior to participation in this study: high frequency of binge eating (once a week or more; HF group; n = 8 and low frequency of binge eating (less than once a week; LF group; n = 5. Two-tailed Mann-Whitney U-test with Bonferroni's correction was performed after the Kruskal-Wallis test to assess differences between HF group, LF group, and healthy controls. Median (quartiles plasma iFGF23 levels were greater in BN patients (35.5 [14.8-65.0] pg/ml than in controls (3.8 [not detected-5.3] pg/ml; p = 0.002. In addition, median (quartiles plasma iFGF23 levels were greater in the HF group (62.3 [44.4-73.4] pg/ml than in controls (p

  15. Reprogramming chromatin

    DEFF Research Database (Denmark)

    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...

  16. Discovery and progress of direct cardiac reprogramming.

    Science.gov (United States)

    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.

  17. Ablation of the Galnt3 gene leads to low-circulating intact fibroblast growth factor 23 (Fgf23) concentrations and hyperphosphatemia despite increased Fgf23 expression.

    Science.gov (United States)

    Ichikawa, Shoji; Sorenson, Andrea H; Austin, Anthony M; Mackenzie, Donald S; Fritz, Timothy A; Moh, Akira; Hui, Siu L; Econs, Michael J

    2009-06-01

    Familial tumoral calcinosis is characterized by ectopic calcifications and hyperphosphatemia. The disease is caused by inactivating mutations in fibroblast growth factor 23 (FGF23), Klotho (KL), and uridine diphosphate-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3). In vitro studies indicate that GALNT3 O-glycosylates a phosphaturic hormone, FGF23, and prevents its proteolytic processing, thereby allowing secretion of intact FGF23. In this study we generated mice lacking the Galnt3 gene, which developed hyperphosphatemia without apparent calcifications. In response to hyperphosphatemia, Galnt3-deficient mice had markedly increased Fgf23 expression in bone. However, compared with wild-type and heterozygous littermates, homozygous mice had only about half of circulating intact Fgf23 levels and higher levels of C-terminal Fgf23 fragments in bone. Galnt3-deficient mice also exhibited an inappropriately normal 1,25-dihydroxyvitamin D level and decreased alkaline phosphatase activity. Furthermore, renal expression of sodium-phosphate cotransporters and Kl were elevated in Galnt3-deficient mice. Interestingly, there were sex-specific phenotypes; only Galnt3-deficient males showed growth retardation, infertility, and significantly increased bone mineral density. In summary, ablation of Galnt3 impaired secretion of intact Fgf23, leading to decreased circulating Fgf23 and hyperphosphatemia, despite increased Fgf23 expression. Our findings indicate that Galnt3-deficient mice have a biochemical phenotype of tumoral calcinosis and provide in vivo evidence that Galnt3 plays an essential role in proper secretion of Fgf23 in mice.

  18. Connective tissue growth factor/CCN2-null mouse embryonic fibroblasts retain intact transforming growth factor-β responsiveness

    International Nuclear Information System (INIS)

    Mori, Yasuji; Hinchcliff, Monique; Wu, Minghua; Warner-Blankenship, Matthew; Lyons, Karen M.; Varga, John

    2008-01-01

    Background: The matricellular protein connective tissue growth factor (CCN2) has been implicated in pathological fibrosis, but its physiologic role remains elusive. In vitro, transforming growth factor-β (TGF-β) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance profibrotic responses elicited by TGF-β, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-β. To explore this notion, we characterized TGF-β-induced activation of fibroblasts from CCN2-null (CCN2 -/- ) mouse embryos. Methods: The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-β signal transduction and regulation of collagen gene expression were examined in CCN2 -/- MEFs by immunohistochemistry, Northern, Western and RT-PCR analysis, immunocytochemistry and transient transfection assays. Results: Bleomycin-induced skin fibrosis in the mouse was associated with substantial CCN2 up-regulation in lesional fibroblasts. Whereas in vitro proliferation rate of CCN2 -/- MEFs was markedly reduced compared to wild type MEFs, TGF-β-induced activation of the Smad pathways, including Smad2 phosphorylation, Smad2/3 and Smad4 nuclear accumulation and Smad-dependent transcriptional responses, were unaffected by loss of CCN2. The stimulation of COL1A2 and fibronectin mRNA expression and promoter activity, and of corresponding protein levels, showed comparable time and dose-response in wild type and CCN2 -/- MEFs, whereas stimulation of alpha smooth muscle actin and myofibroblast transdifferentiation showed subtle impairment in MEFs lacking CCN2. Conclusion: Whereas endogenous CCN2 plays a role in regulation of proliferation and TGF-β-induced myofibroblast transdifferentiation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts

  19. 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.

  20. 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.

  1. 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.

  2. Boosters and barriers for direct cardiac reprogramming.

    Science.gov (United States)

    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.

  3. Metabolic Reprogramming Regulates the Proliferative and Inflammatory Phenotype of Adventitial Fibroblasts in Pulmonary Hypertension Through the Transcriptional Corepressor C-Terminal Binding Protein-1

    Czech Academy of Sciences Publication Activity Database

    Li, M.; Riddle, S.; Zhang, H.; D'Alessandro, A.; Flockton, A.; Serkova, N. J.; Hansen, K. C.; Moldvan, R.; McKeon, B. A.; Frid, M.; Kumar, S.; Li, H.; Liu, H.; Canovas, A.; Medrano, J. F.; Thomas, M. G.; Iloska, D.; Plecitá-Hlavatá, Lydie; Ježek, Petr; Pullamsetti, S.; Fini, M. A.; El Kasmi, K. C.; Zhang, Q. H.; Stenmark, K. R.

    2016-01-01

    Roč. 134, č. 15 (2016), s. 1105-1121 ISSN 0009-7322 R&D Projects: GA MŠk(CZ) LH11055; GA MŠk(CZ) LH15071 Institutional support: RVO:67985823 Keywords : arterial fibroblasts * pulmonary hypertension * metabolism * CtBP1 Subject RIV: ED - Physiology Impact factor: 19.309, year: 2016

  4. 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.

  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. Optimal ROS Signaling Is Critical for Nuclear Reprogramming

    Directory of Open Access Journals (Sweden)

    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.

  7. Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: Evidence that DNA polymerases δ and β are involved in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine

    International Nuclear Information System (INIS)

    Hammond, R.A.; Miller, M.R.; McClung, J.K.

    1990-01-01

    The involvement of DNA polymerases α, β, and δ in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in human fibroblasts (HF). The effects of anti-(DNA polymerase α) monoclonal antibody, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), dideoxythymidine triphosphate (ddTTP), and aphidicolin on MNNG-induced DNA repair synthesis were investigated to dissect the roles of the different DNA polymerases. A subcellular system (permeable cells), in which DNA repair synthesis and DNA replication were differentiated by CsCl gradient centrifugation of BrdUMP density-labeled DNA, was used to examine the effects of the polymerase inhibitors. Another approach investigated the effects of several of these inhibitors of MNNG-induced DNA repair synthesis in intact cells by measuring the amount of [ 3 H]thymidine incorporated into repair DNA as determined by autoradiography and quantitation with an automated video image analysis system. In permeable cells, MNNG-induced DNA repair synthesis was inhibited 56% by 50 μg of aphidicolin/mL, 6% by 10 μM BuPdGTP, 13% by anti-(DNA polymerse α) monoclonal antibodies, and 29% by ddTTP. In intact cells, MNNG-induced DNA repair synthesis was inhibited 57% by 50 μg of aphidicolin/mL and was not significantly inhibited by microinjecting anti-(DNA polymerase α) antibodies into HF nuclei. These results indicate that both DNA polymerase δ and β are involved in repairing DNA damage caused by MNNG

  8. 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...

  9. 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...

  10. 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.

  11. Direct Cardiac Reprogramming: Advances in Cardiac Regeneration

    Directory of Open Access Journals (Sweden)

    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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. Generation of hiPSTZ16 (ISMMSi003-A cell line from normal human foreskin fibroblasts

    Directory of Open Access Journals (Sweden)

    Marion Dejosez

    2018-01-01

    Full Text Available Human foreskin fibroblasts from a commercial source were reprogrammed into induced pluripotent stem cells to establish a clonal stem cell line, hiPSTZ16 (ISMMSi003-A. These cells show a normal karyotype and full differentiation potential in teratoma assays. The described cells provide a useful resource in combination with other iPS cell lines generated from normal human foreskin fibroblasts to study source- and reprogramming method-independent effects in downstream applications.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. 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.

  1. 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 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.

  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. 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

  4. 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.

  5. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  7. Reprogramming cells with synthetic proteins

    Directory of Open Access Journals (Sweden)

    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.

  8. 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

  9. Totipotency, Pluripotency and Nuclear Reprogramming

    Science.gov (United States)

    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.

  10. Re: Epigenetics of Cellular Reprogramming

    Directory of Open Access Journals (Sweden)

    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.

  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. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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...

  18. The cellular memory disc of reprogrammed cells.

    Science.gov (United States)

    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].

  19. Fibroblastic rheumatism

    Directory of Open Access Journals (Sweden)

    Jyoti Ranjan Parida

    2017-01-01

    Full Text Available Fibroblastic rheumatism (FR is a rare dermoarthopathy reported from different parts of the world since 1980. Although the exact cause is unknown, few reports implicate infection may be a triggering event. Patients usually present with multiple skin nodules and polyarthropathy with progressive skin contractures. Laboratory parameters including acute phase reactants are usually normal. The confirmatory diagnosis is based on histopathologic study of skin nodules, which demonstrate fibroblastic proliferation, thickened collagen fibers, dermal fibrosis, and decreased number of elastic fibers. Immunoreactivity for b-catenin, smooth muscle actin, and the monoclonal antibody HHF35 show myofibroblastic differentiation. Treatments with oral prednisolone and other disease-modifying drugs such as methotrexate, infliximab, and interferon have been tried with variable success. In general, skin lesions respond more aptly than joint symptoms indicating that skin fibroblast is more amenable to treatment than synovial fibroblasts. Awareness regarding this orphan disease among clinicians and pathologists will help in more reporting of such cases and finding out optimal treatment regimen.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. Epigenetic reprogramming in the porcine germ line

    DEFF Research Database (Denmark)

    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...

  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. Small Molecules Facilitate Single Factor-Mediated Hepatic Reprogramming

    Directory of Open Access Journals (Sweden)

    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.

  8. 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.

  9. 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.

  10. 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.

  11. 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).

  12. 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.

  13. Understanding the molecular mechanisms of reprogramming

    Energy Technology Data Exchange (ETDEWEB)

    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.

  14. Metabolic Reprogramming in Thyroid Carcinoma

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    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.

  15. 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

  16. 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.

  17. Plasmid-based generation of induced neural stem cells from adult human fibroblasts

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    Philipp Capetian

    2016-10-01

    Full Text Available Direct reprogramming from somatic to neural cell types has become an alternative to induced pluripotent stem cells. Most protocols employ viral expression systems, posing the risk of random genomic integration. Recent developments led to plasmid-based protocols, lowering this risk. However, these protocols either relied on continuous presence of a variety of small molecules or were only able to reprogram murine cells. We therefore established a reprogramming protocol based on vectors containing the Epstein-Barr virus (EBV-derived oriP/EBNA1 as well as the defined expression factors Oct3/4, Sox2, Klf4, L-myc, Lin28, and a small hairpin directed against p53. We employed a defined neural medium in combination with the neurotrophins bFGF, EGF and FGF4 for cultivation without the addition of small molecules. After reprogramming, cells demonstrated a temporary increase in the expression of endogenous Oct3/4. We obtained induced neural stem cells (iNSC 30 days after transfection. In contrast to previous results, plasmid vectors as well as a residual expression of reprogramming factors remained detectable in all cell lines. Cells showed a robust differentiation into neuronal (72% and glial cells (9% astrocytes, 6% oligodendrocytes. Despite the temporary increase of pluripotency-associated Oct3/4 expression during reprogramming, we did not detect pluripotent stem cells or non-neural cells in culture (except occasional residual fibroblasts. Neurons showed electrical activity and functional glutamatergic synapses. Our results demonstrate that reprogramming adult human fibroblasts to iNSC by plasmid vectors and basic neural medium without small molecules is possible and feasible. However, a full set of pluripotency-associated transcription factors may indeed result in the acquisition of a transient (at least partial pluripotent intermediate during reprogramming. In contrast to previous reports, the EBV-based plasmid system remained present and active inside

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

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    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. 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.

  1. Non-Viral Generation of Neural Precursor-like Cells from Adult Human Fibroblasts

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    Maucksch C

    2012-01-01

    Full Text Available Recent studies have reported direct reprogramming of human fibroblasts to mature neurons by the introduction of defined neural genes. This technology has potential use in the areas of neurological disease modeling and drug development. However, use of induced neurons for large-scale drug screening and cell-based replacement strategies is limited due to their inability to expand once reprogrammed. We propose it would be more desirable to induce expandable neural precursor cells directly from human fibroblasts. To date several pluripotent and neural transcription factors have been shown to be capable of converting mouse fibroblasts to neural stem/precursor-like cells when delivered by viral vectors. Here we extend these findings and demonstrate that transient ectopic insertion of the transcription factors SOX2 and PAX6 to adult human fibroblasts through use of non-viral plasmid transfection or protein transduction allows the generation of induced neural precursor (iNP colonies expressing a range of neural stem and pro-neural genes. Upon differentiation, iNP cells give rise to neurons exhibiting typical neuronal morphologies and expressing multiple neuronal markers including tyrosine hydroxylase and GAD65/67. Importantly, iNP-derived neurons demonstrate electrophysiological properties of functionally mature neurons with the capacity to generate action potentials. In addition, iNP cells are capable of differentiating into glial fibrillary acidic protein (GFAP-expressing astrocytes. This study represents a novel virus-free approach for direct reprogramming of human fibroblasts to a neural precursor fate.

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

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    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.

  3. (Photosynthesis in intact plants)

    Energy Technology Data Exchange (ETDEWEB)

    1990-01-01

    Progress in the two years since the last renewal application has been excellent. We have made substantial contributions on both main fronts of the projects, and are particularly happy with the progress of our research on intact plants. The approach of basing our field work on a sound foundation of laboratory studies has enabled is to use methods which provide unambiguous assays of well characterized reactions. We have also made excellent progress in several laboratory studies which will have direct applications in future field work, and have introduced to the laboratory a range of molecular genetics techniques which will allow us to explore new options in the attempt to understand function at the level of molecular structure.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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 ...

  11. 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.

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

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    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. Reprogramming with Small Molecules instead of Exogenous Transcription Factors

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    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.

  14. 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

  15. Cellular Reprogramming Employing Recombinant Sox2 Protein

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    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.

  16. 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.

  17. 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.

  18. Generation of Footprint-Free Induced Pluripotent Stem Cells from Human Fibroblasts Using Episomal Plasmid Vectors.

    Science.gov (United States)

    Ovchinnikov, Dmitry A; Sun, Jane; Wolvetang, Ernst J

    2015-01-01

    Human induced pluripotent stem cells (hiPSCs) have provided novel insights into the etiology of disease and are set to transform regenerative medicine and drug screening over the next decade. The generation of human iPSCs free of a genetic footprint of the reprogramming process is crucial for the realization of these potential uses. Here we describe in detail the generation of human iPSC from control and disease-carrying individuals' fibroblasts using episomal plasmids.

  19. 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...

  20. Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors

    Science.gov (United States)

    Hiramatsu, Kunihiko; Sasagawa, Satoru; Outani, Hidetatsu; Nakagawa, Kanako; Yoshikawa, Hideki; Tsumaki, Noriyuki

    2011-01-01

    Repair of cartilage injury with hyaline cartilage continues to be a challenging clinical problem. Because of the limited number of chondrocytes in vivo, coupled with in vitro de-differentiation of chondrocytes into fibrochondrocytes, which secrete type I collagen and have an altered matrix architecture and mechanical function, there is a need for a novel cell source that produces hyaline cartilage. The generation of induced pluripotent stem (iPS) cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. Here, we show that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts, i.e., the promoters of type I collagen genes were extensively methylated. Although some induced cell lines formed tumors when subcutaneously injected into nude mice, other induced cell lines generated stable homogenous hyaline cartilage–like tissue. Further, the doxycycline-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. Thus, this approach could lead to the preparation of hyaline cartilage directly from skin, without generating iPS cells. PMID:21293062

  1. 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

  2. MicroRNA-124 controls the proliferative, migratory, and inflammatory phenotype of pulmonary vascular fibroblasts.

    Science.gov (United States)

    Wang, Daren; Zhang, Hui; Li, Min; Frid, Maria G; Flockton, Amanda R; McKeon, B Alexandre; Yeager, Michael E; Fini, Mehdi A; Morrell, Nicholas W; Pullamsetti, Soni S; Velegala, Sivareddy; Seeger, Werner; McKinsey, Timothy A; Sucharov, Carmen C; Stenmark, Kurt R

    2014-01-03

    Pulmonary hypertensive remodeling is characterized by excessive proliferation, migration, and proinflammatory activation of adventitial fibroblasts. In culture, fibroblasts maintain a similar activated phenotype. The mechanisms responsible for generation/maintenance of this phenotype remain unknown. We hypothesized that aberrant expression of microRNA-124 (miR-124) regulates this activated fibroblast phenotype and sought to determine the signaling pathways through which miR-124 exerts effects. We detected significant decreases in miR-124 expression in fibroblasts isolated from calves and humans with severe pulmonary hypertension. Overexpression of miR-124 by mimic transfection significantly attenuated proliferation, migration, and monocyte chemotactic protein-1 expression of hypertensive fibroblasts, whereas anti-miR-124 treatment of control fibroblasts resulted in their increased proliferation, migration, and monocyte chemotactic protein-1 expression. Furthermore, the alternative splicing factor, polypyrimidine tract-binding protein 1, was shown to be a direct target of miR-124 and to be upregulated both in vivo and in vitro in bovine and human pulmonary hypertensive fibroblasts. The effects of miR-124 on fibroblast proliferation were mediated via direct binding to the 3' untranslated region of polypyrimidine tract-binding protein 1 and subsequent regulation of Notch1/phosphatase and tensin homolog/FOXO3/p21Cip1 and p27Kip1 signaling. We showed that miR-124 directly regulates monocyte chemotactic protein-1 expression in pulmonary hypertension/idiopathic pulmonary arterial hypertension fibroblasts. Furthermore, we demonstrated that miR-124 expression is suppressed by histone deacetylases and that treatment of hypertensive fibroblasts with histone deacetylase inhibitors increased miR-124 expression and decreased proliferation and monocyte chemotactic protein-1 production. Stable decreases in miR-124 expression contribute to an epigenetically reprogrammed, highly

  3. 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....

  4. 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.

  5. 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.

  6. 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.

  7. Re-patterning of H3K27me3, H3K4me3 and DNA methylation during fibroblast conversion into induced cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Ziqing Liu

    2016-03-01

    Full Text Available Direct conversion of fibroblasts into induced cardiomyocytes (iCMs offers an alternative strategy for cardiac disease modeling and regeneration. During iCM reprogramming, the starting fibroblasts must overcome existing epigenetic barriers to acquire the CM-like chromatin pattern. However, epigenetic dynamics along this reprogramming process have not been studied. Here, we took advantage of our recently generated polycistronic system and determined the dynamics of two critical histone marks, H3K27me3 and H3K4me3, in parallel with gene expression at a set of carefully selected cardiac and fibroblast loci during iCM reprogramming. We observed reduced H3K27me3 and increased H3K4me3 at cardiac promoters as early as day 3, paralleled by a rapid significant increase in their mRNA expression. In contrast, H3K27me3 at loci encoding fibroblast marker genes did not increase until day 10 and H3K4me3 progressively decreased along the reprogramming process; these changes were accompanied by a gradual decrease in the mRNA expression of fibroblast marker genes. Further analyses of fibroblast-enriched transcription factors revealed a similarly late deposition of H3K27me3 and decreased mRNA expression of Sox9, Twist1 and Twist2, three important players in epithelial−mesenchymal transition. Our data suggest early rapid activation of the cardiac program and later progressive suppression of fibroblast fate at both epigenetic and transcriptional levels. Additionally, we determined the DNA methylation states of representative cardiac promoters and found that not every single CpG was equally demethylated during early stages of iCM reprogramming. Rather, there are specific CpGs, whose demethylation states correlated tightly with transcription activation, that we propose are the major contributing CpGs. Our work thus reveals a differential re-patterning of H3K27me3, H3K4me3 at cardiac and fibroblast loci during iCM reprogramming and could provide future genome

  8. 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.

  9. (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.

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

    Directory of Open Access Journals (Sweden)

    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.

  11. 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.

  12. 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.

  13. Direct induction of chondrogenic cells from human dermal fibroblast culture by defined factors.

    Directory of Open Access Journals (Sweden)

    Hidetatsu Outani

    Full Text Available The repair of large cartilage defects with hyaline cartilage continues to be a challenging clinical issue. We recently reported that the forced expression of two reprogramming factors (c-Myc and Klf4 and one chondrogenic factor (SOX9 can induce chondrogenic cells from mouse dermal fibroblast culture without going through a pluripotent state. We here generated induced chondrogenic (iChon cells from human dermal fibroblast (HDF culture with the same factors. We developed a chondrocyte-specific COL11A2 promoter/enhancer lentiviral reporter vector to select iChon cells. The human iChon cells expressed marker genes for chondrocytes but not fibroblasts, and were derived from non-chondrogenic COL11A2-negative cells. The human iChon cells formed cartilage but not tumors in nude mice. This approach could lead to the preparation of cartilage directly from skin in human, without going through pluripotent stem cells.

  14. Deterministic direct reprogramming of somatic cells to pluripotency.

    Science.gov (United States)

    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.

  15. 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.

  16. In vitro generation of renal tubular epithelial cells from fibroblasts: implications for precision and regenerative medicine in nephrology.

    Science.gov (United States)

    Wyatt, Christina M; Dubois, Nicole

    2017-02-01

    Prior efforts to generate renal epithelial cells in vitro have relied on pluripotent or bone marrow-derived mesenchymal stem cells. A recent publication in Nature Cell Biology describes the generation of induced tubular epithelial cells from fibroblasts, potentially offering a novel platform for personalized drug toxicity screening and in vitro disease modeling. This report serves as a promising proof of principle study and opens future research directions, including the optimization of the reprogramming process, efficient translation to adult human fibroblasts, and the generation of highly specific functional renal cell types. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  17. Generation of iPSC line iPSC-FH2.1 in hypoxic conditions from human foreskin fibroblasts

    Directory of Open Access Journals (Sweden)

    María Questa

    2016-03-01

    Full Text Available Human foreskin fibroblasts were used to generate the iPSC line iPSC-FH2.1 using the EF1a-hSTEMCCA-loxP vector expressing OCT4, SOX2, c-MYC and KLF4, in 5% O2 culture conditions. Stemness was confirmed, as was pluripotency both in vivo and in vitro, in normoxia and hypoxia. Human Embryonic Stem Cell (hESC line WA-09 and reprogrammed fibroblast primary culture HFF-FM were used as controls.

  18. Fibroblast growth factor 23

    African Journals Online (AJOL)

    Dr Olaleye

    Systemic phosphate homeostasis is maintained through several hormonal mechanisms which involve fibroblast growth factor 23 (FGF-23), α-klotho, vitamin D and parathyroid hormone. FGF-23 is known to be the major regulator of phosphate balance (Mirams et al., 2004). FGF-23 is a phosphaturic hormone, which is.

  19. 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

  20. 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 ...

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

    African Journals Online (AJOL)

    user

    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-.

  2. 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.

  3. 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...

  4. 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.

  5. Comparative study of human-induced pluripotent stem cells derived from bone marrow cells, hair keratinocytes, and skin fibroblasts.

    Science.gov (United States)

    Streckfuss-Bömeke, Katrin; Wolf, Frieder; Azizian, Azadeh; Stauske, Michael; Tiburcy, Malte; Wagner, Stefan; Hübscher, Daniela; Dressel, Ralf; Chen, Simin; Jende, Jörg; Wulf, Gerald; Lorenz, Verena; Schön, Michael P; Maier, Lars S; Zimmermann, Wolfram H; Hasenfuss, Gerd; Guan, Kaomei

    2013-09-01

    Induced pluripotent stem cells (iPSCs) provide a unique opportunity for the generation of patient-specific cells for use in disease modelling, drug screening, and regenerative medicine. The aim of this study was to compare human-induced pluripotent stem cells (hiPSCs) derived from different somatic cell sources regarding their generation efficiency and cardiac differentiation potential, and functionalities of cardiomyocytes. We generated hiPSCs from hair keratinocytes, bone marrow mesenchymal stem cells (MSCs), and skin fibroblasts by using two different virus systems. We show that MSCs and fibroblasts are more easily reprogrammed than keratinocytes. This corresponds to higher methylation levels of minimal promoter regions of the OCT4 and NANOG genes in keratinocytes than in MSCs and fibroblasts. The success rate and reprogramming efficiency was significantly higher by using the STEMCCA system than the OSNL system. All analysed hiPSCs are pluripotent and show phenotypical characteristics similar to human embryonic stem cells. We studied the cardiac differentiation efficiency of generated hiPSC lines (n = 24) and found that MSC-derived hiPSCs exhibited a significantly higher efficiency to spontaneously differentiate into beating cardiomyocytes when compared with keratinocyte-, and fibroblast-derived hiPSCs. There was no significant difference in the functionalities of the cardiomyocytes derived from hiPSCs with different origins, showing the presence of pacemaker-, atrial-, ventricular- and Purkinje-like cardiomyocytes, and exhibiting rhythmic Ca2+ transients and Ca2+ sparks in hiPSC-derived cardiomyocytes. Furthermore, spontaneously and synchronously beating and force-developing engineered heart tissues were generated. Human-induced pluripotent stem cells can be reprogrammed from all three somatic cell types, but with different efficiency. All analysed iPSCs can differentiate into cardiomyocytes, and the functionalities of cardiomyocytes derived from different cell

  6. Induction of pluripotent stem cells from fibroblast cultures.

    Science.gov (United States)

    Takahashi, Kazutoshi; Okita, Keisuke; Nakagawa, Masato; Yamanaka, Shinya

    2007-01-01

    Clinical application of embryonic stem (ES) cells faces difficulties regarding use of embryos, as well as tissue rejection after implantation. One way to circumvent these issues is to generate pluripotent stem cells directly from somatic cells. Somatic cells can be reprogrammed to an embryonic-like state by the injection of a nucleus into an enucleated oocyte or by fusion with ES cells. However, little is known about the mechanisms underlying these processes. We have recently shown that the combination of four transcription factors can generate ES-like pluripotent stem cells directly from mouse fibroblast cultures. The cells, named induced pluripotent stem (iPS) cells, can be differentiated into three germ layers and committed to chimeric mice. Here we describe detailed methods and tips for the generation of iPS cells.

  7. Direct Neural Conversion from Human Fibroblasts Using Self-Regulating and Nonintegrating Viral Vectors

    Directory of Open Access Journals (Sweden)

    Shong Lau

    2014-12-01

    Full Text Available Summary: Recent findings show that human fibroblasts can be directly programmed into functional neurons without passing via a proliferative stem cell intermediate. These findings open up the possibility of generating subtype-specific neurons of human origin for therapeutic use from fetal cell, from patients themselves, or from matched donors. In this study, we present an improved system for direct neural conversion of human fibroblasts. The neural reprogramming genes are regulated by the neuron-specific microRNA, miR-124, such that each cell turns off expression of the reprogramming genes once the cell has reached a stable neuronal fate. The regulated system can be combined with integrase-deficient vectors, providing a nonintegrative and self-regulated conversion system that rids problems associated with the integration of viral transgenes into the host genome. These modifications make the system suitable for clinical use and therefore represent a major step forward in the development of induced neurons for cell therapy. : Lau et al. now use miRNA targeting to build a self-regulating neural conversion system. Combined with nonintegrating vectors, this system can efficiently drive conversion of human fibroblasts into functional induced neurons (iNs suitable for clinical applications.

  8. 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.

  9. Binding, uptake, and release of nicotine by human gingival fibroblasts

    International Nuclear Information System (INIS)

    Hanes, P.J.; Schuster, G.S.; Lubas, S.

    1991-01-01

    Previous studies of the effects of nicotine on fibroblasts have reported an altered morphology and attachment of fibroblasts to substrates and disturbances in protein synthesis and secretion. This altered functional and attachment response may be associated with changes in the cell membrane resulting from binding of the nicotine, or to disturbances in cell metabolism as a result of high intracellular levels of nicotine. The purpose of the present study, therefore, was to (1) determine whether gingival fibroblasts bound nicotine and if any binding observed was specific or non-specific in nature; (2) determine whether gingival fibroblasts internalized nicotine, and if so, at what rate; (3) determine whether gingival fibroblasts also released nicotine back into the extracellular environment; and (4) if gingival fibroblasts release nicotine intact or as a metabolite. Cultures of gingival fibroblasts were prepared from gingival connective tissue biopsies. Binding was evaluated at 4 degree C using a mixture of 3 H-nicotine and unlabeled nicotine. Specific binding was calculated as the difference between 3 H-nicotine bound in the presence and absence of unlabeled nicotine. The cells bound 1.44 (+/- 0.42) pmols/10(6) cells in the presence of unlabeled nicotine and 1.66 (+/- 0.55) pmols/10(6) cells in the absence of unlabeled nicotine. The difference was not significant. Uptake of nicotine was measured at 37 degree C after treating cells with 3 H-nicotine for time periods up to 4 hours. Uptake in pmols/10(6) cells was 4.90 (+/- 0.34) at 15 minutes, 8.30 (+/- 0.75) at 30 minutes, 12.28 (+/- 2.62) at 1 hour and 26.31 (+/- 1.15) at 4 hours

  10. Metabolic Reprogramming During Multidrug Resistance in Leukemias

    Directory of Open Access Journals (Sweden)

    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.

  11. 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.

  12. High-efficiency generation of induced pluripotent mesenchymal stem cells from human dermal fibroblasts using recombinant proteins.

    Science.gov (United States)

    Chen, Fanfan; Zhang, Guoqiang; Yu, Ling; Feng, Yanye; Li, Xianghui; Zhang, Zhijun; Wang, Yongting; Sun, Dapeng; Pradhan, Sriharsa

    2016-07-30

    Induced pluripotent mesenchymal stem cells (iPMSCs) are novel candidates for drug screening, regenerative medicine, and cell therapy. However, introduction of transcription factor encoding genes for induced pluripotent stem cell (iPSC) generation which could be used to generate mesenchymal stem cells is accompanied by the risk of insertional mutations in the target cell genome. We demonstrate a novel method using an inactivated viral particle to package and deliver four purified recombinant Yamanaka transcription factors (Sox2, Oct4, Klf4, and c-Myc) resulting in reprogramming of human primary fibroblasts. Whole genome bisulfite sequencing was used to analyze genome-wide CpG methylation of human iPMSCs. Western blot, quantitative PCR, immunofluorescence, and in-vitro differentiation were used to assess the pluripotency of iPMSCs. The resulting reprogrammed fibroblasts show high-level expression of stem cell markers. The human fibroblast-derived iPMSC genome showed gains in DNA methylation in low to medium methylated regions and concurrent loss of methylation in previously hypermethylated regions. Most of the differentially methylated regions are close to transcription start sites and many of these genes are pluripotent pathway associated. We found that DNA methylation of these genes is regulated by the four iPSC transcription factors, which functions as an epigenetic switch during somatic reprogramming as reported previously. These iPMSCs successfully differentiate into three embryonic germ layer cells, both in vitro and in vivo. Following multipotency induction in our study, the delivered transcription factors were degraded, leading to an improved efficiency of subsequent programmed differentiation. Recombinant transcription factor based reprogramming and derivatization of iPMSC offers a novel high-efficiency approach for regenerative medicine from patient-derived cells.

  13. 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.

  14. The HIST1 Locus Escapes Reprogramming in Cloned Bovine Embryos

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    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.

  15. 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.

  16. 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.

  17. 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

  18. Strategies for Analyzing Data from Intact Groups.

    Science.gov (United States)

    Cross, Lawrence H.; Lane, Carolyn E.

    Action research often necessitates the use of intact groups for the comparison of educational treatments or programs. This paper considers several analytical methods that might be used for such situations when pretest scores indicate that these intact groups differ significantly initially. The methods considered include gain score analysis of…

  19. Exploratory investigations of hypervelocity intact capture spectroscopy

    Science.gov (United States)

    Tsou, P.; Griffiths, D. J.

    1993-01-01

    The ability to capture hypervelocity projectiles intact opens a new technique available for hypervelocity research. A determination of the reactions taking place between the projectile and the capture medium during the process of intact capture is extremely important to an understanding of the intact capture phenomenon, to improving the capture technique, and to developing a theory describing the phenomenon. The intact capture of hypervelocity projectiles by underdense media generates spectra, characteristic of the material species of projectile and capture medium involved. Initial exploratory results into real-time characterization of hypervelocity intact capture techniques by spectroscopy include ultra-violet and visible spectra obtained by use of reflecting gratings, transmitting gratings, and prisms, and recorded by photographic and electronic means. Spectrometry proved to be a valuable real-time diagnostic tool for hypervelocity intact capture events, offering understanding of the interactions of the projectile and the capture medium during the initial period and providing information not obtainable by other characterizations. Preliminary results and analyses of spectra produced by the intact capture of hypervelocity aluminum spheres in polyethylene (PE), polystyrene (PS), and polyurethane (PU) foams are presented. Included are tentative emission species identifications, as well as gray body temperatures produced in the intact capture process.

  20. Intact glycopeptide characterization using mass spectrometry

    OpenAIRE

    Cao, Li; Qu, Yi; Zhang, Zhaorui; Wang, Zhe; Prykova, Iya; Wu, Si

    2016-01-01

    Glycosylation is one of the most prominent and extensively studied protein post-translational modifications. However, traditional proteomic studies at the peptide level (bottom-up) rarely characterize intact glycopeptides (glycosylated peptides without removing glycans), so no glycoprotein heterogeneity information is retained. Intact glycopeptide characterization, on the other hand, provides opportunities to simultaneously elucidate the glycan structure and the glycosylation site needed to r...

  1. 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

  2. Activation of pluripotency genes in human fibroblast cells by a novel mRNA based approach.

    Directory of Open Access Journals (Sweden)

    Jordan R Plews

    2010-12-01

    Full Text Available Several methods have been used to induce somatic cells to re-enter the pluripotent state. Viral transduction of reprogramming genes yields higher efficiency but involves random insertions of viral sequences into the human genome. Although induced pluripotent stem (iPS cells can be obtained with the removable PiggyBac transposon system or an episomal system, both approaches still use DNA constructs so that resulting cell lines need to be thoroughly analyzed to confirm they are free of harmful genetic modification. Thus a method to change cell fate without using DNA will be very useful in regenerative medicine.In this study, we synthesized mRNAs encoding OCT4, SOX2, cMYC, KLF4 and SV40 large T (LT and electroporated them into human fibroblast cells. Upon transfection, fibroblasts expressed these factors at levels comparable to, or higher than those in human embryonic stem (ES cells. Ectopically expressed OCT4 localized to the cell nucleus within 4 hours after mRNA introduction. Transfecting fibroblasts with a mixture of mRNAs encoding all five factors significantly increased the expression of endogenous OCT4, NANOG, DNMT3β, REX1 and SALL4. When such transfected fibroblasts were also exposed to several small molecules (valproic acid, BIX01294 and 5'-aza-2'-deoxycytidine and cultured in human embryonic stem cell (ES medium they formed small aggregates positive for alkaline phosphatase activity and OCT4 protein within 30 days.Our results demonstrate that mRNA transfection can be a useful approach to precisely control the protein expression level and short-term expression of reprogramming factors is sufficient to activate pluripotency genes in differentiated cells.

  3. Epigenetic Induction of Definitive and Pancreatic Endoderm Cell Fate in Human Fibroblasts

    Directory of Open Access Journals (Sweden)

    Rangarajan Sambathkumar

    2016-01-01

    Full Text Available Reprogramming can occur by the introduction of key transcription factors (TFs as well as by epigenetic changes. We demonstrated that histone deacetylase inhibitor (HDACi Trichostatin A (TSA combined with a chromatin remodeling medium (CRM induced expression of a number of definitive endoderm and early and late pancreatic marker genes. When CRM was omitted, endoderm/pancreatic marker genes were not induced. Furthermore, treatment with DNA methyltransferase inhibitor (DNMTi 5-azacytidine (5AZA CRM did not affect gene expression changes, and when 5AZA was combined with TSA, no further increase in gene expression of endoderm, pancreatic endoderm, and endocrine markers was seen over levels induced with TSA alone. Interestingly, TSA-CRM did not affect expression of pluripotency and hepatocyte genes but induced some mesoderm transcripts. Upon removal of TSA-CRM, the endoderm/pancreatic gene expression profile returned to baseline. Our findings underscore the role epigenetic modification in transdifferentiation of one somatic cell into another. However, full reprogramming of fibroblasts to β-cells will require combination of this approach with TF overexpression and/or culture of the partially reprogrammed cells under β-cell specific conditions.

  4. Bilirubin glucuronidation by intact Gunn rat fibroblasts expressing bilirubin UDP-glucuronosyltransferase

    NARCIS (Netherlands)

    Seppen, J.; Tada, K.; Hellwig, S.; Bakker, C. T.; Prasad, V. R.; Roy Chowdhury, N.; Roy Chowdhury, J.; Bosma, P. J.; Oude Elferink, R. P.

    1996-01-01

    Crigler-Najjar (CN) disease is an inherited disorder of bilirubin metabolism. The disease is caused by a deficiency of the hepatic enzyme bilirubin UDP-glucuronosyltransferase (B-UGT). Patients with CN disease have high serum levels of the toxic compound, unconjugated bilirubin. The only defect in

  5. 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.

  6. Molecular Imaging Of Metabolic Reprogramming In Mutant IDH Cells

    Directory of Open Access Journals (Sweden)

    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.

  7. 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.

  8. 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.

  9. The hallmarks of fibroblast ageing.

    Science.gov (United States)

    Tigges, Julia; Krutmann, Jean; Fritsche, Ellen; Haendeler, Judith; Schaal, Heiner; Fischer, Jens W; Kalfalah, Faiza; Reinke, Hans; Reifenberger, Guido; Stühler, Kai; Ventura, Natascia; Gundermann, Sabrina; Boukamp, Petra; Boege, Fritz

    2014-06-01

    Ageing is influenced by the intrinsic disposition delineating what is maximally possible and extrinsic factors determining how that frame is individually exploited. Intrinsic and extrinsic ageing processes act on the dermis, a post-mitotic skin compartment mainly consisting of extracellular matrix and fibroblasts. Dermal fibroblasts are long-lived cells constantly undergoing damage accumulation and (mal-)adaptation, thus constituting a powerful indicator system for human ageing. Here, we use the systematic of ubiquitous hallmarks of ageing (Lopez-Otin et al., 2013, Cell 153) to categorise the available knowledge regarding dermal fibroblast ageing. We discriminate processes inducible in culture from phenomena apparent in skin biopsies or primary cells from old donors, coming to the following conclusions: (i) Fibroblasts aged in culture exhibit most of the established, ubiquitous hallmarks of ageing. (ii) Not all of these hallmarks have been detected or investigated in fibroblasts aged in situ (in the skin). (iii) Dermal fibroblasts aged in vitro and in vivo exhibit additional features currently not considered ubiquitous hallmarks of ageing. (iv) The ageing process of dermal fibroblasts in their physiological tissue environment has only been partially elucidated, although these cells have been a preferred model of cell ageing in vitro for decades. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  10. 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.

  11. 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

  12. 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.

  13. Intact glycopeptide characterization using mass spectrometry.

    Science.gov (United States)

    Cao, Li; Qu, Yi; Zhang, Zhaorui; Wang, Zhe; Prytkova, Iya; Wu, Si

    2016-05-01

    Glycosylation is one of the most prominent and extensively studied protein post-translational modifications. However, traditional proteomic studies at the peptide level (bottom-up) rarely characterize intact glycopeptides (glycosylated peptides without removing glycans), so no glycoprotein heterogeneity information is retained. Intact glycopeptide characterization, on the other hand, provides opportunities to simultaneously elucidate the glycan structure and the glycosylation site needed to reveal the actual biological function of protein glycosylation. Recently, significant improvements have been made in the characterization of intact glycopeptides, ranging from enrichment and separation, mass spectroscopy (MS) detection, to bioinformatics analysis. In this review, we recapitulated currently available intact glycopeptide characterization methods with respect to their advantages and limitations as well as their potential applications.

  14. Direct conversion of human fibroblasts into functional osteoblasts by defined factors.

    Science.gov (United States)

    Yamamoto, Kenta; Kishida, Tsunao; Sato, Yoshiki; Nishioka, Keisuke; Ejima, Akika; Fujiwara, Hiroyoshi; Kubo, Toshikazu; Yamamoto, Toshiro; Kanamura, Narisato; Mazda, Osam

    2015-05-12

    Osteoblasts produce calcified bone matrix and contribute to bone formation and remodeling. In this study, we established a procedure to directly convert human fibroblasts into osteoblasts by transducing some defined factors and culturing in osteogenic medium. Osteoblast-specific transcription factors, Runt-related transcription factor 2 (Runx2), and Osterix, in combination with Octamer-binding transcription factor 3/4 (Oct4) and L-Myc (RXOL) transduction, converted ∼ 80% of the fibroblasts into osteocalcin-producing cells. The directly converted osteoblasts (dOBs) induced by RXOL displayed a similar gene expression profile as normal human osteoblasts and contributed to bone repair after transplantation into immunodeficient mice at artificial bone defect lesions. The dOBs expressed endogenous Runx2 and Osterix, and did not require continuous expression of the exogenous genes to maintain their phenotype. Another combination, Oct4 plus L-Myc (OL), also induced fibroblasts to produce bone matrix, but the OL-transduced cells did not express Osterix and exhibited a more distant gene expression profile to osteoblasts compared with RXOL-transduced cells. These findings strongly suggest successful direct reprogramming of fibroblasts into functional osteoblasts by RXOL, a technology that may provide bone regeneration therapy against bone disorders.

  15. Modeling the phenotype of spinal muscular atrophy by the direct conversion of human fibroblasts to motor neurons.

    Science.gov (United States)

    Zhang, Qi-Jie; Li, Jin-Jing; Lin, Xiang; Lu, Ying-Qian; Guo, Xin-Xin; Dong, En-Lin; Zhao, Miao; He, Jin; Wang, Ning; Chen, Wan-Jin

    2017-02-14

    Spinal muscular atrophy (SMA) is a lethal autosomal recessive neurological disease characterized by selective degeneration of motor neurons in the spinal cord. In recent years, the development of cellular reprogramming technology has provided an alternative and effective method for obtaining patient-specific neurons in vitro. In the present study, we applied this technology to the field of SMA to acquire patient-specific induced motor neurons that were directly converted from fibroblasts via the forced expression of 8 defined transcription factors. The infected fibroblasts began to grow in a dipolar manner, and the nuclei gradually enlarged. Typical Tuj1-positive neurons were generated at day 23. After day 35, induced neurons with multiple neurites were observed, and these neurons also expressed the hallmarks of Tuj1, HB9, ISL1 and CHAT. The conversion efficiencies were approximately 5.8% and 5.5% in the SMA and control groups, respectively. Additionally, the SMA-induced neurons exhibited a significantly reduced neurite outgrowth rate compared with the control neurons. After day 60, the SMA-induced neurons also exhibited a liability of neuronal degeneration and remarkable fracturing of the neurites was observed. By directly reprogramming fibroblasts, we established a feeder-free conversion system to acquire SMA patient-specific induced motor neurons that partially modeled the phenotype of SMA in vitro.

  16. DNA excision repair in permeable human fibroblasts

    International Nuclear Information System (INIS)

    Kaufmann, W.K.; Bodell, W.J.; Cleaver, J.E.

    1983-01-01

    U.v. irradiation of confluent human fibroblasts activated DNA repair, aspects of which were characterized in the cells after they were permeabilized. Incubation of intact cells for 20 min between irradiation and harvesting was necessary to obtain a maximum rate of reparative DNA synthesis. Cells harvested immediately after irradiation before repair was initiated displayed only a small stimulation of DNA synthesis, indicating that permeable cells have a reduced capacity to recognize pyrimidine dimers and activate repair. The distribution of sizes of DNA strands labeled during 10 min of reparative DNA synthesis resembled that of parental DNA. However, during a 60-min incubation of permeable cells at 37 degrees C, parental DNA and DNA labeled by reparative DNA synthesis were both cleaved to smaller sizes. Cleavage also occurred in unirradiated cells, indicating that endogenous nuclease was active during incubation. Repair patches synthesized in permeable cells displayed increased sensitivity to digestion by micrococcal nuclease. However, the change in sensitivity during a chase with unlabeled DNA precursors was small, suggesting that reassembly of nucleosome structure at sites of repair was impaired. To examine whether this deficiency was due to a preponderance of incomplete or unligated repair patches, 3H-labeled (repaired) DNA was purified, then digested with exonuclease III and nuclease S1 to probe for free 3' ends and single-stranded regions. About 85% of the [3H]DNA synthesized during a 10-min pulse resisted digestion, suggesting that a major fraction of the repair patches that were filled were also ligated. U.v. light-activated DNA synthesis in permeable cells, therefore, appears to represent the continuation of reparative gap-filling at sites of excision repair activated within intact cells. Gap-filling and ligation were comparatively efficient processes in permeable cells

  17. 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.

  18. 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.

  19. Rat embryo fibroblasts require both the cell-binding and the heparin-binding domains of fibronectin for survival

    DEFF Research Database (Denmark)

    Jeong, J; Han, I; Lim, Y

    2001-01-01

    of the cell-binding domain of FN with integrin is sufficient to rescue rat embryo fibroblasts (REFs) from detachment-induced apoptosis. REFs attached and spread normally after plating on substrates coated with either intact FN or a FN fragment, FN120, that contains the cell-binding domain but lacks the C...

  20. Secretion of intact proteins and peptide fragments by lysosomal pathways of protein degradation

    International Nuclear Information System (INIS)

    Isenman, L.D.; Dice, J.F.

    1989-01-01

    We report that degradation of proteins microinjected into human fibroblasts is accompanied by release into the culture medium of peptide fragments and intact proteins as well as single amino acids. For the nine proteins and polypeptides microinjected, acid-precipitable radioactivity, i.e. peptide fragments and/or intact proteins, ranged from 10 to 67% of the total released radioactivity. Peptide fragments and/or intact protein accounted for 60% of the radioactivity released into the medium by cells microinjected with ribonuclease A. Two major radiolabeled peptide fragments were found, and one was of an appropriate size to function as an antigen in antigen-presenting cells. The peptides released from microinjected ribonuclease A were derived from lysosomal pathways of proteolysis based on several lines of evidence. Previous studies have shown that microinjected ribonuclease A is degraded to single amino acids entirely within lysosomes. We show that release of free amino acids and peptide fragments and/or intact protein was equivalently stimulated by serum deprivation and equivalently inhibited by NH4Cl. We also show that lysosomal degradation of endocytosed [3H]ribonuclease A was accompanied by the release of two peptide fragments similar in size and charge to those from microinjected [ 3 H]ribonuclease A. These findings demonstrate that degradation within lysosomes occurs in a manner that spares specific peptides; they also suggest a previously unsuspected pathway by which cells can secrete cytosol-derived polypeptides

  1. Key role of the kidney in the regulation of fibroblast growth factor 23

    DEFF Research Database (Denmark)

    Mace, Maria L; Gravesen, Eva; Hofman-Bang, Jacob

    2015-01-01

    was significantly increased in BNX rats. The rapid rise in FGF23 after BNX was independent of parathyroid hormone or FGF receptor signaling. No evidence of early stimulation of FGF23 gene expression in the bone was found. Furthermore, acute severe hyperphosphatemia or hypercalcemia had no impact on intact FGF23......High circulating levels of fibroblast growth factor 23 (FGF23) have been demonstrated in kidney failure, but mechanisms of this are not well understood. Here we examined the impact of the kidney on the early regulation of intact FGF23 in acute uremia as induced by bilateral or unilateral...

  2. 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.

  3. 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...

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

    Directory of Open Access Journals (Sweden)

    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.

  5. 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

  6. Fibroblast growth factor receptors in breast cancer.

    Science.gov (United States)

    Wang, Shuwei; Ding, Zhongyang

    2017-05-01

    Fibroblast growth factor receptors are growth factor receptor tyrosine kinases, exerting their roles in embryogenesis, tissue homeostasis, and development of breast cancer. Recent genetic studies have identified some subtypes of fibroblast growth factor receptors as strong genetic loci associated with breast cancer. In this article, we review the recent epidemiological findings and experiment results of fibroblast growth factor receptors in breast cancer. First, we summarized the structure and physiological function of fibroblast growth factor receptors in humans. Then, we discussed the common genetic variations in fibroblast growth factor receptors that affect breast cancer risk. In addition, we also introduced the potential roles of each fibroblast growth factor receptors isoform in breast cancer. Finally, we explored the potential therapeutics targeting fibroblast growth factor receptors for breast cancer. Based on the biological mechanisms of fibroblast growth factor receptors leading to the pathogenesis in breast cancer, targeting fibroblast growth factor receptors may provide new opportunities for breast cancer therapeutic strategies.

  7. DNA synthesis in vitro in human fibroblast preparations

    Energy Technology Data Exchange (ETDEWEB)

    Kaufmann, W.K.

    1983-01-01

    When confluent cultures of human fibroblasts were ultraviolet irradiated and either permeabilized or lysed, three types of DNA synthesis were subsequently observed during incubation in vitro: (A) a low level of DNA replication, which ceased after 15-30 min incubation at 37/sup 0/C; (B) radiation-dependent reparative gap-filling, which also ceased after 15 min at 37/sup 0/C; and (C) radiation-independent DNA synthesis, which was not semiconservative and proceeded at a linear rate for 1 hr at 37/sup 0/C. Normal and xeroderma pigmentosum fibroblasts displayed different rates of radiation-dependent reparative gap-filling after lysis but similar rates of radiation-independent DNA synthesis. The rates of DNA replication and radiation-independent DNA synthesis were less in the permeable cell system than in the lysed cell system, whereas radiation-dependent reparative gap-filling was the same in both. Preparations of permeable and lysed cells activated radiation-dependent reparative gap-filling at about 15% of the rate estimated for intact cells. No radiation-dependent DNA strand breaks, as assayed by alkaline elution, were observed in the lysed cell preparation. Some radiation-dependent breaks were observed in the permeable cell preparation, but radiation-dependent DNA breakage was less than that seen in intact cells. This inability to incise DNA at damaged sites could account for the low rate of activation of reparative gap-filling in vitro. DNA strand breaks were produced in fibroblast preparations nonspecifically during lysis or permeabilization and incubation in vitro, and this breakage of DNA probably was responsible for the radiation-independent DNA synthesis.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  10. 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.

  11. Fibroblast Growth Factor 23 and Kidney Disease Progression in Autosomal Dominant Polycystic Kidney Disease.

    Science.gov (United States)

    Chonchol, Michel; Gitomer, Berenice; Isakova, Tamara; Cai, Xuan; Salusky, Isidro; Pereira, Renata; Abebe, Kaleab; Torres, Vicente; Steinman, Theodor I; Grantham, Jared J; Chapman, Arlene B; Schrier, Robert W; Wolf, Myles

    2017-09-07

    Increases in fibroblast growth factor 23 precede kidney function decline in autosomal dominant polycystic kidney disease; however, the role of fibroblast growth factor 23 in autosomal dominant polycystic kidney disease has not been well characterized. We measured intact fibroblast growth factor 23 levels in baseline serum samples from 1002 participants in the HALT-PKD Study A ( n =540; mean eGFR =91±17 ml/min per 1.73 m 2 ) and B ( n =462; mean eGFR =48±12 ml/min per 1.73 m 2 ). We used linear mixed and Cox proportional hazards models to test associations between fibroblast growth factor 23 and eGFR decline, percentage change in height-adjusted total kidney volume, and composite of time to 50% reduction in eGFR, onset of ESRD, or death. Median (interquartile range) intact fibroblast growth factor 23 was 44 (33-56) pg/ml in HALT-PKD Study A and 69 (50-93) pg/ml in Study B. In adjusted models, annualized eGFR decline was significantly faster in the upper fibroblast growth factor 23 quartile (Study A: quartile 4, -3.62; 95% confidence interval, -4.12 to -3.12 versus quartile 1, -2.51; 95% confidence interval, -2.71 to -2.30 ml/min per 1.73 m 2 ; P for trend kidney volume in adjusted models (quartile 4, 6.76; 95% confidence interval, 5.57 to 7.96 versus quartile 1, 6.04; 95% confidence interval, 5.55 to 6.54; P for trend =0.03). In Study B, compared with the lowest quartile, the highest fibroblast growth factor 23 quartile was associated with elevated risk for the composite outcome (hazard ratio, 3.11; 95% confidence interval, 1.84 to 5.25). Addition of fibroblast growth factor 23 to a model of annualized decline in eGFR≥3.0 ml/min per 1.73 m 2 did not improve risk prediction. Higher serum fibroblast growth factor 23 concentration was associated with kidney function decline, height-adjusted total kidney volume percentage increase, and death in patients with autosomal dominant polycystic kidney disease. However, fibroblast growth factor 23 did not substantially

  12. Intacting Integrity in coping with health issues

    DEFF Research Database (Denmark)

    Jepsen, Stine Leegaard; Bastrup Jørgensen, Lene; Fridlund, Bengt

    2017-01-01

    The aim of this study was to develop a formal substantive theory (FST) on the multidimensional behavioral process of coping with health issues. Intacting integrity while coping with health issues emerged as the core category of this FST. People facing health issues strive to safeguard and keep...

  13. Epispadias in boys with an intact prepuce

    NARCIS (Netherlands)

    Bos, E. M. E.; Kuijper, C. F.; Chrzan, R. J.; Dik, P.; Klijn, A. J.; de Jong, T. P. V. M.

    2014-01-01

    To present an overview of the clinical presentation and pathological anatomy, and the results of surgical correction of 7 cases of epispadias with intact prepuce; a rare condition that has only occasionally been reported in literature. A retrospective search was performed in the surgical and

  14. HYDROCARBON VAPOR DIFFUSION IN INTACT CORE SLEEVES

    Science.gov (United States)

    The diffusion of 2,2,4-trimethylpentane (TMP) and 2,2,5-trimethylhexane (TMH) vapors put of residually contaminated sandy soil from the U.S. Environmental Protection Agency (EPA) field research site at Traverse City, Michigan, was measured and modeled. The headspace of an intact ...

  15. Early detection of degraded A14-125I-insulin in human fibroblasts by the use of high performance liquid chromatography

    International Nuclear Information System (INIS)

    Stentz, F.B.; Harris, H.L.; Kitabchi, A.E.

    1983-01-01

    We studied the metabolism of A14-125I-insulin in intact human fibroblasts using high performance liquid chromatography (HPLC) to detect and separate its early degradation products. The high resolving power of HPLC enabled us to separate what has been considered ''intact insulin'' by Sephadex G-50 chromatography or TCA precipitability into two additional peaks that had decreased biochemical properties with respect to immunoprecipitability and receptor binding but not decreased TCA precipitability. We conclude that human fibroblast is capable of metabolizing insulin within 2 min at 37 degrees C into intermediate molecules that can be detected by HPLC but not by TCA precipitability or molecular sieve chromatography

  16. Microprobe analysis of human fibroblasts

    International Nuclear Information System (INIS)

    Allan, G.L.; Zhu, J.; Legge, G.J.F.

    1985-01-01

    The Melbourne Proton Microprobe has been used to study the copper content in human skin fibroblast cells derived from patients with the genetic disease Menkes Syndrome. Both normal and diseased cells have been studied to investigate any elemental differences occurring between the two cell types. This paper details the preparatory techniques necessary for individual cell analysis and presents the elemental information with a new three dimensional contour mapping technique. These maps are used to highlight elemental differences between normal and mutant fibroblasts. The work also confirms the expected copper excess found in the Menkes cell and indicates that the microprobe can be used for rapid identification of a Menkes carrier

  17. Small Particles Intact Capture Experiment (SPICE)

    Science.gov (United States)

    Nishioka, Ken-Ji; Carle, G. C.; Bunch, T. E.; Mendez, David J.; Ryder, J. T.

    1994-01-01

    The Small Particles Intact Capture Experiment (SPICE) will develop technologies and engineering techniques necessary to capture nearly intact, uncontaminated cosmic and interplanetary dust particles (IDP's). Successful capture of such particles will benefit the exobiology and planetary science communities by providing particulate samples that may have survived unaltered since the formation of the solar system. Characterization of these particles may contribute fundamental data to our knowledge of how these particles could have formed into our planet Earth and, perhaps, contributed to the beginnings of life. The term 'uncontaminated' means that captured cosmic and IDP particles are free of organic contamination from the capture process and the term 'nearly intact capture' means that their chemical and elemental components are not materially altered during capture. The key to capturing cosmic and IDP particles that are organic-contamination free and nearly intact is the capture medium. Initial screening of capture media included organic foams, multiple thin foil layers, and aerogel (a silica gel); but, with the exception of aerogel, the requirements of no contamination or nearly intact capture were not met. To ensure no contamination of particles in the capture process, high-purity aerogel was chosen. High-purity aerogel results in high clarity (visual clearness), a useful quality in detection and recovery of embedded captured particles from the aerogel. P. Tsou at the Jet Propulsion Laboratory (JPL) originally described the use of aerogel for this purpose and reported laboratory test results. He has flown aerogel as a 'GAS-can Lid' payload on STS-47 and is evaluating the results. The Timeband Capture Cell Experiment (TICCE), a Eureca 1 experiment, is also flying aerogel and is scheduled for recovery in late April.

  18. Transformation of human mesenchymal cells and skin fibroblasts into hematopoietic cells.

    Directory of Open Access Journals (Sweden)

    David M Harris

    Full Text Available Patients with prolonged myelosuppression require frequent platelet and occasional granulocyte transfusions. Multi-donor transfusions induce alloimmunization, thereby increasing morbidity and mortality. Therefore, an autologous or HLA-matched allogeneic source of platelets and granulocytes is needed. To determine whether nonhematopoietic cells can be reprogrammed into hematopoietic cells, human mesenchymal stromal cells (MSCs and skin fibroblasts were incubated with the demethylating agent 5-azacytidine (Aza and the growth factors (GF granulocyte-macrophage colony-stimulating factor and stem cell factor. This treatment transformed MSCs to round, non-adherent cells expressing T-, B-, myeloid-, or stem/progenitor-cell markers. The transformed cells engrafted as hematopoietic cells in bone marrow of immunodeficient mice. DNA methylation and mRNA array analysis suggested that Aza and GF treatment demethylated and activated HOXB genes. Indeed, transfection of MSCs or skin fibroblasts with HOXB4, HOXB5, and HOXB2 genes transformed them into hematopoietic cells. Further studies are needed to determine whether transformed MSCs or skin fibroblasts are suitable for therapy.

  19. Case report 511: Fibroblastic rheumatism

    International Nuclear Information System (INIS)

    Hernandez, R.J.; Martel, W.; Headington, J.T.; Kaufman, R.A.; Cincinnati Univ., OH

    1989-01-01

    We report a ten-year-old child with the newly described entity of fibroblastic rheumatism. This child developed rapid, progressive, symmetrical polyarthritis, similar to the radiographic appearance of juvenile rheumatoid arthritis, except for the rapidity of progression. The polyarthritis was preceded by the development of skin nodules with characteristic histological changes. (orig./GDG)

  20. Cellular reprogramming through mitogen-activated protein kinases

    Directory of Open Access Journals (Sweden)

    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.

  1. 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

  2. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  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. An easy method for preparation of Cre-loxP regulated fluorescent adenoviral expression vectors and its application for direct reprogramming into hepatocytes

    Directory of Open Access Journals (Sweden)

    Chitose Kurihara

    2016-12-01

    Full Text Available The recombinant adenoviral gene expression system is a powerful tool for gene delivery. However, it is difficult to obtain high titers of infectious virus, principally due to the toxicity of the expressed gene which affects on virus replication in the host HEK293 cells. To avoid these problems, we generated a Cre-loxP-regulated fluorescent universal vector (termed pAxCALRL. This vector produces recombinant adenoviruses that express the red fluorescent protein (RFP instead of the inserted gene during proliferation, which limits toxicity and can be used to monitor viral replication. Expression of the gene of interest is induced by co-infection with an adenovirus that expresses Cre-recombinase (AxCANCre. Recombinant adenovirus produced by this system that express Hnf4α and Foxa2 were used to reprogram mouse embryo fibroblast (MEF into induced-hepatocyte-like cells (iHep following several rounds of infection, demonstrating the efficacy of this new system.

  6. Measurement of diffusive properties of intact rock

    Energy Technology Data Exchange (ETDEWEB)

    Harvey, K B

    1996-12-01

    In the Postclosure Assessment of a Reference System for the Disposal of Canada`s Nuclear Fuel Waste (Goodwin et al. 1994) the disposal vault is assumed to be surrounded by a zone of intact rock, referred to as the `exclusion zone.` A sensitivity analysis of the relative effectiveness of the several engineered and natural barriers that contribute to the safety of the reference disposal system has shown that this zone of intact rock is the most effective of these barriers to the movement of radionuclides through the reference system. Peer review of the geosphere model used in the case study for the EIS (Environmental Impact Statement) of the Canadian Nuclear Fuel Waste Management Program has identified the need to quantify the properties of the intact rock surrounding the disposal vault that would control the transport of radionuclides by diffusion. The Postclosure Assessment also identified the need for appropriate values of the free water diffusion coefficient (D{sub o}) for {sup 129}1 and {sup 14}C. The measurement of rock resistivity allows the calculation of the Formation Factor for a rock This review describes the Formation Factor, diffusivity, permeability, and porosity, and how these properties might be measured or inferred for insitu rock under the conditions that apply to the intact rock surrounding a potential disposal vault. The importance of measuring the intrinsic diffusion coefficient (D{sup i}) of diffusing species under solution salinities simulating those of groundwaters is emphasised, and a method of measurement is described that is independent of the diffusing medium, and which would be appropriate for measurements made in chemically complex media such as groundwaters. (author). 95 refs., 4 tabs., 39 figs.

  7. Measurement of diffusive properties of intact rock

    International Nuclear Information System (INIS)

    Harvey, K.B.

    1996-12-01

    In the Postclosure Assessment of a Reference System for the Disposal of Canada's Nuclear Fuel Waste (Goodwin et al. 1994) the disposal vault is assumed to be surrounded by a zone of intact rock, referred to as the 'exclusion zone.' A sensitivity analysis of the relative effectiveness of the several engineered and natural barriers that contribute to the safety of the reference disposal system has shown that this zone of intact rock is the most effective of these barriers to the movement of radionuclides through the reference system. Peer review of the geosphere model used in the case study for the EIS (Environmental Impact Statement) of the Canadian Nuclear Fuel Waste Management Program has identified the need to quantify the properties of the intact rock surrounding the disposal vault that would control the transport of radionuclides by diffusion. The Postclosure Assessment also identified the need for appropriate values of the free water diffusion coefficient (D o ) for 129 1 and 14 C. The measurement of rock resistivity allows the calculation of the Formation Factor for a rock This review describes the Formation Factor, diffusivity, permeability, and porosity, and how these properties might be measured or inferred for insitu rock under the conditions that apply to the intact rock surrounding a potential disposal vault. The importance of measuring the intrinsic diffusion coefficient (D i ) of diffusing species under solution salinities simulating those of groundwaters is emphasised, and a method of measurement is described that is independent of the diffusing medium, and which would be appropriate for measurements made in chemically complex media such as groundwaters. (author). 95 refs., 4 tabs., 39 figs

  8. 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.

  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. 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 ...

  11. 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.

  12. 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.

  13. Autism Spectrum Disorder and intact executive functioning.

    Science.gov (United States)

    Ferrara, R; Ansermet, F; Massoni, F; Petrone, L; Onofri, E; Ricci, P; Archer, T; Ricci, S

    2016-01-01

    Earliest notions concerning autism (Autism Spectrum Disorders, ASD) describe the disturbance in executive functioning. Despite altered definition, executive functioning, expressed as higher cognitive skills required complex behaviors linked to the prefrontal cortex, are defective in autism. Specific difficulties in children presenting autism or verbal disabilities at executive functioning levels have been identified. Nevertheless, the developmental deficit of executive functioning in autism is highly diversified with huge individual variation and may even be absent. The aim of the present study to examine the current standing of intact executive functioning intact in ASD. Analysis of ASD populations, whether high-functioning, Asperger's or autism Broad Phenotype, studied over a range of executive functions including response inhibition, planning, cognitive flexibility, cognitive inhibition, and alerting networks indicates an absence of damage/impairment compared to the typically-developed normal control subjects. These findings of intact executive functioning in ASD subjects provide a strong foundation on which to construct applications for growth environments and the rehabilitation of autistic subjects.

  14. 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

  15. 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...

  16. 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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    OpenAIRE

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

    2013-01-01

    Nupura S Bhise,1,* Karl J Wahlin,2,* Donald J Zack,2–4 Jordan J Green1,21Department of Biomedical Engineering, Translational Tissue Engineering Center, and Institute for Nanobiotechnology, 2Department of Ophthalmology, The Johns Hopkins University School of Medicine, Baltimore, MD, 3Solomon H Snyder Department of Neuroscience, Department of Molecular Biology and Genetics, and Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; 4Institu...

  19. Physiological Actions of Fibroblast Growth Factor-23

    Directory of Open Access Journals (Sweden)

    Reinhold G. Erben

    2018-05-01

    Full Text Available Fibroblast growth factor-23 (FGF23 is a bone-derived hormone suppressing phosphate reabsorption and vitamin D hormone synthesis in the kidney. At physiological concentrations of the hormone, the endocrine actions of FGF23 in the kidney are αKlotho-dependent, because high-affinity binding of FGF23 to FGF receptors requires the presence of the co-receptor αKlotho on target cells. It is well established that excessive concentrations of intact FGF23 in the blood lead to phosphate wasting in patients with normal kidney function. Based on the importance of diseases associated with gain of FGF23 function such as phosphate-wasting diseases and chronic kidney disease, a large body of literature has focused on the pathophysiological consequences of FGF23 excess. Less emphasis has been put on the role of FGF23 in normal physiology. Nevertheless, during recent years, lessons we have learned from loss-of-function models have shown that besides the paramount physiological roles of FGF23 in the control of 1α-hydroxylase expression and of apical membrane expression of sodium-phosphate co-transporters in proximal renal tubules, FGF23 also is an important stimulator of calcium and sodium reabsorption in distal renal tubules. In addition, there is an emerging role of FGF23 as an auto-/paracrine regulator of alkaline phosphatase expression and mineralization in bone. In contrast to the renal actions of FGF23, the FGF23-mediated suppression of alkaline phosphatase in bone is αKlotho-independent. Moreover, FGF23 may be a physiological suppressor of differentiation of hematopoietic stem cells into the erythroid lineage in the bone microenvironment. At present, there is little evidence for a physiological role of FGF23 in organs other than kidney and bone. The purpose of this mini-review is to highlight the current knowledge about the complex physiological functions of FGF23.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. How can we conserve intact tropical peatlands?

    Science.gov (United States)

    Lawson, Ian; Roucoux, Katherine

    2017-04-01

    The scientific community has, for more than three decades, been expressing increasing alarm about the fate of peatlands in parts of Indonesia and Malaysia, where extensive land-use conversion and drainage for rice and oil palm have greatly compromised peatland hydrology, ecology, biological richness, and carbon storage. The discourse in the literature on these peatlands is now moving on from attempts to preserve the last remaining fragments of peat-swamp forest, towards discussion of how best to restore damaged ecosystems, and whether it is possible to manage plantations more 'sustainably'. It is becoming increasingly clear, however, that peatlands occur quite widely in other parts of the lowland tropics, including parts of Amazonia and the Congo Basin, and many of these peatlands can reasonably be described as 'intact': although few if any parts of the tropics are totally unaffected by human actions, the hydrology and functional ecology of these systems appear to be close to a 'natural' state. The question then arises as to what should be done with the knowledge of their existence. Here we analyse the arguments in favour of protecting intact peatlands, and the potential conflicts with other priorities such as economic development and social justice. We evaluate alternative mechanisms for protecting intact peatlands, focusing on the particular issues raised by peatlands as opposed to other kinds of tropical ecosystem. We identify ways in which natural science agendas can help to inform these arguments, using our own contributions in palaeoecology and carbon mapping as examples. Finally, we argue for a radical reconsideration of research agendas in tropical peatlands, highlighting the potential contribution of methodologies borrowed from the social sciences and humanities.

  8. Inhibition of EGF processing in responsive and nonresponsive human fibroblasts

    International Nuclear Information System (INIS)

    Schaudies, R.P.; Wray, H.L.

    1988-01-01

    We have examined the proteolytic processing of radiolabeled epidermal growth factor (EGF) in EGF growth-responsive human foreskin fibroblasts (HFF) versus EGF nonresponsive human fetal lung fibroblasts (HFL). Previous studies have shown that both cell lines demonstrate similar binding affinities and numbers of binding sites, as well as similar rates of internalization and degradation of the bound, radiolabeled hormone. We have used nondenaturing electrophoresis to compare how these two cell lines process EGF at its carboxy terminus. EGF lacking either one [des-(53)-EGF] or six [des (48-53)-EGF] carboxy terminal amino acids could be distinguished by this method. Chloroquine or leupeptin were added to the incubation system in an attempt to accentuate potential differences in hormonal processing between the responsive and nonresponsive cell lines. In the absence of inhibitors, the responsive and nonresponsive cells generated similar distributions of processed forms of EGF after 30-minutes incubation. However, after 4-hours incubation in the constant presence of 125I-EGF, the electrophoretic profiles of extracted hormone were substantially different. The radiolabel within the responsive cells, as well as that released from them, migrated predominantly at the dye front, indicating complete degradation of EGF. In contrast, the majority of the radiolabel within the nonresponsive cells migrated as partially processed forms of hormone, while the released radiolabel migrated at the dye front. Addition of chloroquine to either cell line inhibited processing of EGF beyond removal of the carboxyl terminal arginine residue. Both intact 125I-EGF, and 125I-EGF lacking the carboxyl terminal arginine were released from chloroquine-treated cells in a ratio equal to that present in the intact cells

  9. Sorption of cesium in intact rock

    International Nuclear Information System (INIS)

    Puukko, E.

    2014-04-01

    The mass distribution coefficient K d is used in performance assessment (PA) to describe sorption of a radionuclide on rock. The R d is determined using crushed rock which causes uncertainty in converting the R d values to K d values for intact rock. This work describes a method to determine the equilibrium of sorption on intact rock. The rock types of the planned Olkiluoto waste disposal site were T-series mica gneiss (T-MGN), T-series tonalite granodiorite granite gneiss (T-TGG), P-series tonalite granodiorite granite gneiss (P-TGG) and pegmatitic granite (PGR). These rocks contain different amount of biotite which is the main sorbing mineral. The sorption of cesium on intact rock slices was studied by applying an electrical field to speed up migration of cesium into the rock. Cesium is in the solution as a noncomplex cation Cs + and it is sorbed by ion exchange. The tracer used in the experiments was 134 Cs. The experimental sorption on the intact rock is compared with values calculated using the in house cation exchange sorption model (HYRL model) in PHREEQC program. The observed sorption on T-MGN and T-TGG rocks was close to the calculated values. Two PGR samples were from a depth of 70 m and three samples were from a depth of 150 m. Cesium sorbed more than predicted on the two 70 m PGR samples. The sorption of Cs on the three 150 m PGR samples was small which was consistent with the calculations. The pegmatitic granite PGR has the smallest content of biotite of the four rock types. In the case of P-TGG rock the observed values of sorption were only half of the calculated values. Two kind of slices were cut from P-TGG drill core. The slices were against and to the direction of the foliation of the biotite rims. The sorption of cesium on P-TGG rock was same in both cases. The results indicated that there was no effect of the directions of the electric field and the foliation of biotite in the P-TGG rock. (orig.)

  10. Quantitative proteomic analysis of intact plastids.

    Science.gov (United States)

    Shiraya, Takeshi; Kaneko, Kentaro; Mitsui, Toshiaki

    2014-01-01

    Plastids are specialized cell organelles in plant cells that are differentiated into various forms including chloroplasts, chromoplasts, and amyloplasts, and fulfill important functions in maintaining the overall cell metabolism and sensing environmental factors such as sunlight. It is therefore important to grasp the mechanisms of differentiation and functional changes of plastids in order to enhance the understanding of vegetality. In this chapter, details of a method for the extraction of intact plastids that makes analysis possible while maintaining the plastid functions are provided; in addition, a quantitative shotgun method for analyzing the composition and changes in the content of proteins in plastids as a result of environmental impacts is described.

  11. Oxidation of molecular tritium by intact soils

    International Nuclear Information System (INIS)

    Sweet, C.W.; Murphy, C.E. Jr.

    1980-01-01

    The effects of environmental factors on the rate of oxidation of molecular tritium (T 2 ) to tritiated water (HTO) were determined for intact soils during field exposures. Maximum deposition velocities of approximately 0.03 cm/sec were measured for T 2 at low wind speeds for a variety of soils over a wide range of conditions. Deposition velocities were slightly inhibited in wet soils and at 0 0 C. In dry soils, oxidation of T 2 to HTO occurred deeper in the soil profile, but deposition velocities were unaffected

  12. Defining Optimized Properties of Modified mRNA to Enhance Virus- and DNA- Independent Protein Expression in Adult Stem Cells and Fibroblasts

    Directory of Open Access Journals (Sweden)

    Frauke Hausburg

    2015-02-01

    Full Text Available Background: By far, most strategies for cell reprogramming and gene therapy are based on the introduction of DNA after viral delivery. To avoid the high risks accompanying these goals, non-viral and DNA-free delivery methods for various cell types are required. Methods: Relying on an initially established PCR-based protocol for convenient template DNA production, we synthesized five differently modified EGFP mRNA (mmRNA species, incorporating various degrees of 5-methylcytidine-5'-triphosphate (5mC and pseudouridine-5'-triphosphate (Ψ. We then investigated their effect on i protein expression efficiencies and ii cell viability for human mesenchymal stem cells (hMSCs and fibroblasts from different origins. Results: Our protocol allows highly efficient mmRNA production in vitro, enabling rapid and stable protein expression after cell transfection. However, our results also demonstrate that the terminally optimal modification needs to be defined in pilot experiments for each particular cell type. Transferring our approach to the conversion of fibroblasts into skeletal myoblasts using mmRNA encoding MyoD, we confirm the huge potential of mmRNA based protein expression for virus- and DNA-free reprogramming strategies. Conclusion: The achieved high protein expression levels combined with good cell viability not only in fibroblasts but also in hMSCs provides a promising option for mmRNA based modification of various cell types including slowly proliferating adult stem cells. Therefore, we are confident that our findings will substantially contribute to the improvement of efficient cell reprogramming and gene therapy approaches.

  13. Fibroblast-matrix interplay: Nintedanib and pirfenidone modulate the effect of IPF fibroblast-conditioned matrix on normal fibroblast phenotype.

    Science.gov (United States)

    Epstein Shochet, Gali; Wollin, Lutz; Shitrit, David

    2018-03-12

    Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. Activated fibroblasts are the key effector cells in fibrosis, producing excessive amounts of collagen and extracellular matrix (ECM) proteins. Whether the ECM conditioned by IPF fibroblasts determines the phenotype of naïve fibroblasts is difficult to explore. IPF-derived primary fibroblasts were cultured on Matrigel and then cleared using ammonium hydroxide, creating an IPF-conditioned matrix (CM). Normal fibroblast CM served as control. Normal fibroblasts were cultured on both types of CM, and cell count, cell distribution and markers of myofibroblast differentiation; transforming growth factor beta (TGFβ) signalling; and ECM expression were assessed. The effects of the anti-fibrotic drugs nintedanib and pirfenidone at physiologically relevant concentrations were also explored. Normal fibroblasts cultured on IPF-CM arranged in large aggregates as a result of increased proliferation and migration. Moreover, increased levels of pSmad3, pSTAT3 (phospho signal transducer and activator of transcription 3), alpha smooth muscle actin (αSMA) and Collagen1a were found, suggesting a differentiation towards a myofibroblast-like phenotype. SB505124 (10 μmol/L) partially reversed these alterations, suggesting a TGFβ contribution. Furthermore, nintedanib at 100 nmol/L and, to a lesser extent, pirfenidone at 100 μmol/L prevented the IPF-CM-induced fibroblast phenotype alterations, suggesting an attenuation of the ECM-fibroblast interplay. IPF fibroblasts alter the ECM, thus creating a CM that further propagates an IPF-like phenotype in normal fibroblasts. This assay demonstrated differences in drug activities for approved IPF drugs at clinically relevant concentrations. Thus, the matrix-fibroblast phenotype interplay might be a relevant assay to explore drug candidates for IPF treatment. © 2018 Asian Pacific Society of Respirology.

  14. Effects of hydrolysed casein, intact casein and intact whey protein on energy expenditure and appetite regulation

    DEFF Research Database (Denmark)

    Bendtsen, Line Quist; Lorenzen, Janne Kunchel; Gomes, Sisse

    2014-01-01

    Casein and whey differ in amino acid composition and in the rate of absorption; however, the absorption rate of casein can be increased to mimic that of whey by exogenous hydrolysis. The objective of the present study was to compare the effects of hydrolysed casein (HC), intact casein (IC......) and intact whey (IW) on energy expenditure (EE) and appetite regulation, and thereby to investigate the influence of amino acid composition and the rate of absorption. In the present randomised cross-over study, twenty-four overweight and moderately obese young men and women consumed three isoenergetic...

  15. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  17. 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.

  18. Xenopus egg cytoplasm with intact actin.

    Science.gov (United States)

    Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts. © 2014 Elsevier Inc. All rights reserved.

  19. Exosome-mediated delivery of miR-9 induces cancer-associated fibroblast-like properties in human breast fibroblasts

    Science.gov (United States)

    Baroni, S; Romero-Cordoba, S; Plantamura, I; Dugo, M; D'Ippolito, E; Cataldo, A; Cosentino, G; Angeloni, V; Rossini, A; Daidone, M G; Iorio, M V

    2016-01-01

    It is established that the interaction between microenvironment and cancer cells has a critical role in tumor development, given the dependence of neoplastic cells on stromal support. However, how this communication promotes the activation of normal (NFs) into cancer-associated fibroblasts (CAFs) is still not well understood. Most microRNA (miRNA) studies focused on tumor cell, but there is increasing evidence of their involvement in reprogramming NFs into CAFs. Here we show that miR-9, upregulated in various breast cancer cell lines and identified as pro-metastatic miRNA, affects the properties of human breast fibroblasts, enhancing the switch to CAF phenotype, thus contributing to tumor growth. Expressed at higher levels in primary triple-negative breast CAFs versus NFs isolated from patients, miR-9 improves indeed migration and invasion capabilities when transfected in immortalized NFs; viceversa, these properties are strongly impaired in CAFs upon miR-9 inhibition. We also demonstrate that tumor-secreted miR-9 can be transferred via exosomes to recipient NFs and this uptake results in enhanced cell motility. Moreover, we observed that this miRNA is also secreted by fibroblasts and in turn able to alter tumor cell behavior, by modulating its direct target E-cadherin, and NFs themselves. Consistently with the biological effects observed, gene expression profiles of NFs upon transient transfection with miR-9 show the modulation of genes mainly involved in cell motility and extracellular matrix remodeling pathways. Finally, we were able to confirm the capability of NFs transiently transfected with miR-9 to promote in vivo tumor growth. Taken together, these data provide new insights into the role of miR-9 as an important player in the cross-talk between cancer cells and stroma. PMID:27468688

  20. 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...

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. EMMPRIN is secreted by human uterine epithelial cells in microvesicles and stimulates metalloproteinase production by human uterine fibroblast cells.

    Science.gov (United States)

    Braundmeier, A G; Dayger, C A; Mehrotra, P; Belton, R J; Nowak, R A

    2012-12-01

    Endometrial remodeling is a physiological process involved in the gynecological disease, endometriosis. Tissue remodeling is directed by uterine fibroblast production of matrix metalloproteinases (MMPs). Several MMPs are regulated directly by the protein extracellular matrix metalloproteinase inducer (EMMPRIN) and also by proinflammatory cytokines such as interleukin (IL)1-α/β. We hypothesized that human uterine epithelial cells (HESs) secrete intact EMMPRIN to stimulate MMPs. Microvesicles from HES cell-conditioned medium (CM) expressed intact EMMPRIN protein. Treatment of HES cells with estradiol or phorbyl 12-myristate-13-acetate increased the release of EMMPRIN-containing microvesicles. The HES CM stimulated MMP-1, -2, and -3 messenger RNA levels in human uterine fibroblasts (HUFs) and EMMPRIN immunodepletion from HES-cell concentrated CM reduced MMP stimulation (P EMMPRIN, in response to ovarian hormones, proinflammatory cytokines as well as activation of protein kinase C.

  6. Fibroblast spheroids as a model to study sustained fibroblast quiescence and their crosstalk with tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Salmenperä, Pertteli, E-mail: pertteli.salmenpera@helsinki.fi [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland); Karhemo, Piia-Riitta [Research Programs Unit, Translational Cancer Biology, and Institute of Biomedicine, University of Helsinki, P.O. Box 63, FIN-00014 (Finland); Räsänen, Kati [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland); Laakkonen, Pirjo [Research Programs Unit, Translational Cancer Biology, and Institute of Biomedicine, University of Helsinki, P.O. Box 63, FIN-00014 (Finland); Vaheri, Antti [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland)

    2016-07-01

    Stromal fibroblasts have an important role in regulating tumor progression. Normal and quiescent fibroblasts have been shown to restrict and control cancer cell growth, while cancer-associated, i. e. activated fibroblasts have been shown to enhance proliferation and metastasis of cancer cells. In this study we describe generation of quiescent fibroblasts in multicellular spheroids and their effects on squamous cell carcinoma (SCC) growth in soft-agarose and xenograft models. Quiescent phenotype of fibroblasts was determined by global down-regulation of expression of genes related to cell cycle and increased expression of p27. Interestingly, microarray analysis showed that fibroblast quiescence was associated with similar secretory phenotype as seen in senescence and they expressed senescence-associated-β-galactosidase. Quiescent fibroblasts spheroids also restricted the growth of RT3 SCC cells both in soft-agarose and xenograft models unlike proliferating fibroblasts. Restricted tumor growth was associated with marginally increased tumor cell senescence and cellular differentiation, showed with senescence-associated-β-galactosidase and cytokeratin 7 staining. Our results show that the fibroblasts spheroids can be used as a model to study cellular quiescence and their effects on cancer cell progression. - Highlights: • Fibroblasts acquire a sustained quiescence when grown as multicellular spheroids. • This quiescence is associated with drastic change in gene expression. • Fibroblasts spheroids secrete various inflammation-linked cytokines and chemokines. • Fibroblasts spheroids reduced growth of RT3 SCC cells in xenograft model.

  7. Protein methylation reactions in intact pea chloroplasts

    International Nuclear Information System (INIS)

    Niemi, K.J.

    1989-01-01

    Post-translational protein methylation was investigated in Pisum sativum chloroplasts. Intact pea chloroplasts were incubated with ( 3 H-methyl)-S-adenosylmethionine under various conditions. The chloroplasts were then separated into stromal and thylakoid fractions and analyzed for radioactivity transferred to protein. Light enhanced the magnitude of labeling in both fractions. One thylakoid polypeptide with an apparent molecular mass of 43 kDa was labeled only in the light. Several other thylakoid and stromal proteins were labeled in both light and dark-labeling conditions. Both base-labile methylation, carboxy-methylesters and base-stable groups, N-methylations were found. Further characterization of the methyl-transfer reactions will be presented

  8. A new infusion pathway intactness monitoring system.

    Science.gov (United States)

    Ogawa, Hidekuni; Yonezawa, Yoshiharu; Maki, Hiromichi; Ninomiya, Ishio; Sata, Koji; Hamada, Shingo; Caldwell, W Morton

    2006-01-01

    A new infusion pathway monitoring system has been developed for hospital and home use. The system consists of linear integrated circuits and a low-power 8-bit single chip microcomputer which constantly monitors the infusion pathway intactness. An AC (alternating current) voltage is induced on the patient's body by electrostatic coupling from the normal 100 volt, 60 Hz AC power line wiring field in the patient's room. The induced AC voltage can be recorded by a main electrode wrapped around the infusion polyvinyl chloride tube. A reference electrode is wrapped on the electrode to monitor the AC voltage around the main electrode. If the injection needle or infusion tube becomes detached, then the system detects changes in the induced AC voltages and alerts the nursing station, via the nurse call system or PHS (personal handy phone system).

  9. Reconciling certification and intact forest landscape conservation.

    Science.gov (United States)

    Kleinschroth, Fritz; Garcia, Claude; Ghazoul, Jaboury

    2018-05-29

    In 2014, the Forest Stewardship Council (FSC) added a new criterion to its principles that requires protection of intact forest landscapes (IFLs). An IFL is an extensive area of forest that lacks roads and other signs of human activity as detected through remote sensing. In the Congo basin, our analysis of road networks in formally approved concessionary logging areas revealed greater loss of IFL in certified than in noncertified concessions. In areas of informal (i.e., nonregulated) extraction, road networks are known to be less detectable by remote sensing. Under the current definition of IFL, companies certified under FSC standards are likely to be penalized relative to the noncertified as well as the informal logging sector on account of their planned road networks, despite an otherwise better standard of forest management. This could ultimately undermine certification and its wider adoption, with implications for the future of sustainable forest management.

  10. 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.

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

    DEFF Research Database (Denmark)

    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...

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

    Directory of Open Access Journals (Sweden)

    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.

  13. 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.

  14. Induction of Pluripotency in Adult Equine Fibroblasts without c-MYC

    Directory of Open Access Journals (Sweden)

    Khodadad Khodadadi

    2012-01-01

    Full Text Available Despite tremendous efforts on isolation of pluripotent equine embryonic stem (ES cells, to date there are few reports about successful isolation of ESCs and no report of in vivo differentiation of this important companion species. We report the induction of pluripotency in adult equine fibroblasts via retroviral transduction with three transcription factors using OCT4, SOX2, and KLF4 in the absence of c-MYC. The cell lines were maintained beyond 27 passages (more than 11 months and characterized. The equine iPS (EiPS cells stained positive for alkaline phosphatase by histochemical staining and expressed OCT4, NANOG, SSEA1, and SSEA4. Gene expression analysis of the cells showed the expression of OCT4, SOX2 NANOG, and STAT3. The cell lines retained a euploid chromosome count of 64 after long-term culture cryopreservation. The EiPS demonstrated differentiation capacity for the three embryonic germ layers both in vitro by embryoid bodies (EBs formation and in vivo by teratoma formation. In conclusion, we report the derivation of iPS cells from equine adult fibroblasts and long-term maintenance using either of the three reprogramming factors.

  15. Paracrine interactions of cancer-associated fibroblasts, macrophages and endothelial cells: tumor allies and foes.

    Science.gov (United States)

    Ronca, Roberto; Van Ginderachter, Jo A; Turtoi, Andrei

    2018-01-01

    Tumor stroma is composed of many cellular subtypes, of which the most abundant are fibroblasts, macrophages and endothelial cells. During the process of tissue injury, these three cellular subtypes must coordinate their activity to efficiently contribute to tissue regeneration. In tumor, this mechanism is hijacked by cancer cells, which rewire the interaction of stromal cells to benefit tumor development. The present review aims at summarizing most relevant information concerning both pro-tumorigenic and anti-tumorigenic actions implicating the three stromal cell subtypes as well as their mutual interactions. Although stromal cells are generally regarded as tumor-supportive and at will manipulated by cancer cells, several novel studies point at many defaults in cancer cell-mediated stromal reprograming. Indeed, parts of initial tissue-protective and homeostatic functions of the stromal cells remain in place even after tumor development. Both tumor-supportive and tumor-suppressive functions have been well described for macrophages, whereas similar results are emerging for fibroblasts and endothelial cells. Recent success of immunotherapies have finally brought the long awaited proof that stroma is key for efficient tumor targeting. However, a better understanding of paracrine stromal interactions is needed in order to encourage drug development not only aiming at disruption of tumor-supportive communication but also re-enforcing, existing, tumor-suppressive mechanisms.

  16. Engraftment potential of dermal fibroblasts following in vivo myogenic conversion in immunocompetent dystrophic skeletal muscle

    Directory of Open Access Journals (Sweden)

    Lindsey A Muir

    2014-01-01

    Full Text Available Autologous dermal fibroblasts (dFbs are promising candidates for enhancing muscle regeneration in Duchenne muscular dystrophy (DMD due to their ease of isolation, immunological compatibility, and greater proliferative potential than DMD satellite cells. We previously showed that mouse fibroblasts, after MyoD-mediated myogenic reprogramming in vivo, engraft in skeletal muscle and supply dystrophin. Assessing the therapeutic utility of this system requires optimization of conversion and transplantation conditions and quantitation of engraftment so that these parameters can be correlated with possible functional improvements. Here, we derived dFbs from transgenic mice carrying mini-dystrophin, transduced them by lentivirus carrying tamoxifen-inducible MyoD, and characterized their myogenic and engraftment potential. After cell transplantation into the muscles of immunocompetent dystrophic mdx4cv mice, tamoxifen treatment drove myogenic conversion and fusion into myofibers that expressed high levels of mini-dystrophin. Injecting 50,000 cells/µl (1 × 106 total cells resulted in a peak of ∼600 mini-dystrophin positive myofibers in tibialis anterior muscle single cross-sections. However, extensor digitorum longus muscles with up to 30% regional engraftment showed no functional improvements; similar limitations were obtained with whole muscle mononuclear cells. Despite the current lack of physiological improvement, this study suggests a viable initial strategy for using a patient-accessible dermal cell population to enhance skeletal muscle regeneration in DMD.

  17. Fibroblasts from long-lived Snell dwarf mice are resistant to oxygen-induced in vitro growth arrest

    DEFF Research Database (Denmark)

    Maynard, Scott P; Miller, Richard A

    2006-01-01

    Snell dwarf mice live longer than controls, and show lower age-adjusted rates of lethal neoplastic diseases. Fibroblast cells from adult dwarf mice are resistant to the lethal effects of oxidative and nonoxidative stresses, including the carcinogen methyl methanesulfonate. We now report that dwar...... in skin fibroblasts by the hormonal milieu of the Snell dwarf lead to resistance to multiple forms of injury, including the oxidative damage that contributes to growth arrest in vitro and neoplasia in intact mice.......Snell dwarf mice live longer than controls, and show lower age-adjusted rates of lethal neoplastic diseases. Fibroblast cells from adult dwarf mice are resistant to the lethal effects of oxidative and nonoxidative stresses, including the carcinogen methyl methanesulfonate. We now report that dwarf...

  18. Radiosensitivity in cultured human fibroblasts

    International Nuclear Information System (INIS)

    Cox, R.; Masson, W.K.

    1980-01-01

    Caution is urged in the use of freshly isolated cultures of human diploid fibroblasts for quantitative studies of radiosensitivity. The distribution of x ray sensitivities of 'normal' human fibroblast cultures of foetal origin (10 subjects, skin or lung biopsy) and post-foetal origin (34 subjects, skin biopsy) are compared with the distribution in 12 patients with ataxia telangiectasia (probability of including any one of these in a normal post-foetal distribution is 0.01%). Cultures from nominally normal subjects showed a broad distribution of D 0 range of 98 +- 160 rad and assuming normal distribution, a mean +- one standard deviation of 122 +- 17 rad. Mean D 0 values for foetal origin cultures were 117 +- 12; values for post-foetal cultures D 0 were 124 +- 18. No systematic variation in D 0 was observed for age of donor, number of cell divisions in culture or for cloning efficiency. For ataxia telangiectasia D 0 values were 46 +- 7 rad. (U.K.)

  19. 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...

  20. Toxicity of silver nanoparticles in mouse embryonic stem cells and chemical based reprogramming of somatic cells to sphere cells

    Science.gov (United States)

    Rajanahalli Krishnamurthy, Pavan

    Abstract 1: Silver nanoparticles (Ag Np's) have an interesting surface chemistry and unique plasmonic properties. They are used in a wide variety of applications ranging from consumer products like socks, medical dressing, computer chips and it is also shown to have antimicrobial, anti bacterial activity and wound healing. Ag Np toxicity studies have been limited to date which needs to be critically addressed due to its wide applications. Mouse embryonic stem (MES) cells represent a unique cell population with the ability to undergo both self renewal and differentiation. They exhibit very stringent and tightly regulated mechanisms to circumvent DNA damage and stress response. We used 10 nm coated (polysaccharide) and uncoated Ag Np's to test its toxic effects on MES cells. MES cells and embryoid bodies (EB's) were treated with two concentrations of Ag Np's: 5 microg/ml and 50 ug/ml and exposed for 24, 48 and 72 hours. Increased cell death, ROS production and loss of mitochondrial membrane potential and alkaline phosphatase (AP) occur in a time and a concentration dependant manner. Due to increased cell death, there is a progressive increase in Annexin V (apoptosis) and Propidium Iodide (PI) staining (necrosis). Oct4 and Nanog undergo ubiquitination and dephosphorylation post-translational modifications in MES cells thereby altering gene expression of pluripotency factors and differentiation of EB's into all the three embryonic germ layers with specific growth factors were also inhibited after Ag Np exposure. Flow cytometry analysis revealed Ag Np's treated cells had altered cell cycle phases correlating with altered self renewal capacity. Our results suggest that Ag Np's effect MES cell self renewal, pluripotency and differentiation and serves as a perfect model system for studying toxicity induced by engineered Ag Np's. Abstract 2: The reprogramming of fibroblasts to pluripotent stem cells and the direct conversion of fibroblasts to functional neurons has been

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  3. 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.

  4. 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)

  5. 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.

  6. Fibroblasts Cultured on Nanowires Exhibit Low Motility, Impaired Cell Division, and DNA Damage

    DEFF Research Database (Denmark)

    Persson, H.; Købler, Carsten; Mølhave, Kristian

    2013-01-01

    beam milling and scanning electron microscopy, highly curved but intact nuclear membranes are observed, showing no direct contact between the nanowires and the DNA. The nanowires possibly induce cellular stress and high respiration rates, which trigger the formation of ROS, which in turn results in DNA......Nanowires are commonly used as tools for interfacing living cells, acting as biomolecule-delivery vectors or electrodes. It is generally assumed that the small size of the nanowires ensures a minimal cellular perturbation, yet the effects of nanowires on cell migration and proliferation remain...... largely unknown. Fibroblast behaviour on vertical nanowire arrays is investigated, and it is shown that cell motility and proliferation rate are reduced on nanowires. Fibroblasts cultured on long nanowires exhibit failed cell division, DNA damage, increased ROS content and respiration. Using focused ion...

  7. Transcriptome comparison of human neurons generated using induced pluripotent stem cells derived from dental pulp and skin fibroblasts.

    Science.gov (United States)

    Chen, Jian; Lin, Mingyan; Foxe, John J; Pedrosa, Erika; Hrabovsky, Anastasia; Carroll, Reed; Zheng, Deyou; Lachman, Herbert M

    2013-01-01

    Induced pluripotent stem cell (iPSC) technology is providing an opportunity to study neuropsychiatric disorders through the capacity to grow patient-specific neurons in vitro. Skin fibroblasts obtained by biopsy have been the most reliable source of cells for reprogramming. However, using other somatic cells obtained by less invasive means would be ideal, especially in children with autism spectrum disorders (ASD) and other neurodevelopmental conditions. In addition to fibroblasts, iPSCs have been developed from cord blood, lymphocytes, hair keratinocytes, and dental pulp from deciduous teeth. Of these, dental pulp would be a good source for neurodevelopmental disorders in children because obtaining material is non-invasive. We investigated its suitability for disease modeling by carrying out gene expression profiling, using RNA-seq, on differentiated neurons derived from iPSCs made from dental pulp extracted from deciduous teeth (T-iPSCs) and fibroblasts (F-iPSCs). This is the first RNA-seq analysis comparing gene expression profiles in neurons derived from iPSCs made from different somatic cells. For the most part, gene expression profiles were quite similar with only 329 genes showing differential expression at a nominally significant p-value (pdisease-modeling neuropsychiatric disorder and may have some advantages over those derived from F-iPSCs.

  8. Comparison of Intact PTH and Bio-Intact PTH Assays Among Non-Dialysis Dependent Chronic Kidney Disease Patients.

    Science.gov (United States)

    Einbinder, Yael; Benchetrit, Sydney; Golan, Eliezer; Zitman-Gal, Tali

    2017-09-01

    The third-generation bio-intact parathyroid hormone (PTH) (1-84) assay was designed to overcome problems associated with the detection of C-terminal fragments by the second-generation intact PTH assay. The two assays have been compared primarily among dialysis populations. The present study evaluated the correlations and differences between these two PTH assays among patients with chronic kidney disease (CKD) stages 3 to 5 not yet on dialysis. Blood samples were collected from 98 patients with CKD stages 3 to 5. PTH concentrations were measured simultaneously by using the second-generation - PTH intact-STAT and third-generation bio-intact 1-84 PTH assays. Other serum biomarkers of bone mineral disorders were also assessed. CKD stage was calculated by using the CKD-Epidemiology Collaboration (EPI) formula. Serum bio-intact PTH concentrations were strongly correlated but significantly lower than the intact PTH concentrations (r=0.963, Pbio-intact PTH) positively correlated with urea (r=0.523, r=0.504; P=0.002, respectively), phosphorus (r=0.532, r=0.521; Pbio-intact PTH assay detected significantly lower PTH concentrations compared with intact PTH assay. Additional studies that correlate the diagnosis and management of CKD mineral and bone disorders with bone histomorphometric findings are needed to determine whether bio-intact PTH assay results are better surrogate markers in these early stages of CKD. © The Korean Society for Laboratory Medicine

  9. Pathophysiology of preterm labor with intact membranes.

    Science.gov (United States)

    Talati, Asha N; Hackney, David N; Mesiano, Sam

    2017-11-01

    Preterm labor with intact membranes is a major cause of spontaneous preterm birth (sPTB). To prevent sPTB a clear understanding is needed of the hormonal interactions that initiate labor. The steroid hormone progesterone acting via its nuclear progesterone receptors (PRs) in uterine cells is essential for the establishment and maintenance of pregnancy and disruption of PR signaling (i.e., functional progesterone/PR withdrawal) is key trigger for labor. The process of parturition is also associated with inflammation within the uterine tissues and it is now generally accepted that inflammatory stimuli from multiple extrinsic and intrinsic sources induce labor. Recent studies suggest inflammatory stimuli induce labor by affecting PR transcriptional activity in uterine cells to cause functional progesterone/PR withdrawal. Advances in understanding the functional interaction of inflammatory load on the pregnancy uterus and progesterone/PR signaling is opening novel areas of research and may lead to rational therapeutic strategies to effectively prevent sPTB. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Excitons in intact cells of photosynthetic bacteria.

    Science.gov (United States)

    Freiberg, Arvi; Pajusalu, Mihkel; Rätsep, Margus

    2013-09-26

    Live cells and regular crystals seem fundamentally incompatible. Still, effects characteristic to ideal crystals, such as coherent sharing of excitation, have been recently used in many studies to explain the behavior of several photosynthetic complexes, especially the inner workings of the light-harvesting apparatus of the oldest known photosynthetic organisms, the purple bacteria. To this date, there has been no concrete evidence that the same effects are instrumental in real living cells, leaving a possibility that this is an artifact of unnatural study conditions, not a real effect relevant to the biological operation of bacteria. Hereby, we demonstrate survival of collective coherent excitations (excitons) in intact cells of photosynthetic purple bacteria. This is done by using excitation anisotropy spectroscopy for tracking the temperature-dependent evolution of exciton bands in light-harvesting systems of increasing structural complexity. The temperature was gradually raised from 4.5 K to ambient temperature, and the complexity of the systems ranged from detergent-isolated complexes to complete bacterial cells. The results provide conclusive evidence that excitons are indeed one of the key elements contributing to the energetic and dynamic properties of photosynthetic organisms.

  11. 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.

  12. Establishment of human induced pluripotent stem cell lines from normal fibroblast TIG-1.

    Science.gov (United States)

    Kumazaki, Tsutomu; Kurata, Sayaka; Matsuo, Taira; Mitsui, Youji; Takahashi, Tomoko

    2011-06-01

    Normal human cells have a replicative life span and therefore senesce. Usually, normal human cell strains are differentiated cells and reach a terminally differentiated state after a number of cell divisions. At present, definitive differences are not known between replicative senescence and terminal differentiation. TIG-1 is a human fibroblast strain established from fetal lung and has been used extensively in studies of cellular senescence, and numerous data were accumulated at the molecular level. Recently, a method for generating induced pluripotent stem cells (iPSCs) was developed. Using the method, we introduced four reprogramming genes to TIG-1 fibroblasts and succeeded in isolating colonies that had embryonic stem cell (ESC)-like morphologies. They showed alkaline phosphatase activity and expressed ESC markers, as shown by immunostaining of OCT4, SOX2, SSEA4, and TRA-1-81 as well as reverse-transcription polymerase chain reaction (RT-PCR) for OCT4 and NANOG transcripts. Thus, we succeeded in establishing iPSC clones from TIG-1. The iPSC clones could differentiate to cells originated from all three germ-cell layers, as shown by RT-PCR, for messenger RNA (mRNA) expression of α-fetoprotein (endoderm), MSX1 (mesoderm) and microtubule-associated protein 2 (ectoderm), and by immunostaining for α-fetoprotein (endoderm), α-smooth muscle actin (mesoderm), and β-III-tubulin (ectoderm). The iPSCs formed teratoma containing the structures developed from all three germ-cell layers in severe combined immune-deficiency mice. Thus, by comparing the aging process of parental TIG-1 cells and the differentiation process of iPSC-derived fibrocytes to fibroblasts, we can reveal the exact differences in processes between senescence and terminal differentiation.

  13. Positive Youth Development, Life Satisfaction and Problem Behaviors of Adolescents in Intact and Non-Intact Families in Hong Kong

    Directory of Open Access Journals (Sweden)

    Daniel Tan Lei Shek

    2013-08-01

    Full Text Available This study investigated whether Chinese adolescents living in intact and non-intact families differed in their positive development, life satisfaction, and risk behavior. A total of 3,328 Secondary 1 students responded to measures of positive youth development (such as resilience and psychosocial competencies, life satisfaction, and risk behavior (substance abuse, delinquency, Internet addiction, consumption of pornographic materials, self-harm, and behavioral intention to engage in problem behavior. Findings revealed that adolescents growing up in intact families reported higher levels of positive developmental outcomes and life satisfaction as compared with adolescents from non-intact families. Adolescents in non-intact families also reported higher levels of risk behaviors than those growing up in intact families.

  14. Generation of human-induced pluripotent stem cells from burn patient-derived skin fibroblasts using a non-integrative method.

    Science.gov (United States)

    Fu, Shangfeng; Ding, Jianwu; Liu, Dewu; Huang, Heping; Li, Min; Liu, Yang; Tu, Longxiang; Liu, Deming

    2018-01-01

    Patient specific induced pluripotent stem cells (iPSCs) have been recognized as a possible source of cells for skin tissue engineering. They have the potential to greatly benefit patients with large areas of burned skin or skin defects. However, the integration virus-based reprogramming method is associated with a high risk of genetic mutation and mouse embryonic fibroblast feeder-cells may be a pollutant. In the present study, human skin fibroblasts (HSFs) were successfully harvested from patients with burns and patient-specific iPSCs were generated using a non-integration method with a feeder-free approach. The octamer-binding transcription factor 4 (OCT4), sex-determining region Y box 2 (SOX2) and NANOG transcription factors were delivered using Sendai virus vectors. iPSCs exhibited representative human embryonic stem cell-like morphology and proliferation characteristics. They also expressed pluripotent markers, including OCT4, NANOG, SOX2, TRA181, stage-specific embryonic antigen 4 and TRA-160, and exhibited a normal karyotype. Teratoma and embryoid body formation revealed that iPSCs were able to differentiate into cells of all three germ layers in vitro and in vivo. The results of the present study demonstrate that HSFs derived from patients with burns, may be reprogrammed into stem cells with pluripotency, which provides a basis for cell‑based skin tissue engineering in the future.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. Impact of matrix stiffness on fibroblast function

    Energy Technology Data Exchange (ETDEWEB)

    El-Mohri, Hichem; Wu, Yang; Mohanty, Swetaparna; Ghosh, Gargi, E-mail: gargi@umich.edu

    2017-05-01

    Chronic non-healing wounds, caused by impaired production of growth factors and reduced vascularization, represent a significant burden to patients, health care professionals, and health care system. While several wound dressing biomaterials have been developed, the impact of the mechanical properties of the dressings on the residing cells and consequently on the healing of the wounds is largely overlooked. The primary focus of this study is to explore whether manipulation of the substrate mechanics can regulate the function of fibroblasts, particularly in the context of their angiogenic activity. A photocrosslinkable hydrogel platform with orthogonal control over gel modulus and cell adhesive sites was developed to explore the quantitative relationship between ECM compliance and fibroblast function. Increase in matrix stiffness resulted in enhanced fibroblast proliferation and stress fiber formation. However, the angiogenic activity of fibroblasts was found to be optimum when the cells were seeded on compliant matrices. Thus, the observations suggest that the stiffness of the wound dressing material may play an important role in the progression of wound healing. - Highlights: • Proliferation and stress fiber formation of fibroblasts increase with increasing matrix mechanics. • Cell area correlates with the growth of fibroblasts. • Angiogenic activity of fibroblasts optimum when cells seeded on compliant gels.

  4. 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.

  5. 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

  6. Transduction of Oct6 or Oct9 gene concomitant with Myc family gene induced osteoblast-like phenotypic conversion in normal human fibroblasts.

    Science.gov (United States)

    Mizoshiri, N; Kishida, T; Yamamoto, K; Shirai, T; Terauchi, R; Tsuchida, S; Mori, Y; Ejima, A; Sato, Y; Arai, Y; Fujiwara, H; Yamamoto, T; Kanamura, N; Mazda, O; Kubo, T

    2015-11-27

    Osteoblasts play essential roles in bone formation and regeneration, while they have low proliferation potential. Recently we established a procedure to directly convert human fibroblasts into osteoblasts (dOBs). Transduction of Runx2 (R), Osterix (X), Oct3/4 (O) and L-myc (L) genes followed by culturing under osteogenic conditions induced normal human fibroblasts to express osteoblast-specific genes and produce calcified bone matrix both in vitro and in vivo Intriguingly, a combination of only two factors, Oct3/4 and L-myc, significantly induced osteoblast-like phenotype in fibroblasts, but the mechanisms underlying the direct conversion remains to be unveiled. We examined which Oct family genes and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. As result Oct3/4, Oct6 and Oct9, among other Oct family members, had the capability, while N-myc was the most effective Myc family gene. The Oct9 plus N-myc was the best combination to induce direct conversion of human fibroblasts into osteoblast-like cells. The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Isolation of intact elastin fibers devoid of microfibrils.

    NARCIS (Netherlands)

    Daamen, W.F.; Hafmans, T.G.M.; Veerkamp, J.H.; Kuppevelt, A.H.M.S.M. van

    2005-01-01

    Purification protocols for elastin generally result in greatly damaged elastin fibers and this likely influences the biological response. We here describe a novel protocol for the isolation of elastin whereby the fibers stay intact, and introduce the term "elastin fiber" for intact elastic fibers

  8. 50 CFR 622.38 - Landing fish intact.

    Science.gov (United States)

    2010-10-01

    ... that is operating under the respective trip limits. Such cut-off fish also may be sold. A maximum of... 50 Wildlife and Fisheries 8 2010-10-01 2010-10-01 false Landing fish intact. 622.38 Section 622.38... Landing fish intact. The operator of a vessel that fishes in the EEZ is responsible for ensuring that fish...

  9. Generation of HEXA-deficient hiPSCs from fibroblasts of a Tay-Sachs disease patient.

    Science.gov (United States)

    Liu, Zhong; Zhao, Rui

    2016-09-01

    Human iPSC line TSD-01-hiPSC was generated from fibroblasts of a patient with infantile Tay-Sachs disease (TSD). The patient is compound heterozygous at the HEXA gene by carrying a 1278insTATC allele and an IVS12+1G>C allele. STEMCCA lentivirus, which expresses OCT4, SOX2, KLF4, and c-MYC from a polycistronic transcript, were used for reprogramming. TSD-01-hiPSC express pluripotency markers such as OCT4, SOX2, NANOG, Tra-1-60, and alkaline phosphatase, and can differentiate into tissues from all the three embryonic germ layers. This TSD patient-derived hiPSC line may serve as a valuable in vitro tool for disease modeling and drug test. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  10. Generation of HEXA-deficient hiPSCs from fibroblasts of a Tay-Sachs disease patient

    Directory of Open Access Journals (Sweden)

    Zhong Liu

    2016-09-01

    Full Text Available Human iPSC line TSD-01-hiPSC was generated from fibroblasts of a patient with infantile Tay-Sachs disease (TSD. The patient is compound heterozygous at the HEXA gene by carrying a 1278insTATC allele and an IVS12+1G>C allele. STEMCCA lentivirus, which expresses OCT4, SOX2, KLF4, and c-MYC from a polycistronic transcript, were used for reprogramming. TSD-01-hiPSC express pluripotency markers such as OCT4, SOX2, NANOG, Tra-1-60, and alkaline phosphatase, and can differentiate into tissues from all the three embryonic germ layers. This TSD patient-derived hiPSC line may serve as a valuable in vitro tool for disease modeling and drug test.

  11. Quantitative Proteomic Analysis of Mouse Embryonic Fibroblasts and Induced Pluripotent Stem Cells Using 16O /18O labeling

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Xin; Tian, Changhai; Liu, Miao; Wang, Yongxiang; Tolmachev, Aleksey V.; Sharma, Seema; Yu, Fang; Fu, Kai; Zheng, Jialin; Ding, Shi-Jian

    2012-04-06

    Induced pluripotent stem cells (iPSC) hold great promise for regenerative medicine as well as for investigations into the pathogenesis and treatment of various diseases. Understanding of key intracellular signaling pathways and protein targets that control development of iPSC from somatic cells is essential for designing new approaches to improve reprogramming efficiency. Here we report the development and application of an integrated quantitative proteomics platform for investigating differences in protein expressions between mouse embryonic fibroblasts (MEF) and MEF-derived iPSC. This platform consists of 16O/18O labeling, multidimensional peptide separation coupled with tandem mass spectrometry, and data analysis with UNiquant software. Using this platform a total of 2,481 proteins were identified and quantified from the 16O/18O-labeled MEF-iPSC proteome mixtures with a false discovery rate of 0.01. Among them, 218 proteins were significantly upregulated, while 247 proteins were significantly downregulated in iPSC compared to MEF. Many nuclear proteins, including Hdac1, Dnmt1, Pcna, Ccnd1, Smarcc1, and subunits in DNA replication and RNA polymerase II complex were found to be enhanced in iPSC. Protein network analysis revealed that Pcna functions as a hub orchestrating complicated mechanisms including DNA replication, epigenetic inheritance (Dnmt1) and chromatin remodeling (Smarcc1) to reprogram MEF and maintain stemness of iPSC.

  12. Resistance to 1,25-dihydroxyvitamin D. Association with heterogeneous defects in cultured skin fibroblasts

    International Nuclear Information System (INIS)

    Liberman, U.A.; Eil, C.; Marx, S.J.

    1983-01-01

    We evaluated the interaction of [ 3 H]1,25(OH) 2 D 3 with skin fibroblasts cultured from normal subjects or from affected members of six kindreds with rickets and resistance to 1-alpha, 25(OH) 2 D [1,25(OH) 2 D]. We analyzed two aspects of the radioligand interaction; nuclear uptake with dispersed, intact cells at 37 degrees C and binding at 0 degrees C with soluble extract (cytosol) prepared from cells disrupted in buffer containing 300 mM KCl and 10 mM sodium molybdate. With normal fibroblasts the affinity and capacity of nuclear uptake of [ 3 H]1,25(OH) 2 D 3 were 0.5 nM and 10,300 sites per cell, respectively; for binding with cytosol these were 0.13 nM and 8,900 sites per cell, respectively. In all cases where the radioligand bound with high affinity in nucleus or cytosol, the nucleus- or cytosol-associated radioligand exhibited normal sedimentation velocity on sucrose density gradients. When two kindreds exhibited similar patterns (i.e. pattern a or c) with the analyses of cultured fibroblasts, clinical features in affected members suggested that the underlying genetic defects were not identical. In conclusion: (a) Fibroblasts cultured from human skin manifest nuclear uptake and cytosol binding of [ 3 H]1,25(OH) 2 D 3 that is an expression of the genes determining these processes in target tissues. (b) Based upon data from clinical evaluations and from analyses of cultured fibroblasts, severe resistance to 1,25(OH) 2 D resulted from five or six distinct genetic mutations in six kindreds

  13. Resistance to 1,25-dihydroxyvitamin D. Association with heterogeneous defects in cultured skin fibroblasts

    International Nuclear Information System (INIS)

    Liberman, U.A.; Eil, C.; Marx, S.J.

    1983-01-01

    The authors evaluated the interaction of [ 3 H]1,25(OH) 2 D3 with skin fibroblasts cultured from normal subjects or from affected members of six kindreds with rickets and resistance to 1-alpha, 25(OH) 2 D [1,25(OH) 2 D]. They analyzed two aspects of the radioligand interaction; nuclear uptake with dispersed, intact cells at 37 degrees C and binding at 0 degrees C with soluble extract (cytosol) prepared from cells disrupted in buffer. With normal fibroblasts the affinity and capacity of nuclear uptake of [ 3 H]1,25(OH) 2 D3 were 0.5 nM and 10,300 sites per cell, respectively; for binding with cytosol these were 0.13 nM and 8,900 sites per cell, respectively. The following four patterns of interaction with [ 3 H]1,25(OH) 2 D3 were observed with cells cultured from affected patients. In all cases where the radioligand bound with high affinity in nucleus or cytosol, the nucleus- or cytosol-associated radioligand exhibited normal sedimentation velocity on sucrose density gradients. When two kindreds exhibited similar patterns (i.e. pattern a or c) with the analyses of cultured fibroblasts, clinical features in affected members suggested that the underlying genetic defects were not identical. In conclusion: (a) Fibroblasts cultured from human skin manifest nuclear uptake and cytosol binding of [ 3 H]1,25(OH) 2 D3 that is an expression of the genes determining these processes in target tissues. (b) Based upon data from clinical evaluations and from analyses of cultured fibroblasts, severe resistance to 1,25(OH) 2 D resulted from five or six distinct genetic mutations in six kindreds

  14. Transduction of Oct6 or Oct9 gene concomitant with Myc family gene induced osteoblast-like phenotypic conversion in normal human fibroblasts

    International Nuclear Information System (INIS)

    Mizoshiri, N.; Kishida, T.; Yamamoto, K.; Shirai, T.; Terauchi, R.; Tsuchida, S.; Mori, Y.; Ejima, A.; Sato, Y.; Arai, Y.; Fujiwara, H.; Yamamoto, T.; Kanamura, N.; Mazda, O.; Kubo, T.

    2015-01-01

    Introduction: Osteoblasts play essential roles in bone formation and regeneration, while they have low proliferation potential. Recently we established a procedure to directly convert human fibroblasts into osteoblasts (dOBs). Transduction of Runx2 (R), Osterix (X), Oct3/4 (O) and L-myc (L) genes followed by culturing under osteogenic conditions induced normal human fibroblasts to express osteoblast-specific genes and produce calcified bone matrix both in vitro and in vivo Intriguingly, a combination of only two factors, Oct3/4 and L-myc, significantly induced osteoblast-like phenotype in fibroblasts, but the mechanisms underlying the direct conversion remains to be unveiled. Materials and Methods: We examined which Oct family genes and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. Results: As result Oct3/4, Oct6 and Oct9, among other Oct family members, had the capability, while N-myc was the most effective Myc family gene. The Oct9 plus N-myc was the best combination to induce direct conversion of human fibroblasts into osteoblast-like cells. Discussion: The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases. - Highlights: • Introducing L-myc in a combination with either Oct3/4, Oct6 or Oct9 enables the conversion of fibroblasts to osteoblasts. • A combination of L-myc with Oct3/4 or Oct9 can induce the cells to a phenotype closer to normal osteoblasts. • N-myc was considered the most appropriate Myc family gene for induction of osteoblast-like phenotype in fibroblasts. • The combination of Oct9 plus N-myc has the strongest capability of inducing osteoblast-like phenotype.

  15. Transduction of Oct6 or Oct9 gene concomitant with Myc family gene induced osteoblast-like phenotypic conversion in normal human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Mizoshiri, N. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Kishida, T. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Yamamoto, K. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto (Japan); Shirai, T.; Terauchi, R.; Tsuchida, S. [Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Mori, Y. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Ejima, A. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Sato, Y. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto (Japan); Arai, Y.; Fujiwara, H. [Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Yamamoto, T.; Kanamura, N. [Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto (Japan); Mazda, O., E-mail: mazda@koto.kpu-m.ac.jp [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Kubo, T. [Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan)

    2015-11-27

    Introduction: Osteoblasts play essential roles in bone formation and regeneration, while they have low proliferation potential. Recently we established a procedure to directly convert human fibroblasts into osteoblasts (dOBs). Transduction of Runx2 (R), Osterix (X), Oct3/4 (O) and L-myc (L) genes followed by culturing under osteogenic conditions induced normal human fibroblasts to express osteoblast-specific genes and produce calcified bone matrix both in vitro and in vivo Intriguingly, a combination of only two factors, Oct3/4 and L-myc, significantly induced osteoblast-like phenotype in fibroblasts, but the mechanisms underlying the direct conversion remains to be unveiled. Materials and Methods: We examined which Oct family genes and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. Results: As result Oct3/4, Oct6 and Oct9, among other Oct family members, had the capability, while N-myc was the most effective Myc family gene. The Oct9 plus N-myc was the best combination to induce direct conversion of human fibroblasts into osteoblast-like cells. Discussion: The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases. - Highlights: • Introducing L-myc in a combination with either Oct3/4, Oct6 or Oct9 enables the conversion of fibroblasts to osteoblasts. • A combination of L-myc with Oct3/4 or Oct9 can induce the cells to a phenotype closer to normal osteoblasts. • N-myc was considered the most appropriate Myc family gene for induction of osteoblast-like phenotype in fibroblasts. • The combination of Oct9 plus N-myc has the strongest capability of inducing osteoblast-like phenotype.

  16. Fibroblast cultures in duchenne muscular dystrophy

    International Nuclear Information System (INIS)

    Ionasescu, V.; Lara-Braud, C.; Zellweger, H.; Ionasescu, R.; Burmeister, L.

    1977-01-01

    Primary skin fibroblast cultures were grown from forearm pinch skin biopsies obtained from 24 patients with Duchenne muscular dystrophy (DMD) and ten normal controls matched for sex and age. The first subcultures were grown for 7 days and incubated with L-( 3 H)-proline for 24 hours. Intracellular collagen incoption was significantly decreased (2.2 X) and extracellular collagen incorporation significantly increased (1.8 X) in fibroblast cultures from patients with DMD by both collagenase assay and polyacrylamide gel electrophoresis. The synthesis of noncollagen proteins showed low values from the DMD fibroblast cultures. The alterations in synthesis and secretion of collagen and noncollagen proteins were characteristic only for the log phase of DMD fibroblasts. (author)

  17. 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.

  18. ADP-ribosylation of actins in fibroblasts and myofibroblasts by botulinum C2 toxin: Influence on microfilament morphology and migratory behavior

    DEFF Research Database (Denmark)

    Rønnov-Jessen, Lone; Petersen, Ole William

    1996-01-01

    botulinum C2 toxin. The substrate for C2 toxin is globular actin, which upon ribosylation cannot incorporate into microfilaments. The pattern of actin ADP-ribosylation in (myo)fibroblasts in the presence of [32P]NAD was analyzed by isoelectric focusing, fluorography and immunoblotting. The influence of C2...... toxin on microfilaments in intact cells was further assessed by immunofluorescence, and motility was measured in a mass migration assay and by computerized video time-lapse microscopy. We show here that C2 toxin specifically ribosylates - and -actin in both fibroblasts and myofibroblasts. Whereas...

  19. Serum steroid levels in intact and endocrine ablated BALB/c nude mice and their intact littermates

    DEFF Research Database (Denmark)

    Brünner, N; Svenstrup, B; Spang-Thomsen, M

    1986-01-01

    An investigation was made of the serum steroid levels found in intact and endocrine ablated nude mice of both sexes and in their intact homozygous littermates. The results showed that nude mice have a normal steroidogenesis, but with decreased levels of circulating steroids compared to those...

  20. 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.

  1. Fibroblasts in fibrosis: novel roles and mediators

    Directory of Open Access Journals (Sweden)

    Ryan Thomas Kendall

    2014-05-01

    Full Text Available Fibroblasts are the most common cell type of the connective tissues found throughout the body and the principal source of the extensive extracellular matrix (ECM characteristic of these tissues. They are also the central mediators of the pathological fibrotic accumulation of ECM and the cellular proliferation and differentiation that occurs in response to prolonged tissue injury and chronic inflammation. The transformation of the fibroblast cell lineage involves classical developmental signaling programs and includes a surprisingly diverse range of precursor cell types—most notably, myofibroblasts that are the apex of the fibrotic phenotype. Myofibroblasts display exaggerated ECM production; constitutively secrete and are hypersensitive to chemical signals such as cytokines, chemokines, and growth factors; and are endowed with a contractile apparatus allowing them to manipulate the ECM fibers physically to close open wounds. In addition to ECM production, fibroblasts have multiple concomitant biological roles, such as in wound healing, inflammation, and angiogenesis, which are each interwoven with the process of fibrosis. We now recognize many common fibroblast-related features across various physiological and pathological protracted processes. Indeed, a new appreciation has emerged for the role of noncancerous fibroblast interactions with tumors in cancer progression. Although the predominant current clinical treatments of fibrosis involve nonspecific immunosuppressive and anti-proliferative drugs, a variety of potential therapies under investigation specifically target fibroblast biology.

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

    Science.gov (United States)

    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

  3. Human diploid fibroblasts have receptors for the globular domain of C1Q

    International Nuclear Information System (INIS)

    Bordin, S.; Page, R.C.

    1986-01-01

    The authors showed that mass cultures of fibroblasts grown from gingival explants in DB medium with 10% human serum are enriched in a phenotype that binds C1q with an affinity much higher than the rest of the population. Because of potential biologic importance of C1q receptors, the authors studied whether the interaction between C1q and this phenotype was mediated by the globular or collagenous domains of the molecule. Globular fragments were prepared by digesting C1q with collagenase, and collagenous fragments obtained after pepsin treatment. C1q binding on cells in suspension was determined by reaction with 125 I-C1q as reported. Competition experiments were performed under conditions in which intact 125 I-C1q binding saturated all available receptors. The results showed that collagenous fragments inhibited 20% of the 125 I-C1q binding to high affinity receptors, whereas inhibition by globular fragments was 70%. Unlabeled intact C1q and collagen type 1 were used as controls, and inhibited 92% and 17% of C1q binding, respectively. These studies show that C1q interacts with the fibroblast phenotype expressing high affinity receptors through its globular domain. The authors suggest that at sites of trauma, native C1 may bind to the surface of these cells via the globular domain of C1q, and that this unique phenotype may play an important role in tissue repair

  4. Zymosterol is located in the plasma membrane of cultured human fibroblasts

    International Nuclear Information System (INIS)

    Echevarria, F.; Norton, R.A.; Nes, W.D.; Lange, Y.

    1990-01-01

    Zymosterol (5 alpha-cholesta-8(9),24-dien-3 beta-ol) comprised a negligible fraction of the mass of sterol in cultured human fibroblasts but was well labeled biosynthetically with radioactive acetate. Treatment of cells with triparanol, a potent inhibitor of sterol delta 24-reductase, led to a marked increase in labeled zymosterol while its mass rose to 1 mol% of total sterol. All of this sterol could be chased into cholesterol. Furthermore, cell homogenates converted exogenous radiolabeled zymosterol to cholesterol. Three lines of evidence suggested that biosynthetically labeled zymosterol was associated with the plasma membrane. (1) About 80% of radiolabeled zymosterol was oxidized by the impermeant enzyme, cholesterol oxidase, in glutaraldehyde-fixed intact cells. (2) Sucrose density gradient analysis of homogenates showed that the equilibrium buoyant density profile of newly synthesized zymosterol was identical with that of the plasma membrane. (3) Newly synthesized zymosterol was transferred as readily from fixed intact fibroblasts to exogenous acceptors as was cholesterol. Given that cholesterol is synthesized within the cell, it is unclear why most of the zymosterol is in the plasma membrane. The pathway of cholesterol biosynthesis may compel zymosterol to flux through the plasma membrane. Alternatively, plasma membrane zymosterol may represent a separate pool, in equilibrium with the zymosterol in the intracellular biosynthetic pool

  5. 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

  6. 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.

  7. 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.

  8. 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].

  9. 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.

  10. Intracellular insulin-receptor dissociation and segregation in a rat fibroblast cell line transfected with a human insulin receptor gene

    International Nuclear Information System (INIS)

    Levy, J.R.; Olefsky, J.M.

    1988-01-01

    The cellular processing of insulin and insulin receptors was studied using a rat fibroblast cell line that had been transfected with a normal human insulin receptor gene, expressing approximately 500 times the normal number of native fibroblasts insulin receptors. These cells bind and internalize insulin normally. Biochemically assays based on the selective precipitation by polyethylene glycol of intact insulin-receptor complexes but not of free intracellular insulin were developed to study the time course of intracellular insulin-receptor dissociation. Fibroblasts were incubated with radiolabeled insulin at 4 0 C, and internalization of insulin-receptor complexes was initiated by warming the cells to 37 0 C. Within 2 min, 90% of the internalized radioactivity was composed of intact insulin-receptor complexes. The dissociation of insulin from internalized insulin-receptor complexes was markedly inhibited by monensin and chloroquine. Furthermore, chloroquine markedly increased the number of cross-linkable intracellular insulin-receptor complexes, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. These findings suggest that acidification of intracellular vesicles is responsible for insulin-receptor dissociation. Physical segregation of dissociated intracellular insulin from its receptor was monitored. The results are consistent with the view that segregation of insulin and receptor occurs 5-10 min after initiation of dissociation. These studies demonstrate the intracellular itinerary of insulin-receptor complexes, including internalization, dissociation of insulin from the internalized receptor within an acidified compartment, segregation of insulin from the receptor, and subsequent ligand degradation

  11. N-cadherin is overexpressed in Crohn's stricture fibroblasts and promotes intestinal fibroblast migration.

    LENUS (Irish Health Repository)

    Burke, John P

    2012-02-01

    BACKGROUND: Intestinal fibroblasts mediate stricture formation in Crohn\\'s disease (CD). Transforming growth factor-beta (TGF-beta) is important in fibroblast activation, while cell attachment and migration is regulated by the adhesion molecule N-cadherin. The aim of this study was to investigate the expression and function of N-cadherin in intestinal fibroblasts in patients with fibrostenosing CD. METHODS: Intestinal fibroblasts were cultured from seromuscular biopsies from patients undergoing resection for terminal ileal fibrostenosing CD (n = 14) or controls patients (n = 8). N-cadherin expression was assessed using Western blot and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Fibroblasts were stimulated with TGF-beta and selective pathway inhibitors Y27632, PD98050, and LY294002 were used to examine the Rho\\/ROCK, ERK-1\\/2, and Akt signaling pathways, respectively. Cell migration was assessed using a scratch wound assay. N-cadherin was selectively overexpressed using a plasmid. RESULTS: Fibroblasts from fibrostenosing CD express increased constitutive N-cadherin mRNA and protein and exhibit enhanced basal cell migration relative to those from directly adjacent normal bowel. Control fibroblasts treated with TGF-beta induced N-cadherin in a dose-dependent manner which was inhibited by Rho\\/ROCK and Akt pathway modulation. Control fibroblasts exhibited enhanced cell migration in response to treatment with TGF-beta or transfection with an N-cadherin plasmid. CONCLUSIONS: Fibroblasts from strictures in CD express increased constitutive N-cadherin and exhibit enhanced basal cell migration. TGF-beta is a potent inducer of N-cadherin in intestinal fibroblasts resulting in enhanced cell migration. The TGF-beta-mediated induction of N-cadherin may potentiate Crohn\\'s stricture formation.

  12. Dependence of u.v.-induced DNA excision repair on deoxyribonucleoside triphosphate concentrations in permeable human fibroblasts: a model for the inhibition of repair by hydroxyurea

    International Nuclear Information System (INIS)

    Hunting, D.J.; Dresler, S.L.

    1985-01-01

    We have tested the hypothesis that the inhibition by hydroxyurea of repair patch ligation and chromatin rearrangement during u.v.-induced DNA excision repair results from a reduction in cellular deoxyribonucleotide concentrations and not from a direct effect of hydroxyurea on the repair process. Using permeable human fibroblasts, we have shown that hydroxyurea has no direct effect on either repair synthesis or repair patch ligation. We also have shown that by reducing the deoxyribonucleoside triphosphate concentrations in the permeable cell reaction mixture, we can mimic the inhibition of repair patch ligation and chromatin rearrangement seen when u.v.-damaged intact confluent fibroblasts are treated with hydroxyurea. Our results are consistent with the concept that hydroxyurea inhibits DNA repair in intact cells by inhibiting deoxyribonucleotide synthesis through its effect on ribonucleotide reductase and, conversely, that continued deoxyribonucleotide synthesis is required for the excision repair of u.v.-induced DNA damage even in resting cells

  13. Rapid and efficient CRISPR/Cas9 gene inactivation in human neurons during human pluripotent stem cell differentiation and direct reprogramming.

    Science.gov (United States)

    Rubio, Alicia; Luoni, Mirko; Giannelli, Serena G; Radice, Isabella; Iannielli, Angelo; Cancellieri, Cinzia; Di Berardino, Claudia; Regalia, Giulia; Lazzari, Giovanna; Menegon, Andrea; Taverna, Stefano; Broccoli, Vania

    2016-11-18

    The CRISPR/Cas9 system is a rapid and customizable tool for gene editing in mammalian cells. In particular, this approach has widely opened new opportunities for genetic studies in neurological disease. Human neurons can be differentiated in vitro from hPSC (human Pluripotent Stem Cells), hNPCs (human Neural Precursor Cells) or even directly reprogrammed from fibroblasts. Here, we described a new platform which enables, rapid and efficient CRISPR/Cas9-mediated genome targeting simultaneously with three different paradigms for in vitro generation of neurons. This system was employed to inactivate two genes associated with neurological disorder (TSC2 and KCNQ2) and achieved up to 85% efficiency of gene targeting in the differentiated cells. In particular, we devised a protocol that, combining the expression of the CRISPR components with neurogenic factors, generated functional human neurons highly enriched for the desired genome modification in only 5 weeks. This new approach is easy, fast and that does not require the generation of stable isogenic clones, practice that is time consuming and for some genes not feasible.

  14. Genome-wide DNA methylation reprogramming in response to inorganic arsenic links inhibition of CTCF binding, DNMT expression and cellular transformation

    Science.gov (United States)

    Rea, Matthew; Eckstein, Meredith; Eleazer, Rebekah; Smith, Caroline; Fondufe-Mittendorf, Yvonne N.

    2017-02-01

    Chronic low dose inorganic arsenic (iAs) exposure leads to changes in gene expression and epithelial-to-mesenchymal transformation. During this transformation, cells adopt a fibroblast-like phenotype accompanied by profound gene expression changes. While many mechanisms have been implicated in this transformation, studies that focus on the role of epigenetic alterations in this process are just emerging. DNA methylation controls gene expression in physiologic and pathologic states. Several studies show alterations in DNA methylation patterns in iAs-mediated pathogenesis, but these studies focused on single genes. We present a comprehensive genome-wide DNA methylation analysis using methyl-sequencing to measure changes between normal and iAs-transformed cells. Additionally, these differential methylation changes correlated positively with changes in gene expression and alternative splicing. Interestingly, most of these differentially methylated genes function in cell adhesion and communication pathways. To gain insight into how genomic DNA methylation patterns are regulated during iAs-mediated carcinogenesis, we show that iAs probably targets CTCF binding at the promoter of DNA methyltransferases, regulating their expression. These findings reveal how CTCF binding regulates DNA methyltransferase to reprogram the methylome in response to an environmental toxin.

  15. Influence of the Tumor Microenvironment on Cancer Cells Metabolic Reprogramming

    Directory of Open Access Journals (Sweden)

    Victoire Gouirand

    2018-04-01

    Full Text Available As with castles, tumor cells are fortified by surrounding non-malignant cells, such as cancer-associated fibroblasts, immune cells, but also nerve fibers and extracellular matrix. In most cancers, this fortification creates a considerable solid pressure which limits oxygen and nutrient delivery to the tumor cells and causes a hypoxic and nutritional stress. Consequently, tumor cells have to adapt their metabolism to survive and proliferate in this harsh microenvironment. To satisfy their need in energy and biomass, tumor cells develop new capacities to benefit from metabolites of the microenvironment, either by their uptake through the macropinocytosis process or through metabolite transporters, or by a cross-talk with stromal cells and capture of extracellular vesicles that are released by the neighboring cells. However, the microenvironments of primary tumor and metastatic niches differ tremendously in their cellular/acellular components and available nutrients. Therefore, cancer cells must develop a metabolic flexibility conferring on them the ability to satisfy their biomass and energetic demands at both primary and metastasis sites. In this review, we propose a brief overview of how proliferating cancer cells take advantage of their surrounding microenvironment to satisfy their high metabolic demand at both primary and metastasis sites.

  16. 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

  17. 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

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

    Science.gov (United States)

    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.

  20. 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,

  1. 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

  2. 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.

  3. 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

  4. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  7. 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.

  8. 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.

  9. Testosterone metabolism of fibroblasts grown from prostatic carcinoma, benign prostatic hyperplasia and skin fibroblasts

    International Nuclear Information System (INIS)

    Schweikert, H.U.; Hein, H.J.; Romijn, J.C.; Schroeder, F.H.

    1982-01-01

    The metabolism of [1,2,6,7-3H]testosterone was assessed in fibroblast monolayers derived from tissue of 5 prostates with benign hyperplasia (BPH), 4 prostates with carcinoma (PC), and 3 biopsy samples of skin, 2 nongenital skin (NG) and 1 genital skin. The following metabolites could be identified: androstanedione androstenedione, dihydrotestosterone, androsterone, epiandrosterone, androstane-3 alpha, 17 beta-diol and androstane-3 beta, 17 beta-diol. Testosterone was metabolized much more rapidly in fibroblasts originating from prostatic tissue than in fibroblasts derived from NG. A significantly higher formation of 5 alpha-androstanes and 3 alpha-hydroxysteroids could be observed in fibroblasts from BPH as compared to PC. 17-ketosteroid formation exceeded 5 alpha-androstane formation in BPH, whereas 5 alpha-reduction was the predominant pathway in fibroblasts grown from PC and NG. Since testosterone metabolism in fibroblasts of prostatic origin therefore resembles in many aspects that in whole prostatic tissue, fibroblasts grown from prostatic tissues might be a valuable tool for further investigation of the pathogenesis of human BPH and PC

  10. Hedgehog signaling contributes to basic fibroblast growth factor-regulated fibroblast migration

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Zhong Xin [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Sun, Cong Cong [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Wenzhou People' s Hospital, Wenzhou, Zhejiang (China); Ting Zhu, Yu; Wang, Ying; Wang, Tao; Chi, Li Sha; Cai, Wan Hui [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Zheng, Jia Yong [Wenzhou People' s Hospital, Wenzhou, Zhejiang (China); Zhou, Xuan [Ningbo First Hospital, Ningbo, Zhejiang (China); Cong, Wei Tao [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Li, Xiao Kun, E-mail: proflxk@163.com [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Jin, Li Tai, E-mail: jin_litai@126.com [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China)

    2017-06-15

    Fibroblast migration is a central process in skin wound healing, which requires the coordination of several types of growth factors. bFGF, a well-known fibroblast growth factor (FGF), is able to accelerate fibroblast migration; however, the underlying mechanism of bFGF regulation fibroblast migration remains unclear. Through the RNA-seq analysis, we had identified that the hedgehog (Hh) canonical pathway genes including Smoothened (Smo) and Gli1, were regulated by bFGF. Further analysis revealed that activation of the Hh pathway via up-regulation of Smo promoted fibroblast migration, invasion, and skin wound healing, but which significantly reduced by GANT61, a selective antagonist of Gli1/Gli2. Western blot analyses and siRNA transfection assays demonstrated that Smo acted upstream of phosphoinositide 3-kinase (PI3K)-c-Jun N-terminal kinase (JNK)-β-catenin to promote cell migration. Moreover, RNA-seq and qRT-PCR analyses revealed that Hh pathway genes including Smo and Gli1 were under control of β-catenin, suggesting that β-catenin turn feedback activates Hh signaling. Taken together, our analyses identified a new bFGF-regulating mechanism by which Hh signaling regulates human fibroblast migration, and the data presented here opens a new avenue for the wound healing therapy. - Highlights: • bFGF regulates Hedgehog (Hh) signaling in fibroblasts. • The Smo and Gli two master regulators of Hh signaling positively regulate fibroblast migration. • Smo facilitates β-catenin nuclear translocation via activation PI3K/JNK/GSK3β. • β-catenin positively regulates fibroblast cell migration and the expression of Hh signaling genes including Smo and Gli.

  11. Hedgehog signaling contributes to basic fibroblast growth factor-regulated fibroblast migration

    International Nuclear Information System (INIS)

    Zhu, Zhong Xin; Sun, Cong Cong; Ting Zhu, Yu; Wang, Ying; Wang, Tao; Chi, Li Sha; Cai, Wan Hui; Zheng, Jia Yong; Zhou, Xuan; Cong, Wei Tao; Li, Xiao Kun; Jin, Li Tai

    2017-01-01

    Fibroblast migration is a central process in skin wound healing, which requires the coordination of several types of growth factors. bFGF, a well-known fibroblast growth factor (FGF), is able to accelerate fibroblast migration; however, the underlying mechanism of bFGF regulation fibroblast migration remains unclear. Through the RNA-seq analysis, we had identified that the hedgehog (Hh) canonical pathway genes including Smoothened (Smo) and Gli1, were regulated by bFGF. Further analysis revealed that activation of the Hh pathway via up-regulation of Smo promoted fibroblast migration, invasion, and skin wound healing, but which significantly reduced by GANT61, a selective antagonist of Gli1/Gli2. Western blot analyses and siRNA transfection assays demonstrated that Smo acted upstream of phosphoinositide 3-kinase (PI3K)-c-Jun N-terminal kinase (JNK)-β-catenin to promote cell migration. Moreover, RNA-seq and qRT-PCR analyses revealed that Hh pathway genes including Smo and Gli1 were under control of β-catenin, suggesting that β-catenin turn feedback activates Hh signaling. Taken together, our analyses identified a new bFGF-regulating mechanism by which Hh signaling regulates human fibroblast migration, and the data presented here opens a new avenue for the wound healing therapy. - Highlights: • bFGF regulates Hedgehog (Hh) signaling in fibroblasts. • The Smo and Gli two master regulators of Hh signaling positively regulate fibroblast migration. • Smo facilitates β-catenin nuclear translocation via activation PI3K/JNK/GSK3β. • β-catenin positively regulates fibroblast cell migration and the expression of Hh signaling genes including Smo and Gli.

  12. Unchanged thymidine triphosphate pools and thymidine metabolism in two lines of thymidine kinase 2-mutated fibroblasts.

    Science.gov (United States)

    Frangini, Miriam; Rampazzo, Chiara; Franzolin, Elisa; Lara, Mari-Carmen; Vilà, Maya R; Martí, Ramon; Bianchi, Vera

    2009-02-01

    Mitochondrial thymidine kinase (TK2) catalyzes the phosphorylation of thymidine in mitochondria. Its function becomes essential for dTTP synthesis in noncycling cells, where cytosolic dTTP synthesis via R1/R2 ribonucleotide reductase and thymidine kinase 1 is turned down. Mutations in the nuclear gene for TK2 cause a fatal mtDNA depletion syndrome. Only selected cell types are affected, suggesting that the other cells compensate for the TK2 deficiency by adapting the enzyme network that regulates dTTP synthesis outside S-phase. Here we looked for such metabolic adaptation in quiescent cultures of fibroblasts from two TK2-deficient patients with a slow-progressing syndrome. In cell extracts, we measured the activities of TK2, deoxycytidine kinase, thymidine phosphorylase, deoxynucleotidases and the amounts of the three ribonucleotide reductase subunits. Patient cells contained 40% or 5% TK2 activity and unchanged activities of the other enzymes. However, their mitochondrial and cytosolic dTTP pools were unchanged, and also the overall composition of the dNTP pools was normal. TK2-dependent phosphorylation of [(3)H]thymidine in intact cells and the turnover of the dTTP pool showed that even the fibroblasts with 5% residual TK2 activity synthesized dTTP at an almost normal rate. Normal fibroblasts apparently contain more TK2 than needed to maintain dTTP during quiescence, which would explain why TK2-mutated fibroblasts do not manifest mtDNA depletion despite their reduced TK2 activity.

  13. 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.

  14. 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

  15. A fibroblast-associated antigen: Characterization in fibroblasts and immunoreactivity in smooth muscle differentiated stromal cells

    DEFF Research Database (Denmark)

    Rønnov-Jessen, Lone; Celis, Julio E.; van Deurs, Bo

    1992-01-01

    major brands migrating at apparent Mr of 38,000, 45,000, and 80,000, in addition to many minor bands between Mr 45,000 and 97,000, including Mr 52,000. The Mr 45,000 and 38,000 were associated with the cell membrane and Mr 52,000 as well as Mr 38,000 were associated with the lysosomes. The 1B10......Fibroblasts with smooth muscle differentiation are frequently derived from human breast tissue. Immunofluorescence cytochemistry of a fibroblast-associated antigen recognized by a monoclonal antibody (MAb), 1B10, was analyzed with a view to discriminating smooth muscle differentiated fibroblasts...

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

    Directory of Open Access Journals (Sweden)

    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.

  17. 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

  18. Role of periodontal ligament fibroblasts in osteoclastogenesis: a review

    NARCIS (Netherlands)

    Sokos, D.; Everts, V.; de Vries, T.J.

    2015-01-01

    During the last decade it has become clear that periodontal ligament fibroblasts may contribute to the in vitro differentiation of osteoclasts. We surveyed the current findings regarding their osteoclastogenesis potential. Periodontal ligament fibroblasts have the capacity to select and attract

  19. Fibroblast growth factor 23 - et fosfatregulerende hormon

    DEFF Research Database (Denmark)

    Beck-Nielsen, Signe; Pedersen, Susanne Møller; Kassem, Moustapha

    2010-01-01

    Fibroblast growth factor 23 (FGF23) er et nyligt identificeret fosfatonin. FGF23's fysiologiske hovedfunktion er at opretholde normalt serumfosfat og at virke som et D-vitaminmodregulatorisk hormon. Sygdomme, der er koblet til forhøjet serum FGF23, er hypofosfatæmisk rakitis, fibrøs dysplasi og t...

  20. Cryopreservation of canine ovarian and testicular fibroblasts.

    Science.gov (United States)

    Yu, Il-Jeoung; Leibo, S P; Songsasen, Nucharin; Dresser, Betsy L; Kim, In-Shik

    2009-01-01

    To derive a practical procedure to store canine somatic cells, fibroblasts isolated from testicular or ovarian tissues were cryopreserved in 1.2 M ethylene glycol or in 1.2 M dimethylsulfoxide prepared in Dulbecco's Modified Eagle Medium as cryoprotectants, and were frozen either in plastic straws or vials. Thawed cells were cultured for 24 hr at 38.5 degree C in a humidified atmosphere of 5 percent CO2 95 percent air, and then their membrane integrity was assayed with a double fluorescent stain, Fertilight. In addition, frozen-thawed fibroblasts were cultured for 4 days, and then their functional survival was measured after staining small colonies with trypan blue. After freezing and thawing, membrane integrity of testicular fibroblasts was 55-70 percent and functional survival ranged from 20-40 percent. With frozen-thawed ovarian cells, the average membrane integrity was 55-75 percent and the average functional survival was 35-40 percent. When frozen in ethylene glycol, functional survival of ovarian fibroblasts was significantly higher than that of testicular cells (P less than 0.05). These methods should prove useful to preserve cells collected from canids in the wild.

  1. Assessment of geomechanical properties of intact Opalinus Clay - Expert report

    International Nuclear Information System (INIS)

    Amann, F.; Vogelhuber, M.

    2015-11-01

    This comprehensive report published by the Swiss Federal Nuclear Safety Inspectorate ENSI presents an expert report published on the assessment of the geomechanical properties of intact Opalinus Clay. This review report addresses the conceptual constitutive framework for repositories in Opalinus Clay. The author addresses the geomechanical fundamentals that are necessary in order to adequately judge experiments on intact Opalinus Clay and the interpretation of the results. The report assesses in detail the various test series on intact Opalinus Clay carried out along with the interpretations made by experts and NAGRA. Further assessments are quoted including those on sample geometries tested, effective strength properties, undrained shear strength properties and elastic properties. The results of work done by other experts are also presented and discussed. The report is completed with a list of relevant literature

  2. Assessment of geomechanical properties of intact Opalinus Clay - Expert report

    Energy Technology Data Exchange (ETDEWEB)

    Amann, F. [Eidgenössische Technische Hochschule ETHZ, Zürich (Switzerland); Vogelhuber, M. [Dr. von Moos AG, Geotechnisches Büro, Zürich (Switzerland)

    2015-11-15

    This comprehensive report published by the Swiss Federal Nuclear Safety Inspectorate ENSI presents an expert report published on the assessment of the geomechanical properties of intact Opalinus Clay. This review report addresses the conceptual constitutive framework for repositories in Opalinus Clay. The author addresses the geomechanical fundamentals that are necessary in order to adequately judge experiments on intact Opalinus Clay and the interpretation of the results. The report assesses in detail the various test series on intact Opalinus Clay carried out along with the interpretations made by experts and NAGRA. Further assessments are quoted including those on sample geometries tested, effective strength properties, undrained shear strength properties and elastic properties. The results of work done by other experts are also presented and discussed. The report is completed with a list of relevant literature.

  3. Nuclear targeting of IGF-1 receptor in orbital fibroblasts from Graves' disease: apparent role of ADAM17.

    Directory of Open Access Journals (Sweden)

    Neil Hoa

    Full Text Available Insulin-like growth factor-1 receptor (IGF-1R comprises two subunits, including a ligand binding domain on extra- cellular IGF-1Rα and a tyrosine phosphorylation site located on IGF-1Rβ. IGF-1R is over-expressed by orbital fibroblasts in the autoimmune syndrome, Graves' disease (GD. When activated by IGF-1 or GD-derived IgG (GD-IgG, these fibroblasts produce RANTES and IL-16, while those from healthy donors do not. We now report that IGF-1 and GD-IgG provoke IGF-1R accumulation in the cell nucleus of GD fibroblasts where it co-localizes with chromatin. Nuclear IGF-1R is detected with anti-IGF-1Rα-specific mAb and migrates to approximately 110 kDa, consistent with its identity as an IGF-1R fragment. Nuclear IGF-1R migrating as a 200 kDa protein and consistent with an intact receptor was undetectable when probed with either anti-IGF-1Rα or anti-IGF-1Rβ mAbs. Nuclear redistribution of IGF-1R is absent in control orbital fibroblasts. In GD fibroblasts, it can be abolished by an IGF-1R-blocking mAb, 1H7 and by physiological concentrations of glucocorticoids. When cell-surface IGF-1R is cross-linked with (125I IGF-1, (125I-IGF-1/IGF-1R complexes accumulate in the nuclei of GD fibroblasts. This requires active ADAM17, a membrane associated metalloproteinase, and the phosphorylation of IGF-1R. In contrast, virally encoded IGF-1Rα/GFP fusion protein localizes equivalently in nuclei in both control and GD fibroblasts. This result suggests that generation of IGF-1R fragments may limit the accumulation of nuclear IGF-1R. We thus identify a heretofore-unrecognized behavior of IGF-1R that appears limited to GD-derived fibroblasts. Nuclear IGF-1R may play a role in disease pathogenesis.

  4. 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

  5. The effect of tranilast on fibroblast activation protein α (FAP-α expression in normal and keloid fibroblasts in vitro

    Directory of Open Access Journals (Sweden)

    Paweł P. Antończak

    2017-07-01

    Full Text Available Introduction . Tranilast (N-(3’,4’-demethoxycinnamoyl-anthranilic acid is an anti-allergic drug. Its mechanism of action is based on the inhibition of antigen-induced release of chemical mediators from mast cells and basophils. It also reveals antifibroproliferative activities. These properties of tranilast are used in the treatment of hypertrophic scars and keloids. Keloids are characterized by incorrect extracellular matrix components turnover. Fibroblasts derived from keloids reveal overproduction of collagen type I and decreased degradation of extracellular matrix in comparison with normal fibroblasts. Fibroblast activation protein α (FAP-α may play an important role in remodeling of extracellular matrix and the invasive properties of keloids. Objective . In the present study, the effect of tranilast on expression of FAP-α gene and its protein was evaluated in normal human dermal fibroblasts and fibroblasts derived from keloids cultured in vitro . Materials and methods. In the first stage of the study, the influence of tranilast on cell viability was estimated. The second stage of the study included the quantitative evaluation of FAP-α mRNA expression in normal and keloid fibroblasts treated with tranilast. The third stage of the study comprised fibroblast activation protein α expression analysis in the examined cells treated with tranilast. Results and conclusions . The expression of FAP-α gene and fibroblast activation protein α is higher in keloid fibroblasts. Tranilast at concentrations of 3 μM and 30 μM up-regulated mRNA FAP-α expression in normal fibroblasts but did not influence keloid fibroblasts. The drug, at concentrations of 30 μM and 300 μM up-regulated fibroblast activation protein α expression in normal fibroblasts and did not influence keloid fibroblasts. Tranilast antiproliferative effect is not associated with FAP-α expression in keloid fibroblasts.

  6. Bioavailability and in vivo metabolism of intact glucosinolates

    DEFF Research Database (Denmark)

    Sørensen, Jens Christian; Frandsen, Heidi Blok; Jensen, Søren Krogh

    2016-01-01

    Health benefits associated with consumption of cruciferous vegetables have received considerable attention with a hitherto focus on the role and bioactivity of glucosinolate degradation products. We investigated the in vivo metabolism of intact glucosinolates by following their fate in digesta an...

  7. Capillary zone electrophoresis-mass spectromet of intact proteins

    NARCIS (Netherlands)

    Domínguez-Vega, Elena; Haselberg, Rob; Somsen, Govert W.

    2016-01-01

    Capillary electrophoresis (CE) coupled with mass spectrometry (MS) has proven to be a powerful analytical tool for the characterization of intact proteins. It combines the high separation efficiency, short analysis time, and versatility of CE with the mass selectivity and sensitivity offered by MS

  8. Intact collagen and atelocollagen sponges: Characterization and ESEM observation

    International Nuclear Information System (INIS)

    Ruozi, Barbara; Tosi, Giovanni; Leo, Eliana; Parma, Bruna; Vismara, Susanna; Forni, Flavio; Vandelli, Maria Angela

    2007-01-01

    In this study we have investigated the chemical-physical and morphological properties of intact and atelocollagen sponges used for tissue engineering. The porous sponges were prepared by lyophilization and their physico-chemical characteristics (water binding capacity, denaturing temperature, amino group content) were investigated. Considering the importance of the 'in vivo' interactions between these sponges and the tissue, our attention was addressed (a) to clarify the relationships between the morphology and the amount of water absorbed and (b) to evaluate the influence of pepsin-alkaline treatment on the reorganization of the atelocollagen fibres. Conventional scanning electron microscopy (SEM) and environmental scanning electron microscopy (ESEM) were employed to study the morphology and wetting behaviour of the intact and atelocollagen sponges. The observations by SEM indicated remarkable differences both in the structure and dimension of the pores between intact and atelocollagen sponges. At the data are related to a different water binding capacity. However, the ESEM observations, achieved by changing the relative humidity in the operative chamber, demonstrated that the water adsorbed can be removed with major difficulty from atelocollagen sponges than from intact ones

  9. Phosphorylation of intact erythrocytes in human muscular dystrophy

    International Nuclear Information System (INIS)

    Johnson, R.M.; Nigro, M.

    1986-01-01

    The uptake of exogenous 32 Pi into the membrane proteins of intact erythrocytes was measured in 8 patients with Duchenne muscular dystrophy. No abnormalities were noted after autoradiographic analysis. This contrasts with earlier results obtained when isolated membranes were phosphorylated with gamma-[ 32 P]ATP, and suggests a possible reinterpretation of those experiments

  10. Behavior Management Style of Single Parents and Intact Families.

    Science.gov (United States)

    Smith, Douglas K.; And Others

    Studies examining the behavior management styles of parents as a function of family intactness and parent employment status are lacking. To assess parental style of behavior management, the Parental Management Questionnaire (PMQ) was completed by 1,957 parents of elementary school children (50% response rate). The PMQ is based on Aronfreed's…

  11. Non-intact zona improves development of murine preimplantation ...

    African Journals Online (AJOL)

    ajl5

    2012-09-25

    Sep 25, 2012 ... 2College of Animal Science and Technology, Northwest A & F University, Yangling, ... Key words: Mouse, non-intact zona embryos, adenovirus vector with green fluorescent protein (pAd-GFP), .... Based on microscopic examination, the ZP of some ..... permeable structure of ZP that allowed penetration of.

  12. Pharmacokinetics of erythropoietin in intact and anephric dogs

    International Nuclear Information System (INIS)

    Fu, J.S.; Lertora, J.J.; Brookins, J.; Rice, J.C.; Fisher, J.W.

    1988-01-01

    The present studies were performed to determine the pharmacokinetic parameters of erythropoietin in intact and anephric dogs by use of unlabeled crude native erythropoietin (nEp) and iodine 125-labeled purified recombinant erythropoietin (rEp) given by intravenous infusion for 15 minutes. Sephadex G-75 gel filtration was used to confirm that the 125I-rEp molecule remained iodinated in dog plasma during the 24-hour period of these studies. The plasma disappearance of erythropoietin conformed to a biexponential equation for both nEp and 125I-rEp, with the central compartment being larger than the peripheral compartment. The mean distribution half-life of 75.3 +/- 21.2 minutes for nEp was significantly (p less than 0.05) longer than that of 125I-rEp (23.7 +/- 5.0 minutes) in intact dogs. The intercompartmental clearance (CIic) for nEp (0.018 +/- 0.006 L/kg/hr) was significantly smaller than that of 125I-rEp (0.068 +/- 0.018 L/kg/hr) in intact dogs (p less than 0.05). There were no significant differences in apparent volume of distribution, elimination half-life, and elimination clearance (CIe) for nEp and rEp in intact dogs. The mean elimination half-life for 125I-rEp in intact dogs (9.0 +/- 0.6 hours) and anephric dogs (13.8 +/- 1.4 hours) was significantly different (p less than 0.05). The CIe for 125I-rEp in anephric dogs (0.008 +/- 0.001 L/kg/hr) was significantly (p less than 0.05) smaller than that of 125I-rEp in intact dogs (0.011 +/- 0.001 L/kg/hr). There were no significant differences in apparent volume of distribution, distribution half-life, and CIic for 125I-rEp in intact and anephric dogs

  13. 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.

  14. 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.

  15. Threats to intact tropical peatlands and opportunities for their conservation.

    Science.gov (United States)

    Roucoux, K H; Lawson, I T; Baker, T R; Del Castillo Torres, D; Draper, F C; Lähteenoja, O; Gilmore, M P; Honorio Coronado, E N; Kelly, T J; Mitchard, E T A; Vriesendorp, C F

    2017-12-01

    Large, intact areas of tropical peatland are highly threatened at a global scale by the expansion of commercial agriculture and other forms of economic development. Conserving peatlands on a landscape scale, with their hydrology intact, is of international conservation importance to preserve their distinctive biodiversity and ecosystem services and maintain their resilience to future environmental change. We explored threats to and opportunities for conserving remaining intact tropical peatlands; thus, we excluded peatlands of Indonesia and Malaysia, where extensive deforestation, drainage, and conversion to plantations means conservation in this region can protect only small fragments of the original ecosystem. We focused on a case study, the Pastaza-Marañón Foreland Basin (PMFB) in Peru, which is among the largest known intact tropical peatland landscapes in the world and is representative of peatland vulnerability. Maintenance of the hydrological conditions critical for carbon storage and ecosystem function of peatlands is, in the PMFB, primarily threatened by expansion of commercial agriculture linked to new transport infrastructure that is facilitating access to remote areas. There remain opportunities in the PMFB and elsewhere to develop alternative, more sustainable land-use practices. Although some of the peatlands in the PMFB fall within existing legally protected areas, this protection does not include the most carbon-dense (domed pole forest) areas. New carbon-based conservation instruments (e.g., REDD+, Green Climate Fund), developing markets for sustainable peatland products, transferring land title to local communities, and expanding protected areas offer pathways to increased protection for intact tropical peatlands in Amazonia and elsewhere, such as those in New Guinea and Central Africa which remain, for the moment, broadly beyond the frontier of commercial development. © 2017 The Authors. Conservation Biology published by Wiley Periodicals, Inc

  16. 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

  17. 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...

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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-...

  5. 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...

  6. 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.

  7. 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).

  8. 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.

  9. Transfer of an expression YAC into goat fetal fibroblasts by cell fusion for mammary gland bioreactor

    International Nuclear Information System (INIS)

    Zhang Xufeng; Wu Guoxiang; Chen, Jian-Quan; Zhang Aimin; Liu Siguo; Jiao Binghua; Cheng Guoxiang

    2005-01-01

    Yeast artificial chromosomes (YACs) as transgenes in transgenic animals are likely to ensure optimal expression levels. Microinjection of YACs is the exclusive technique used to produce YACs transgenic livestock so far. However, low efficiency and high cost are its critical restrictive factors. In this study, we presented a novel procedure to produce YACs transgenic livestock as mammary gland bioreactor. A targeting vector, containing the gene of interest-a human serum albumin minigene (intron 1, 2), yeast selectable marker (G418R), and mammalian cell resistance marker (neo r ), replaced the α-lactalbumin gene in a 210 kb human α-lactalbumin YAC by homogeneous recombination in yeasts. The chimeric YAC was introduced into goat fetal fibroblasts using polyethylene glycol-mediated spheroplast fusion. PCR and Southern analysis showed that intact YAC was integrated in the genome of resistant cells. Perhaps, it may offer a cell-based route by nuclear transfer to produce YACs transgenic livestock

  10. Generation and periodontal differentiation of human gingival fibroblasts-derived integration-free induced pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Xiaohui [Department of Periodontology, School and Hospital of Stomatology, Peking University, 22 South Avenue Zhong-Guan-Cun, Beijing 100081 (China); Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Li, Yang [Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Li, Jingwen [Department of Periodontology, School and Hospital of Stomatology, Peking University, 22 South Avenue Zhong-Guan-Cun, Beijing 100081 (China); Li, Peng [Faculty of Dentistry, The University of Hong Kong, 34 Hospital Road, Hong Kong SAR (China); Liu, Yinan [Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Wen, Jinhua, E-mail: jhwen@bjmu.edu.cn [Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Luan, Qingxian, E-mail: kqluanqx@126.com [Department of Periodontology, School and Hospital of Stomatology, Peking University, 22 South Avenue Zhong-Guan-Cun, Beijing 100081 (China)

    2016-05-06

    Induced pluripotent stem cells (iPSCs) have been recognized as a promising cell source for periodontal tissue regeneration. However, the conventional virus-based reprogramming approach is associated with a high risk of genetic mutation and limits their therapeutic utility. Here, we successfully generated iPSCs from readily accessible human gingival fibroblasts (hGFs) through an integration-free and feeder-free approach via delivery of reprogramming factors of Oct4, Sox2, Klf4, L-myc, Lin28 and TP53 shRNA with episomal plasmid vectors. The iPSCs presented similar morphology and proliferation characteristics as embryonic stem cells (ESCs), and expressed pluripotent markers including Oct4, Tra181, Nanog and SSEA-4. Additionally, these cells maintained a normal karyotype and showed decreased CpG methylation ratio in the promoter regions of Oct4 and Nanog. In vivo teratoma formation assay revealed the development of tissues representative of three germ layers, confirming the acquisition of pluripotency. Furthermore, treatment of the iPSCs in vitro with enamel matrix derivative (EMD) or growth/differentiation factor-5 (GDF-5) significantly up-regulated the expression of periodontal tissue markers associated with bone, periodontal ligament and cementum respectively. Taken together, our data demonstrate that hGFs are a valuable cell source for generating integration-free iPSCs, which could be sequentially induced toward periodontal cells under the treatment of EMD and GDF-5. - Highlights: • Integration-free iPSCs are successfully generated from hGFs via an episomal approach. • EMD promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • GDF-5 promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • hGFs-derived iPSCs could be a promising cell source for periodontal regeneration.

  11. Generation and periodontal differentiation of human gingival fibroblasts-derived integration-free induced pluripotent stem cells

    International Nuclear Information System (INIS)

    Yin, Xiaohui; Li, Yang; Li, Jingwen; Li, Peng; Liu, Yinan; Wen, Jinhua; Luan, Qingxian

    2016-01-01

    Induced pluripotent stem cells (iPSCs) have been recognized as a promising cell source for periodontal tissue regeneration. However, the conventional virus-based reprogramming approach is associated with a high risk of genetic mutation and limits their therapeutic utility. Here, we successfully generated iPSCs from readily accessible human gingival fibroblasts (hGFs) through an integration-free and feeder-free approach via delivery of reprogramming factors of Oct4, Sox2, Klf4, L-myc, Lin28 and TP53 shRNA with episomal plasmid vectors. The iPSCs presented similar morphology and proliferation characteristics as embryonic stem cells (ESCs), and expressed pluripotent markers including Oct4, Tra181, Nanog and SSEA-4. Additionally, these cells maintained a normal karyotype and showed decreased CpG methylation ratio in the promoter regions of Oct4 and Nanog. In vivo teratoma formation assay revealed the development of tissues representative of three germ layers, confirming the acquisition of pluripotency. Furthermore, treatment of the iPSCs in vitro with enamel matrix derivative (EMD) or growth/differentiation factor-5 (GDF-5) significantly up-regulated the expression of periodontal tissue markers associated with bone, periodontal ligament and cementum respectively. Taken together, our data demonstrate that hGFs are a valuable cell source for generating integration-free iPSCs, which could be sequentially induced toward periodontal cells under the treatment of EMD and GDF-5. - Highlights: • Integration-free iPSCs are successfully generated from hGFs via an episomal approach. • EMD promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • GDF-5 promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • hGFs-derived iPSCs could be a promising cell source for periodontal regeneration.

  12. Oral fibroblasts produce more HGF and KGF than skin fibroblasts in response to co-culture with keratinocytes

    DEFF Research Database (Denmark)

    Grøn, Birgitte; Stoltze, Kaj; Andersson, Anders

    2002-01-01

    The production of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) in subepithelial fibroblasts from buccal mucosa, periodontal ligament, and skin was determined after co-culture with keratinocytes. The purpose was to detect differences between the fibroblast subpopulations...... days by ELISA. When cultured on polystyrene, the constitutive level of KGF and HGF in periodontal fibroblasts was higher than the level in buccal and skin fibroblasts. In the presence of keratinocytes, all three types of fibroblasts in general increased their HGF and KGF production 2-3 times. When...... cells were maintained in collagen, the level of HGF and KGF was decreased mainly in skin cultures. However, in oral fibroblasts, induction after stimulation was at a similar level in collagen compared to on polystyrene. Skin fibroblasts maintained in collagen produced almost no HGF whether...

  13. Lung Fibroblasts, Aging, and Idiopathic Pulmonary Fibrosis.

    Science.gov (United States)

    Pardo, Annie; Selman, Moisés

    2016-12-01

    Idiopathic pulmonary fibrosis (IPF) is an aging-associated, progressive, and irreversible lung disease of unknown etiology, elusive pathogenesis, and very limited therapeutic options. The hallmarks of IPF are aberrant activation of alveolar epithelial cells and accumulation of fibroblasts and myofibroblasts along with excessive production of extracellular matrix. The linkage of aging with this disorder is uncertain, but a number of changes associated with aging, including telomere attrition, cell senescence, and mitochondrial dysfunction, have been revealed in IPF lungs. Also, aging seems to confer a profibrotic phenotype upon fibroblasts and to increase the severity of the fibrogenic response in non-IPF fibrotic lung disorders. Better knowledge of the pathophysiological mechanisms linking aging to IPF will advance understanding of its pathogenesis and may provide new therapeutic windows to treatment of this devastating disease.

  14. The Multilevel Mixed Intact Group Analysis: A Mixed Method to Seek, Detect, Describe, and Explain Differences Among Intact Groups

    Science.gov (United States)

    Schoonenboom, Judith

    2016-01-01

    Educational innovations often involve intact subgroups, such as school classes or university departments. In small-scale educational evaluation research, typically involving 1 to 20 subgroups, differences among these subgroups are often neglected. This article presents a mixed method from a qualitative perspective, in which differences among…

  15. The Multilevel Mixed Intact Group Analysis: A Mixed Method to Seek, Detect, Describe and Explain Differences Between Intact Groups

    NARCIS (Netherlands)

    Schoonenboom, J.I.

    2014-01-01

    Educational innovations often involve intact subgroups, such as school classes or university departments. In small-scale educational evaluation research, typically involving 1 to 20 subgroups, differences among these subgroups are often neglected. This article presents a mixed method from a

  16. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. 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.

  20. 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)

  1. 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.

  2. Gene targeting in adult rhesus macaque fibroblasts

    Directory of Open Access Journals (Sweden)

    Wolf Don P

    2008-03-01

    Full Text Available Abstract Background Gene targeting in nonhuman primates has the potential to produce critical animal models for translational studies related to human diseases. Successful gene targeting in fibroblasts followed by somatic cell nuclear transfer (SCNT has been achieved in several species of large mammals but not yet in primates. Our goal was to establish the protocols necessary to achieve gene targeting in primary culture of adult rhesus macaque fibroblasts as a first step in creating nonhuman primate models of genetic disease using nuclear transfer technology. Results A primary culture of adult male fibroblasts was transfected with hTERT to overcome senescence and allow long term in vitro manipulations. Successful gene targeting of the HPRT locus in rhesus macaques was achieved by electroporating S-phase synchronized cells with a construct containing a SV40 enhancer. Conclusion The cell lines reported here could be used for the production of null mutant rhesus macaque models of human genetic disease using SCNT technology. In addition, given the close evolutionary relationship and biological similarity between rhesus macaques and humans, the protocols described here may prove useful in the genetic engineering of human somatic cells.

  3. Pathways of sphingomyelin metabolism in cultured fibroblasts from normal and sphingomyelin lipidosis subjects.

    Science.gov (United States)

    Spence, M W; Clarke, J T; Cook, H W

    1983-07-25

    The metabolism of endogenous sphingomyelin labeled with 32P or [methyl-3H]choline and of exogenous [choline-methyl-3H], [32P]-, or [N-acyl-1-14C]sphingomyelin was studied in normal and Niemann-Pick Type A (NP-A) cultured fibroblasts. Despite a greater than 96% decrease in lysosomal sphingomyelinase activity in the NP-A cells, they were able to degrade endogenously produced [32P]- or [methyl-3H]sphingomyelin at normal or near normal rates. Exogenous [methyl-3H]-, [methyl-3H, 32P]-, and [methyl-3H, N-acyl-1-14C] sphingomyelin was taken up intact by normal and NP-A cells, with NP-A cells accumulating 4-8 times more lipid. By 20 h, 50% of the control cell-associated 3H and 32P was recovered in lecithin, and the ratio of activities (3H/32P) indicated most of the phosphorylcholine derived from sphingomyelin had been transferred intact. By comparison in NP-A cells, after a 40-h incubation only 20% of the labeled phosphorylcholine derived from sphingomyelin was recovered in lecithin. With both cell lines, 20 to 50 times more sphingomyelin was hydrolyzed than was taken up by the cells; the reaction products in the medium were ceramide and a mixture of water-soluble compounds such as phosphorylcholine and choline. These results indicate that there are at least two metabolic pathways for sphingomyelin modification in cultured fibroblasts in addition to degradation by the lysosomal acid sphingomyelinase. One route is hydrolysis by a cellular sphingomyelinase. The second is the hydrolysis and/or transfer of phosphorylcholine from sphingomyelin and results in the synthesis of lecithin.

  4. Structural determination of intact proteins using mass spectrometry

    Science.gov (United States)

    Kruppa, Gary [San Francisco, CA; Schoeniger, Joseph S [Oakland, CA; Young, Malin M [Livermore, CA

    2008-05-06

    The present invention relates to novel methods of determining the sequence and structure of proteins. Specifically, the present invention allows for the analysis of intact proteins within a mass spectrometer. Therefore, preparatory separations need not be performed prior to introducing a protein sample into the mass spectrometer. Also disclosed herein are new instrumental developments for enhancing the signal from the desired modified proteins, methods for producing controlled protein fragments in the mass spectrometer, eliminating complex microseparations, and protein preparatory chemical steps necessary for cross-linking based protein structure determination.Additionally, the preferred method of the present invention involves the determination of protein structures utilizing a top-down analysis of protein structures to search for covalent modifications. In the preferred method, intact proteins are ionized and fragmented within the mass spectrometer.

  5. Does human leukocyte elastase degrade intact skin elastin?

    DEFF Research Database (Denmark)

    Schmelzer, Christian E H; Jung, Michael C; Wohlrab, Johannes

    2012-01-01

    This study aimed to investigate the susceptibility of intact fibrillar human elastin to human leukocyte elastase and cathepsin G. Elastin is a vital protein of the extracellular matrix of vertebrates, and provides exceptional properties including elasticity and tensile strength to many tissues...... and organs, including the aorta, lung, cartilage, elastic ligaments and skin, and is thus critical for their long-term function. Mature elastin is an insoluble and extremely durable protein that undergoes very little turnover, but sustained exposure to proteases may lead to irreversible and severe damage......, and thus to functional loss of the elastic fiber network. Hence, it is a key issue to understand which enzymes actually initiate elastolysis under certain pathological conditions or during intrinsic aging. In this paper, we provide a complete workflow for isolation of pure and intact elastin from very...

  6. SOLITARY CHEMORECEPTOR CELL SURVIVAL IS INDEPENDENT OF INTACT TRIGEMINAL INNERVATION

    Science.gov (United States)

    Gulbransen, Brian; Silver, Wayne; Finger, Tom

    2008-01-01

    Nasal solitary chemoreceptor cells (SCCs) are a population of specialized chemosensory epithelial cells presumed to broaden trigeminal chemoreceptivity in mammals (Finger et al., 2003). SCCs are innervated by peptidergic trigeminal nerve fibers (Finger et al., 2003) but it is currently unknown if intact innervation is necessary for SCC development or survival. We tested the dependence of SCCs on innervation by eliminating trigeminal nerve fibers during development with neurogenin-1 knockout mice, during early postnatal development with capsaicin desensitization, and during adulthood with trigeminal lesioning. Our results demonstrate that elimination of innervation at any of these times does not result in decreased SCC numbers. In conclusion, neither SCC development nor mature cell maintenance is dependent on intact trigeminal innervation. PMID:18300260

  7. Direct detection of radicals in intact soybean nodules

    DEFF Research Database (Denmark)

    Mathieu, C; Moreau, S; Frendo, P

    1998-01-01

    Electron paramagnetic resonance spectroscopy has been employed to examine the nature of the metal ions and radicals present in intact root nodules of soybean plants grown in the absence of nitrate. The spectra obtained from nodules of different ages using this non-invasive technique show dramatic...... differences, suggesting that there are both qualitative and quantitative changes in the metal ion and radical species present. A major component of the spectra obtained from young nodules is assigned to a complex (Lb-NO) of nitric oxide (NO.) with the heme protein leghemoglobin (Lb). This Lb-NO species, which...... has not been previously detected in intact root nodules of plants grown in the absence of nitrate, is thought to be formed by reaction of nitric oxide with iron(II) leghemoglobin. The nitric oxide may be generated from arginine via a nitric oxide synthase-like activity present in the nodules...

  8. Isolation and Properties of Intact Chromoplasts from Tomato Fruits

    OpenAIRE

    Norio, Iwatsuki; Ryuichi, Moriyama; Tadashi, Asahi; Laboratory of Biochemistry, Faculty of Agriculture, Nagoya University; Laboratory of Biochemistry, Faculty of Agriculture, Nagoya University; Laboratory of Biochemistry, Faculty of Agriculture, Nagoya University

    1984-01-01

    Intact chromoplasts were isolated from tomato fruits at different ripening stages by Percoll density gradient centrifugation. The isolated chromoplast fractions were contaminated very little by other organelles, although the fraction from fully ripened fruits contained some mitochondria and microbodies. As the transformation of chloroplasts to chromoplasts proceeded, the density of the plastids decreased from 1.096 to 1.075g・cm^ and the decrease was related to a decrease in chlorophyll and an...

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  11. 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

  12. Global forest loss disproportionately erodes biodiversity in intact landscapes.

    Science.gov (United States)

    Betts, Matthew G; Wolf, Christopher; Ripple, William J; Phalan, Ben; Millers, Kimberley A; Duarte, Adam; Butchart, Stuart H M; Levi, Taal

    2017-07-27

    Global biodiversity loss is a critical environmental crisis, yet the lack of spatial data on biodiversity threats has hindered conservation strategies. Theory predicts that abrupt biodiversity declines are most likely to occur when habitat availability is reduced to very low levels in the landscape (10-30%). Alternatively, recent evidence indicates that biodiversity is best conserved by minimizing human intrusion into intact and relatively unfragmented landscapes. Here we use recently available forest loss data to test deforestation effects on International Union for Conservation of Nature Red List categories of extinction risk for 19,432 vertebrate species worldwide. As expected, deforestation substantially increased the odds of a species being listed as threatened, undergoing recent upgrading to a higher threat category and exhibiting declining populations. More importantly, we show that these risks were disproportionately high in relatively intact landscapes; even minimal deforestation has had severe consequences for vertebrate biodiversity. We found little support for the alternative hypothesis that forest loss is most detrimental in already fragmented landscapes. Spatial analysis revealed high-risk hot spots in Borneo, the central Amazon and the Congo Basin. In these regions, our model predicts that 121-219 species will become threatened under current rates of forest loss over the next 30 years. Given that only 17.9% of these high-risk areas are formally protected and only 8.9% have strict protection, new large-scale conservation efforts to protect intact forests are necessary to slow deforestation rates and to avert a new wave of global extinctions.

  13. Antimicrobial activity of a new intact skin antisepsis formulation.

    Science.gov (United States)

    Russo, Antonello; Viotti, Pier Luigi; Vitali, Matteo; Clementi, Massimo

    2003-04-01

    Different antiseptic formulations have shown limitations when applied to disinfecting intact skin, notably short-term tolerability and/or efficacy. The purpose of this study was optimizing a new antiseptic formulation specifically targeted at intact skin disinfection and evaluating its in vitro microbicidal activity and in vivo efficacy. The biocidal properties of the antiseptic solution containing 0.5% chloramine-T diluted in 50% isopropyl alcohol (Cloral; Eurospital SpA Trieste, Italy) were measured in vitro versus gram-positive-, gram-negative-, and acid-alcohol-resistant germs and fungi with standard suspension tests in the presence of fetal bovine serum. Virus-inhibiting activity was evaluated in vitro against human cytomegalovirus, herpes simplex virus, poliovirus, hepatitis B virus, and hepatitis C virus. Tests used different methods for the different biologic and in vitro replication capacity of these human viruses. Lastly, Cloral tolerability and skin colonization retardation efficacy after disinfection were studied in vivo. The antiseptic under review showed fast and sustained antimicrobial activity. The efficacy of Cloral against clinically important bacterial and viral pathogens and fungi was highlighted under the experimental conditions described in this article. Finally, microbial regrowth lag and no side effects were documented in vivo after disinfection of 11 volunteers. A stable chloramine-T solution in isopropyl alcohol may be suggested for intact skin antisepsis.

  14. Chromosome aberration induction in human diploid fibroblast and epithelial cells

    International Nuclear Information System (INIS)

    Scott, D.

    1986-01-01

    The relative sensitivity of cultured human fibroblasts and epithelial cells to radiation-induced chromosomal aberrations was investigated. Lung fibroblast and kidney epithelial cells from the same fetus were compared, as were skin fibroblasts and epithelial keratinocytes from the same foreskin sample. After exposure of proliferating fetal cells to 1.5 Gy X-rays there was a very similar aberration yield in the fibroblasts and epithelial cells. Observations of either little or no difference in chromosomal sensitivity between human fibroblasts and epithelial cells give added confidence that quantitative cytogenetic data obtained from cultured fibroblasts are relevant to the question of sensitivity of epithelial cells which are the predominant cell type in human cancers. (author)

  15. Improved Fibroblast Functionalities by Microporous Pattern Fabricated by Microelectromechanical Systems

    OpenAIRE

    Wei, Hongbo; Zhao, Lingzhou; Chen, Bangdao; Bai, Shizhu; Zhao, Yimin

    2014-01-01

    Fibroblasts, which play an important role in biological seal formation and maintenance, determine the long-term success of percutaneous implants. In this study, well-defined microporous structures with micropore diameters of 10–60 µm were fabricated by microelectromechanical systems and their influence on the fibroblast functionalities was observed. The results show that the microporous structures with micropore diameters of 10–60 µm did not influence the initial adherent fibroblast number; ...

  16. Galvanic microparticles increase migration of human dermal fibroblasts in a wound-healing model via reactive oxygen species pathway.

    Science.gov (United States)

    Tandon, Nina; Cimetta, Elisa; Villasante, Aranzazu; Kupferstein, Nicolette; Southall, Michael D; Fassih, Ali; Xie, Junxia; Sun, Ying; Vunjak-Novakovic, Gordana

    2014-01-01

    Electrical signals have been implied in many biological mechanisms, including wound healing, which has been associated with transient electrical currents not present in intact skin. One method to generate electrical signals similar to those naturally occurring in wounds is by supplementation of galvanic particles dispersed in a cream or gel. We constructed a three-layered model of skin consisting of human dermal fibroblasts in hydrogel (mimic of dermis), a hydrogel barrier layer (mimic of epidermis) and galvanic microparticles in hydrogel (mimic of a cream containing galvanic particles applied to skin). Using this model, we investigated the effects of the properties and amounts of Cu/Zn galvanic particles on adult human dermal fibroblasts in terms of the speed of wound closing and gene expression. The collected data suggest that the effects on wound closing are due to the ROS-mediated enhancement of fibroblast migration, which is in turn mediated by the BMP/SMAD signaling pathway. These results imply that topical low-grade electric currents via microparticles could enhance wound healing. © 2013 Elsevier Inc. All rights reserved.

  17. Tumor-secreted LOXL2 activates fibroblasts through FAK signaling

    DEFF Research Database (Denmark)

    Barker, Holly E; Bird, Demelza; Lang, Georgina

    2013-01-01

    models. Here, we discovered that tumor-derived LOXL2 directly activated stromal fibroblasts in the tumor microenvironment. Genetic manipulation or antibody inhibition of LOXL2 in orthotopically grown mammary tumors reduced the expression of α-smooth muscle actin (α-SMA). Using a marker for reticular....... Importantly, in vitro assays revealed that tumor-derived LOXL2 and a recombinant LOXL2 protein induced fibroblast branching on collagen matrices, as well as increased fibroblast-mediated collagen contraction and invasion of fibroblasts through extracellular matrix. Moreover, LOXL2 induced the expression of α...

  18. 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.

  19. Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

    International Nuclear Information System (INIS)

    Fahrer, Joerg; Wagner, Silvia; Buerkle, Alexander; Koenigsrainer, Alfred

    2009-01-01

    Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.

  20. Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

    Energy Technology Data Exchange (ETDEWEB)

    Fahrer, Joerg, E-mail: joerg.fahrer@uni-ulm.de [Molecular Toxicology Group, Department of Biology, University of Konstanz (Germany); Wagner, Silvia [Clinic of General, Visceral- and Transplantation Surgery, ZMF, University Hospital Tuebingen (Germany); Buerkle, Alexander [Molecular Toxicology Group, Department of Biology, University of Konstanz (Germany); Koenigsrainer, Alfred [Clinic of General, Visceral- and Transplantation Surgery, ZMF, University Hospital Tuebingen (Germany)

    2009-08-14

    Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.

  1. Immortalization of Werner syndrome and progeria fibroblasts

    International Nuclear Information System (INIS)

    Saito, H.; Moses, R.E.

    1991-01-01

    Human fibroblast cells from two different progeroid syndromes, Werner syndrome (WS) and progeria, were established as immortalized cell lines by transfection with plasmid DNA containing the SV40 early region. The lineage of each immortalized cell line was confirmed by VNTR analysis. Each of the immortalized cell lines maintained its original phenotype of slow growth. DNA repair ability of these cells was also studied by measuring sensitivity to killing by uv or the DNA-damaging drugs methyl methansulfonate, bleomycin, and cis-dichlorodiamine platinum. The results showed that both WS and progeria cells have normal sensitivity to these agents

  2. Coupling of cytoskeleton functions for fibroblast locomotion

    DEFF Research Database (Denmark)

    Couchman, J R; Lenn, M; Rees, D A

    1985-01-01

    caused visible protrusions in projected positions at the leading edge. We conclude that fibroblast locomotion may be driven coordinately by a common set of motility mechanisms and that this coordination may be lost as a result of physical or pharmacological disturbance. Taking our evidence with results...... from other Laboratories, we propose the following cytoskeleton functions. (i) Protrusive activity, probably based on solation--gelation cycles of the actin based cytoskeleton and membrane recycling which provides cellular and membrane components for streaming through the cell body to the leading edge...

  3. Differences in motility pattern between human buccal fibroblasts and periodontal and skin fibroblasts

    DEFF Research Database (Denmark)

    Lepekhin, Eugene; Grøn, Birgitte; Berezin, Vladimir

    2002-01-01

    at these sites can be explained by differences in the motile behavior of their respective fibroblast populations. The migratory characteristics were studied in a two-dimensional culture system. The migration of single cells was time-lapse video recorded at intervals of 15 min for a period of 6 h using a computer...

  4. Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

    International Nuclear Information System (INIS)

    Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B.; Altran, Silvana C.; Isaac, Cesar

    2011-01-01

    Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

  5. Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Altran, Silvana C.; Isaac, Cesar [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Medicina. Lab. de Microcirurgia Plastica; Esteves-Pedro, Natalia M. [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Ciencias Farmaceuticas. Lab. de Controle Biologico; Herson, Marisa R. [DonorTissue Bank of Victoria (Australia)

    2011-07-01

    Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

  6. Toll-like receptor 9 mediated responses in cardiac fibroblasts.

    Directory of Open Access Journals (Sweden)

    Ingrid Kristine Ohm

    Full Text Available Altered cardiac Toll-like receptor 9 (TLR9 signaling is important in several experimental cardiovascular disorders. These studies have predominantly focused on cardiac myocytes or the heart as a whole. Cardiac fibroblasts have recently been attributed increasing significance in mediating inflammatory signaling. However, putative TLR9-signaling through cardiac fibroblasts remains non-investigated. Thus, our aim was to explore TLR9-signaling in cardiac fibroblasts and investigate the consequence of such receptor activity on classical cardiac fibroblast cellular functions. Cultivated murine cardiac fibroblasts were stimulated with different TLR9 agonists (CpG A, B and C and assayed for the secretion of inflammatory cytokines (tumor necrosis factor α [TNFα], CXCL2 and interferon α/β. Expression of functional cardiac fibroblast TLR9 was proven as stimulation with CpG B and -C caused significant CXCL2 and TNFα-release. These responses were TLR9-specific as complete inhibition of receptor-stimulated responses was achieved by co-treatment with a TLR9-antagonist (ODN 2088 or chloroquine diphosphate. TLR9-stimulated responses were also found more potent in cardiac fibroblasts when compared with classical innate immune cells. Stimulation of cardiac fibroblasts TLR9 was also found to attenuate migration and proliferation, but did not influence myofibroblast differentiation in vitro. Finally, results from in vivo TLR9-stimulation with subsequent fractionation of specific cardiac cell-types (cardiac myocytes, CD45+ cells, CD31+ cells and cardiac fibroblast-enriched cell-fractions corroborated our in vitro data and provided evidence of differentiated cell-specific cardiac responses. Thus, we conclude that cardiac fibroblast may constitute a significant TLR9 responder cell within the myocardium and, further, that such receptor activity may impact important cardiac fibroblast cellular functions.

  7. 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.

  8. 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.

  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. 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.

  11. 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.

  12. Feasible pickup from intact ossicular chain with floating piezoelectric microphone.

    Science.gov (United States)

    Kang, Hou-Yong; Na, Gao; Chi, Fang-Lu; Jin, Kai; Pan, Tie-Zheng; Gao, Zhen

    2012-02-22

    Many microphones have been developed to meet with the implantable requirement of totally implantable cochlear implant (TICI). However, a biocompatible one without destroying the intactness of the ossicular chain still remains under investigation. Such an implantable floating piezoelectric microphone (FPM) has been manufactured and shows an efficient electroacoustic performance in vitro test at our lab. We examined whether it pick up sensitively from the intact ossicular chain and postulated whether it be an optimal implantable one. Animal controlled experiment: five adult cats (eight ears) were sacrificed as the model to test the electroacoustic performance of the FPM. Three groups were studied: (1) the experiment group (on malleus): the FPM glued onto the handle of the malleus of the intact ossicular chains; (2) negative control group (in vivo): the FPM only hung into the tympanic cavity; (3) positive control group (Hy-M30): a HiFi commercial microphone placed close to the site of the experiment ear. The testing speaker played pure tones orderly ranged from 0.25 to 8.0 kHz. The FPM inside the ear and the HiFi microphone simultaneously picked up acoustic vibration which recorded as .wav files to analyze. The FPM transformed acoustic vibration sensitively and flatly as did the in vitro test across the frequencies above 2.0 kHz, whereas inefficiently below 1.0 kHz for its overloading mass. Although the HiFi microphone presented more efficiently than the FPM did, there was no significant difference at 3.0 kHz and 8.0 kHz. It is feasible to develop such an implantable FPM for future TICIs and TIHAs system on condition that the improvement of Micro Electromechanical System and piezoelectric ceramic material technology would be applied to reduce its weight and minimize its size.

  13. Feasible pickup from intact ossicular chain with floating piezoelectric microphone

    Directory of Open Access Journals (Sweden)

    Kang Hou-Yong

    2012-02-01

    Full Text Available Abstract Objectives Many microphones have been developed to meet with the implantable requirement of totally implantable cochlear implant (TICI. However, a biocompatible one without destroying the intactness of the ossicular chain still remains under investigation. Such an implantable floating piezoelectric microphone (FPM has been manufactured and shows an efficient electroacoustic performance in vitro test at our lab. We examined whether it pick up sensitively from the intact ossicular chain and postulated whether it be an optimal implantable one. Methods Animal controlled experiment: five adult cats (eight ears were sacrificed as the model to test the electroacoustic performance of the FPM. Three groups were studied: (1 the experiment group (on malleus: the FPM glued onto the handle of the malleus of the intact ossicular chains; (2 negative control group (in vivo: the FPM only hung into the tympanic cavity; (3 positive control group (Hy-M30: a HiFi commercial microphone placed close to the site of the experiment ear. The testing speaker played pure tones orderly ranged from 0.25 to 8.0 kHz. The FPM inside the ear and the HiFi microphone simultaneously picked up acoustic vibration which recorded as .wav files to analyze. Results The FPM transformed acoustic vibration sensitively and flatly as did the in vitro test across the frequencies above 2.0 kHz, whereas inefficiently below 1.0 kHz for its overloading mass. Although the HiFi microphone presented more efficiently than the FPM did, there was no significant difference at 3.0 kHz and 8.0 kHz. Conclusions It is feasible to develop such an implantable FPM for future TICIs and TIHAs system on condition that the improvement of Micro Electromechanical System and piezoelectric ceramic material technology would be applied to reduce its weight and minimize its size.

  14. The uptake of radioactive iodine in rat intact Graafian follicles

    International Nuclear Information System (INIS)

    Lieberman, L.M.; Lieberman, G.L.; Lieberman, M.E.

    1984-01-01

    The concentration of iodine-131 in the ovaries of mammals has important implications in the use of I-131 for the diagnosis and treatment of thyroid disease in women. The authors studied the I-131 uptake in whole ovaries and in isolated Graafian follicles of sexually mature rats. Adult female Sprague-Dawley rats, in groups of 5-6 animals, were injected IP with 10-50 μCi of I-131, at 3, 12, and 24 hrs prior to the day of proestrus and killed on the day of proestrus. The thyroid gland and ovaries were removed intact and these organs, as well as eight other tissue specimens, were weighed. The large preovulatory follicles (6-9/ovary) were then isolated under a dissecting microscope and the remaining ovary weighed. All samples were counted in a gamma well counter and the % dose/g estimated. The thyroid gland showed 23.7% dose/organ at 24 hrs. Blood decreased from 1.6% dose/g at 3 hrs to 0.5% dose/g at 24 hrs with the uterus showing 1.1% dose/g and 0.4% dose/g at the same times. Ovarian tissue was 0.5, 0.1, and 0.1% dose/g at 3,12, and 24 hrs respectively, while the intact Graafian follicles had from one-tenth to one-third the concentration of the ovary at the same times. (0.05, 0.03, and 0.03% dose/g). The authors found that the intact Graafian follicle concentrates approximately one-thirtieth to one-sixteenth of the I-131 in the blood and one-tenth to one-third of the I-131 in the ovary. This suggests that there is no active uptake of I-131 in the follicle or follicular fluid

  15. Radionuclide sorption on crushed and intact granitic rock

    International Nuclear Information System (INIS)

    Eriksen, Tryggve E.; Locklund, Birgitta

    1989-05-01

    The specific surface areas and distribution ratios for sorption of 85 Sr, 137 Cs and 152 Eu were measured for crushed and intact granite rock. The experimental data can be accommodated by a sorption model encompassing sorption on outer and inner surface. It is clearly demonstrated that the time required to obtain reliable Kd-values for the sorption of strongly sorbing radionuclides like 152 Eu is very long due to solution depletion and slow diffusion into the rock. A combination of surface area measurements and batch sorption with small particles may therefore be preferable when studying strongly sorbing nuclides. (authors) (17 figs., 6 tabs.)

  16. Radioiodination of an outer membrane protein in intact Rickettsia prowazekii

    International Nuclear Information System (INIS)

    Smith, D.K.; Winkler, H.H.

    1980-01-01

    Intact Rickettsia prowazekii was radiolabeled with the glucose oxidase-lactoperoxidase method of iodination. Separation of the rickettsial extract into cytoplasmic, outer and inner membrane fractions demonstrated that the outer membrane was preferentially labeled. Analysis of the polypeptides of these fractions on high-resolution slab polyacrylamide gels showed that most of the 125 I was in polypeptide T49, an outer membrane constituent. Additional outer membrane polypeptides were iodinated in broken envelope preparations, demonstrating that T49 is uniquely accessible to the external environment and the asymmetric polypeptide organization of the outer membrane

  17. 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

  18. 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.

  19. Myogenic conversion of bladder fibroblasts by construction and ...

    African Journals Online (AJOL)

    The cultured primary bladder fibroblasts were transfected by pEGFP-Myod1 with Lipofection 2000 reagent. The results showed that expression of Myod1 could cause myogenic differentiation of bladder fibroblasts. These findings support the possibility of an alternative approach to exploit the capacity of Myod1 to activate ...

  20. Rac inhibition reverses the phenotype of fibrotic fibroblasts.

    Directory of Open Access Journals (Sweden)

    Shi-wen Xu

    Full Text Available BACKGROUND: Fibrosis, the excessive deposition of scar tissue by fibroblasts, is one of the largest groups of diseases for which there is no therapy. Fibroblasts from lesional areas of scleroderma patients possess elevated abilities to contract matrix and produce alpha-smooth muscle actin (alpha-SMA, type I collagen and CCN2 (connective tissue growth factor, CTGF. The basis for this phenomenon is poorly understood, and is a necessary prerequisite for developing novel, rational anti-fibrotic strategies. METHODS AND FINDINGS: Compared to healthy skin fibroblasts, dermal fibroblasts cultured from lesional areas of scleroderma (SSc patients possess elevated Rac activity. NSC23766, a Rac inhibitor, suppressed the persistent fibrotic phenotype of lesional SSc fibroblasts. NSC23766 caused a decrease in migration on and contraction of matrix, and alpha-SMA, type I collagen and CCN2 mRNA and protein expression. SSc fibroblasts possessed elevated Akt phosphorylation, which was also blocked by NSC23766. Overexpression of rac1 in normal fibroblasts induced matrix contraction and alpha-SMA, type I collagen and CCN2 mRNA and protein expression. Rac1 activity was blocked by PI3kinase/Akt inhibition. Basal fibroblast activity was not affected by NSC23766. CONCLUSION: Rac inhibition may be considered as a novel treatment for the fibrosis observed in SSc.

  1. Mesenchymal stem cells induce dermal fibroblast responses to injury

    International Nuclear Information System (INIS)

    Smith, Andria N.; Willis, Elise; Chan, Vincent T.; Muffley, Lara A.; Isik, F. Frank; Gibran, Nicole S.; Hocking, Anne M.

    2010-01-01

    Although bone marrow-derived mesenchymal stem cells have been shown to promote repair when applied to cutaneous wounds, the mechanism for this response remains to be determined. The aim of this study was to determine the effects of paracrine signaling from mesenchymal stem cells on dermal fibroblast responses to injury including proliferation, migration and expression of genes important in wound repair. Dermal fibroblasts were co-cultured with bone marrow-derived mesenchymal stem cells grown in inserts, which allowed for paracrine interactions without direct cell contact. In this co-culture model, bone marrow-derived mesenchymal stem cells regulate dermal fibroblast proliferation, migration and gene expression. When co-cultured with mesenchymal stem cells, dermal fibroblasts show increased proliferation and accelerated migration in a scratch assay. A chemotaxis assay also demonstrated that dermal fibroblasts migrate towards bone marrow-derived mesenchymal stem cells. A PCR array was used to analyze the effect of mesenchymal stem cells on dermal fibroblast gene expression. In response to mesenchymal stem cells, dermal fibroblasts up-regulate integrin alpha 7 expression and down-regulate expression of ICAM1, VCAM1 and MMP11. These observations suggest that mesenchymal stem cells may provide an important early signal for dermal fibroblast responses to cutaneous injury.

  2. 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...

  3. Collagen expression in fibroblasts with a novel LMNA mutation

    International Nuclear Information System (INIS)

    Nguyen, Desiree; Leistritz, Dru F.; Turner, Lesley; MacGregor, David; Ohson, Kamal; Dancey, Paul; Martin, George M.; Oshima, Junko

    2007-01-01

    Laminopathies are a group of genetic disorders caused by LMNA mutations; they include muscular dystrophies, lipodystrophies, and progeroid syndromes. We identified a novel heterozygous LMNA mutation, L59R, in a patient with the general appearance of mandibuloacral dysplasia and progeroid features. Examination of the nuclei of dermal fibroblasts revealed the irregular morphology characteristic of LMNA mutant cells. The nuclear morphological abnormalities of LMNA mutant lymphoblastoid cell lines were less prominent compared to those of primary fibroblasts. Since it has been reported that progeroid features are associated with increased extracellular matrix in dermal tissues, we compared a subset of these components in fibroblast cultures from LMNA mutants with those of control fibroblasts. There was no evidence of intracellular accumulation or altered mobility of collagen chains, or altered conversion of procollagen to collagen, suggesting that skin fibroblast-mediated matrix production may not play a significant role in the pathogenesis of this particular laminopathy

  4. Novel therapeutic strategies targeting fibroblasts and fibrosis in heart disease

    Science.gov (United States)

    Gourdie, Robert G.; Dimmeler, Stefanie; Kohl, Peter

    2016-01-01

    Our understanding of cardiac fibroblast functions has moved beyond their roles in heart structure and extracellular matrix generation, and now includes contributions to paracrine, mechanical and electrical signalling during ontogenesis and normal cardiac activity. Fibroblasts have central roles in pathogenic remodelling during myocardial ischaemia, hypertension and heart failure. As key contributors to scar formation, they are crucial for tissue repair after interventions including surgery and ablation. Novel experimental approaches targeting cardiac fibroblasts are promising potential therapies for heart disease. Indeed, several existing drugs act, at least partially, through effects on cardiac connective tissue. This Review outlines the origins and roles of fibroblasts in cardiac development, homeostasis and disease; illustrates the involvement of fibroblasts in current and emerging clinical interventions; and identifies future targets for research and development. PMID:27339799

  5. Metabolism of inhaled ethane and pentane by the intact rat

    International Nuclear Information System (INIS)

    Daugherty, M.S.; Luddent, T.M.; Burk, R.F.

    1986-01-01

    Measurement of exhaled ethane or pentane is a noninvasive technique for studying in vivo lipid peroxidation. Many past studies have assumed that pentane and ethane are not metabolized. Radiolabeled ( 14 C) ethane and pentane were used to study the disposition of these compounds in intact rats. Rats were placed for 8 h in a closed plexiglass chamber fitted with a system for replenishing chamber atmospheric O 2 . Evolved CO 2 was trapped by recirculating chamber air through 3 N NaOH contained in a vessel external to the chamber. Radiolabeled ethane or pentane was injected into the chamber at the start of each experiment. The percent of 14 C-activity added to the chamber recovered in the CO 2 trap, urine, and chamber air at the end of the experiment (8 h) in the [ 14 C]-ethane (n=5) and [ 14 C]-pentane (n=4) studies are presented. Results indicate that both ethane and pentane are metabolized to CO 2 in the intact rat. Possible changes in ethane and pentane metabolism must be considered if the exhalation rates of these hydrocarbons are to be used as indices of in vivo lipid peroxidation

  6. Surface plasmon resonance sensing: from purified biomolecules to intact cells.

    Science.gov (United States)

    Su, Yu-Wen; Wang, Wei

    2018-04-12

    Surface plasmon resonance (SPR) has become a well-recognized label-free technique for measuring the binding kinetics between biomolecules since the invention of the first SPR-based immunosensor in 1980s. The most popular and traditional format for SPR analysis is to monitor the real-time optical signals when a solution containing ligand molecules is flowing over a sensor substrate functionalized with purified receptor molecules. In recent years, rapid development of several kinds of SPR imaging techniques have allowed for mapping the dynamic distribution of local mass density within single living cells with high spatial and temporal resolutions and reliable sensitivity. Such capability immediately enabled one to investigate the interaction between important biomolecules and intact cells in a label-free, quantitative, and single cell manner, leading to an exciting new trend of cell-based SPR bioanalysis. In this Trend Article, we first describe the principle and technical features of two types of SPR imaging techniques based on prism and objective, respectively. Then we survey the intact cell-based applications in both fundamental cell biology and drug discovery. We conclude the article with comments and perspectives on the future developments. Graphical abstract Recent developments in surface plasmon resonance (SPR) imaging techniques allow for label-free mapping the mass-distribution within single living cells, leading to great expansions in biomolecular interactions studies from homogeneous substrates functionalized with purified biomolecules to heterogeneous substrates containing individual living cells.

  7. Direct Conversion of Human Fibroblasts into Schwann Cells that Facilitate Regeneration of Injured Peripheral Nerve In Vivo.

    Science.gov (United States)

    Sowa, Yoshihiro; Kishida, Tsunao; Tomita, Koichi; Yamamoto, Kenta; Numajiri, Toshiaki; Mazda, Osam

    2017-04-01

    Schwann cells (SCs) play pivotal roles in the maintenance and regeneration of the peripheral nervous system. Although transplantation of SCs enhances repair of experimentally damaged peripheral and central nerve tissues, it is difficult to prepare a sufficient number of functional SCs for transplantation therapy without causing adverse events for the donor. Here, we generated functional SCs by somatic cell reprogramming procedures and demonstrated their capability to promote peripheral nerve regeneration. Normal human fibroblasts were phenotypically converted into SCs by transducing SOX10 and Krox20 genes followed by culturing for 10 days resulting in approximately 43% directly converted Schwann cells (dSCs). The dSCs expressed SC-specific proteins, secreted neurotrophic factors, and induced neuronal cells to extend neurites. The dSCs also displayed myelin-forming capability both in vitro and in vivo. Moreover, transplantation of the dSCs into the transected sciatic nerve in mice resulted in significantly accelerated regeneration of the nerve and in improved motor function at a level comparable to that with transplantation of the SCs obtained from a peripheral nerve. The dSCs induced by our procedure may be applicable for novel regeneration therapy for not only peripheral nerve injury but also for central nerve damage and for neurodegenerative disorders related to SC dysfunction. Stem Cells Translational Medicine 2017;6:1207-1216. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  8. Doubling potential of fibroblasts from different species after ionising radiation

    International Nuclear Information System (INIS)

    Macieira-Coelho, A.; Diatloff, C.; Malaise, E.

    1976-01-01

    It is stated that whereas chicken fibroblasts invariably die after a certain number of doublings in vitro, and this fact is never altered by chemical or physical agents, mouse fibroblasts invariably acquire spontaneously an infinite growth potential. In the human species fibroblasts never acquire spontaneously the capacity to divide for ever, although they can become permanent cell lines after treatment with certain viruses. This behaviour of fibroblasts in vitro has been attributed to different nutritional requirements. Experiments are described with human and mouse fibroblasts in which it was found that the response to ionising radiation matches the relative tendencies of the fibroblasts to yield permanent cell lines. Irradiation was commenced during the phase of active proliferation. Human fibroblast cultures irradiated with 100 R stopped dividing earlier than the controls, whereas cultures irradiated with 200, 300 and 500 R had the same lifespan as the control cultures. Cultures irradiated with 400 R showed the longest survival. With mouse fibroblasts the growth curves of the irradiated cells were of the same type as in the controls, but recovery occurred earlier. The results indicated that ionising radiation accelerates a natural phenomenon; in cells with a limited growth potential (chicken) it shortens the lifespan, whereas in cells that can acquire an unlimited growth potential (mouse) it accelerates acquisition of this potential; human fibroblasts showed an intermediate response, since ionising radiation neither established the cultures as with mouse cells nor reduced the number of cells produced as with chicken fibroblasts. Possible explanations for the different behaviour of the species are offered. (U.K.)

  9. 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

  10. 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

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

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    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.

  12. 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.

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

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    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.

  14. 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

  15. 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

  16. 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.

  17. 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.

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

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    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.

  19. 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

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

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    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.

  1. 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

  2. LXA4 actions direct fibroblast function and wound closure

    International Nuclear Information System (INIS)

    Herrera, Bruno S.; Kantarci, Alpdogan; Zarrough, Ahmed; Hasturk, Hatice; Leung, Kai P.; Van Dyke, Thomas E.

    2015-01-01

    Timely resolution of inflammation is crucial for normal wound healing. Resolution of inflammation is an active biological process regulated by specialized lipid mediators including the lipoxins and resolvins. Failure of resolution activity has a major negative impact on wound healing in chronic inflammatory diseases that is manifest as excess fibrosis and scarring. Lipoxins, including Lipoxin A 4 (LXA 4 ), have known anti-fibrotic and anti-scarring properties. The goal of this study was to elucidate the impact of LXA 4 on fibroblast function. Mouse fibroblasts (3T3 Mus musculus Swiss) were cultured for 72 h in the presence of TGF-β1, to induce fibroblast activation. The impact of exogenous TGF-β1 (1 ng/mL) on LXA 4 receptor expression (ALX/FPR2) was determined by flow cytometry. Fibroblast proliferation was measured by bromodeoxyuridine (BrdU) labeling and migration in a “scratch” assay wound model. Expression of α-smooth muscle actin (α-SMA), and collagen types I and III were measured by Western blot. We observed that TGF-β1 up-regulates LXA 4 receptor expression, enhances fibroblast proliferation, migration and scratch wound closure. α-SMA levels and Collagen type I and III deposition were also enhanced. LXA 4 slowed fibroblast migration and scratch wound closure at early time points (24 h), but wound closure was equal to TGF-β1 alone at 48 and 72 h. LXA 4 tended to slow fibroblast proliferation at both concentrations, but had no impact on α-SMA or collagen production by TGF-β1 stimulated fibroblasts. The generalizability of the actions of resolution molecules was examined in experiments repeated with resolvin D2 (RvD2) as the agonist. The activity of RvD2 mimicked the actions of LXA 4 in all assays, through an as yet unidentified receptor. The results suggest that mediators of resolution of inflammation enhance wound healing and limit fibrosis in part by modulating fibroblast function. - Highlights: • TGF-β1 up-regulates LXA 4 receptor (ALX

  3. Radiation-induced adaptive response in the intact mouse

    International Nuclear Information System (INIS)

    Yonezawa, Morio

    2009-01-01

    The author and coworkers have revealed that radiation adaptive response (AR) is seen also in the bone marrow of the intact mouse, of which details are described here. First, SPF ICR mice were pre-irradiated (PI) with 0-0.1 Gy of X-ray and after 2 months, subsequently irradiated (SI) with 7.75 Gy. Survival rates at 30 days after SI were about 14% in mice with PI 0-0.025 Gy whereas 40% or more in animals with PI 0.05-0.1 Gy: bone marrow death was found significantly suppressed in this effective PI dose range. The death 2 weeks after SI was found also inhibited at PI 0.3-0.5 Gy. Second, PI doses and interval between PI and SI for acquiring the radio-resistance (RR) were studied and third, the PI 0.3-0.5 Gy with SI 8.0 Gy at 9-17 days later revealed that regional PI of the head (central nervous system) was found unnecessary for RR and of abdomen (systems of hemopoiesis, immunity and digestion), essential. Fourth, strain difference of RR was shown by the fact that RR was observed only in C57BL mouse as well, but neither in BALB/c nor C3H strain. Next, at 12 days after SI 4.25-6.75 Gy (PI 0.5 Gy at 14 days before), mouse spleen cells were subjected to colony formation analysis by counting the endogenous hemopoietic stem cells, which revealed that those cells were increased to about 5 times by PI. Suppression of SI-induced hemorrhage was found in mice with PI by the decreased fecal hemoglobin content. Finally, AR was similarly studied in p53 +/+ and its knockout C57BL mice and was not found in the latter animal, indicating the participation of p53 in AR of the intact mouse. Elucidation of AR mechanisms in the intact animal seems to require somewhat different aspect from that in cells. The results were controvertible to the general concept that radiation risk is proportional to cumulative dose, suggesting that low dose radiation differs from high dose one in biological effect. (K.T.)

  4. Ultrastructure and Light Microscope Analysis of Intact Skin after a Varying Number of Low Level Laser Irradiations in Mice

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    Mamie Mizusaki Iyomasa

    2014-01-01

    Full Text Available Low level laser therapy (LLLT has been used to relieve pain, inflammation, and wound healing processes. Thus, the skin is overexposed to laser and this effect is not completely understood. This study analyzed the effects of the number of laser applications (three, six, and 10 on the intact skin of the masseteric region in mice of strain HRS/J. The animals (n=30 were equally divided into control (0 J/cm2 and irradiated (20 J/cm2, and each of these groups was further equally divided according to the number of laser applications (three, six, and 10 and underwent LLLT on alternate days. Samples were analyzed by light microscopy and transmission electron microscope (TEM. The animals receiving applications exhibited open channels more dilated between the keratinocytes and photobiomodulation effect on endothelial cells and fibroblasts by TEM. Under the light microscope after 10 laser applications, the type I collagen decreased (P<0.05 compared to the three and six applications. Under these experimental conditions, all numbers of applications provided photobiomodulatory effect on the epidermis and dermis, without damage. More studies are needed to standardize the energy density and number of applications recommended for laser therapy to have a better cost-benefit ratio associated with treatment.

  5. Transport of indoleacetic acid in intact corn coleoptiles

    International Nuclear Information System (INIS)

    Parker, K.E.; Briggs, W.R.

    1990-01-01

    We have characterized the transport of [ 3 H]indoleacetic acid (IAA) in intact corn (Zea mays L.) coleoptiles. We have used a wide range of concentrations of added IAA (28 femtomoles to 100 picomoles taken up over 60 minutes). The shape of the transport curve varies with the concentration of added IAA, although the rate of movement of the observed front of tracer is invariant with concentration. At the lowest concentration of tracer used, the labeled IAA in the transport stream is not detectably metabolized or immobilized, curvature does not develop as a result of tracer application, and normal phototropic and gravitropic responsiveness are not affected. Therefore we believe we are observing the transport of true tracer quantities of labeled auxin at this lowest concentration

  6. Simple Genome Editing of Rodent Intact Embryos by Electroporation.

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    Takehito Kaneko

    Full Text Available The clustered regularly interspaced short palindromic repeat (CRISPR/CRISPR-associated (Cas system is a powerful tool for genome editing in animals. Recently, new technology has been developed to genetically modify animals without using highly skilled techniques, such as pronuclear microinjection of endonucleases. Technique for animal knockout system by electroporation (TAKE method is a simple and effective technology that produces knockout rats by introducing endonuclease mRNAs into intact embryos using electroporation. Using TAKE method and CRISPR/Cas system, the present study successfully produced knockout and knock-in mice and rats. The mice and rats derived from embryos electroporated with Cas9 mRNA, gRNA and single-stranded oligodeoxynucleotide (ssODN comprised the edited targeted gene as a knockout (67% of mice and 88% of rats or knock-in (both 33%. The TAKE method could be widely used as a powerful tool to produce genetically modified animals by genome editing.

  7. Selective dansylation of M protein within intact influenza virions

    Energy Technology Data Exchange (ETDEWEB)

    Robertson, B.H.; Bennett, J.C.; Compans, R.W.

    1982-12-01

    Exposure of purified influenza virions to (/sup 14/C)dansyl chloride resulted in the covalent attachment of the dansyl chromophore to the virion. Gel electrophoresis revealed that the dansyl chromophore was specifically coupled to the internal membrane (M) protein. Purification of the M protein by gel filtration followed by cyanogen bromide cleavage and peptide fractionation revealed that four of six peptide peaks contained dansyl label. Acid hydrolysis of the separated peptide peaks followed by thin-layer chromatography revealed that dansyl label was coupled to lysine residues present in these peptides. The results of these investigations have demonstrated that the M protein molecule is the major viral polypeptide labeled when intact virions are exposed to dansyl chloride.

  8. Selective dansylation of M protein within intact influenza virions

    International Nuclear Information System (INIS)

    Robertson, B.H.; Bennett, J.C.; Compans, R.W.

    1982-01-01

    Exposure of purified influenza virions to [ 14 C]dansyl chloride resulted in the covalent attachment of the dansyl chromophore to the virion. Gel electrophoresis revealed that the dansyl chromophore was specifically coupled to the internal membrane (M) protein. Purification of the M protein by gel filtration followed by cyanogen bromide cleavage and peptide fractionation revealed that four of six peptide peaks contained dansyl label. Acid hydrolysis of the separated peptide peaks followed by thin-layer chromatography revealed that dansyl label was coupled to lysine residues present in these peptides. The results of these investigations have demonstrated that the M protein molecule is the major viral polypeptide labeled when intact virions are exposed to dansyl chloride

  9. LIF Mediates Proinvasive Activation of Stromal Fibroblasts in Cancer

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    Jean Albrengues

    2014-06-01

    Full Text Available Signaling crosstalk between tumor cells and fibroblasts confers proinvasive properties to the tumor microenvironment. Here, we identify leukemia inhibitory factor (LIF as a tumor promoter that mediates proinvasive activation of stromal fibroblasts independent of alpha-smooth muscle actin (α-SMA expression. We demonstrate that a pulse of transforming growth factor β (TGF-β establishes stable proinvasive fibroblast activation by inducing LIF production in both fibroblasts and tumor cells. In fibroblasts, LIF mediates TGF-β-dependent actomyosin contractility and extracellular matrix remodeling, which results in collective carcinoma cell invasion in vitro and in vivo. Accordingly, carcinomas from multiple origins and melanomas display strong LIF upregulation, which correlates with dense collagen fiber organization, cancer cell collective invasion, and poor clinical outcome. Blockade of JAK activity by Ruxolitinib (JAK inhibitor counteracts fibroblast-dependent carcinoma cell invasion in vitro and in vivo. These findings establish LIF as a proinvasive fibroblast producer independent of α-SMA and may open novel therapeutic perspectives for patients with aggressive primary tumors.

  10. Isolation of intact sub-dermal secretory cavities from Eucalyptus

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    Goodger Jason QD

    2010-09-01

    Full Text Available Abstract Background The biosynthesis of plant natural products in sub-dermal secretory cavities is poorly understood at the molecular level, largely due to the difficulty of physically isolating these structures for study. Our aim was to develop a protocol for isolating live and intact sub-dermal secretory cavities, and to do this, we used leaves from three species of Eucalyptus with cavities that are relatively large and rich in essential oils. Results Leaves were digested using a variety of commercially available enzymes. A pectinase from Aspergillus niger was found to allow isolation of intact cavities after a relatively short incubation (12 h, with no visible artifacts from digestion and no loss of cellular integrity or cavity contents. Several measurements indicated the potential of the isolated cavities for further functional studies. First, the cavities were found to consume oxygen at a rate that is comparable to that estimated from leaf respiratory rates. Second, mRNA was extracted from cavities, and it was used to amplify a cDNA fragment with high similarity to that of a monoterpene synthase. Third, the contents of the cavity lumen were extracted, showing an unexpectedly low abundance of volatile essential oils and a sizeable amount of non-volatile material, which is contrary to the widely accepted role of secretory cavities as predominantly essential oil repositories. Conclusions The protocol described herein is likely to be adaptable to a range of Eucalyptus species with sub-dermal secretory cavities, and should find wide application in studies of the developmental and functional biology of these structures, and the biosynthesis of the plant natural products they contain.

  11. Intact calcium signaling in adrenergic-deficient embryonic mouse hearts.

    Science.gov (United States)

    Peoples, Jessica N; Taylor, David G; Katchman, Alexander N; Ebert, Steven N

    2018-01-22

    Mouse embryos that lack the ability to produce the adrenergic hormones, norepinephrine (NE) and epinephrine (EPI), due to disruption of the dopamine beta-hydroxylase (Dbh -/- ) gene inevitably perish from heart failure during mid-gestation. Since adrenergic stimulation is well-known to enhance calcium signaling in developing as well as adult myocardium, and impairments in calcium signaling are typically associated with heart failure, we hypothesized that adrenergic-deficient embryonic hearts would display deficiencies in cardiac calcium signaling relative to adrenergic-competent controls at a developmental stage immediately preceding the onset of heart failure, which first appears beginning or shortly after mouse embryonic day 10.5 (E10.5). To test this hypothesis, we used ratiometric fluorescent calcium imaging techniques to measure cytosolic calcium transients, [Ca 2+ ] i in isolated E10.5 mouse hearts. Our results show that spontaneous [Ca 2+ ] i oscillations were intact and robustly responded to a variety of stimuli including extracellular calcium (5 mM), caffeine (5 mM), and NE (100 nM) in a manner that was indistinguishable from controls. Further, we show similar patterns of distribution (via immunofluorescent histochemical staining) and activity (via patch-clamp recording techniques) for the major voltage-gated plasma membrane calcium channel responsible for the L-type calcium current, I Ca,L , in adrenergic-deficient and control embryonic cardiac cells. These results demonstrate that despite the absence of vital adrenergic hormones that consistently leads to embryonic lethality in vivo, intracellular and extracellular calcium signaling remain essentially intact and functional in embryonic mouse hearts through E10.5. These findings suggest that adrenergic stimulation is not required for the development of intracellular calcium oscillations or extracellular calcium signaling through I Ca,L and that aberrant calcium signaling does not likely contribute

  12. Optimized Planning Target Volume for Intact Cervical Cancer

    International Nuclear Information System (INIS)

    Khan, Alvin; Jensen, Lindsay G.; Sun Shuai; Song, William Y.; Yashar, Catheryn M.; Mundt, Arno J.; Zhang Fuquan; Jiang, Steve B.; Mell, Loren K.

    2012-01-01

    Purpose: To model interfraction clinical target volume (CTV) variation in patients with intact cervical cancer and design a planning target volume (PTV) that minimizes normal tissue dose while maximizing CTV coverage. Methods and Materials: We analyzed 50 patients undergoing external-beam radiotherapy for intact cervical cancer using daily online cone-beam computed tomography (CBCT). The CBCTs (n = 972) for each patient were rigidly registered to the planning CT. The CTV was delineated on the planning CT (CTV 0 ) and the set of CBCTs ({CTV 1 –CTV 25 }). Manual (n = 98) and automated (n = 668) landmarks were placed over the surface of CTV 0 with reference to defined anatomic structures. Normal vectors were extended from each landmark, and the minimum length required for a given probability of encompassing CTV 1 –CTV 25 was computed. The resulting expansions were used to generate an optimized PTV. Results: The mean (SD; range) normal vector length to ensure 95% coverage was 4.3 mm (2.7 mm; 1–16 mm). The uniform expansion required to ensure 95% probability of CTV coverage was 13 mm. An anisotropic margin of 20 mm anteriorly and posteriorly and 10 mm superiorly, inferiorly, and laterally also would have ensured a 95% probability of CTV coverage. The volume of the 95% optimized PTV (1470 cm 3 ) was significantly lower than both the anisotropic PTV (2220 cm 3 ) and the uniformly expanded PTV (2110 cm 3 ) (p 0 , 5–10 mm along the interfaces of CTV 0 with the bladder and rectum, and 10–14 mm along the anterior surface of CTV 0 at the level of the uterus. Conclusion: Optimizing PTV definition according to surface landmarking resulted in a high probability of CTV coverage with reduced PTV volumes. Our results provide data justifying planning margins to use in practice and clinical trials.

  13. Bacterial lipopolysaccharide promotes profibrotic activation of intestinal fibroblasts.

    LENUS (Irish Health Repository)

    Burke, J P

    2012-02-01

    BACKGROUND: Fibroblasts play a critical role in intestinal wound healing. Lipopolysaccharide (LPS) is a cell wall component of commensal gut bacteria. The effects of LPS on intestinal fibroblast activation were characterized. METHODS: Expression of the LPS receptor, toll-like receptor (TLR) 4, was assessed in cultured primary human intestinal fibroblasts using flow cytometry and confocal microscopy. Fibroblasts were treated with LPS and\\/or transforming growth factor (TGF) beta1. Nuclear factor kappaB (NFkappaB) pathway activation was assessed by inhibitory kappaBalpha (IkappaBalpha) degradation and NFkappaB promoter activity. Fibroblast contractility was measured using a fibroblast-populated collagen lattice. Smad-7, a negative regulator of TGF-beta1 signalling, and connective tissue growth factor (CTGF) expression were assessed using reverse transcriptase-polymerase chain reaction and western blot. The NFkappaB pathway was inhibited by IkappaBalpha transfection. RESULTS: TLR-4 was present on the surface of intestinal fibroblasts. LPS treatment of fibroblasts induced IkappaBalpha degradation, enhanced NFkappaB promoter activity and increased collagen contraction. Pretreatment with LPS (before TGF-beta1) significantly increased CTGF production relative to treatment with TGF-beta1 alone. LPS reduced whereas TGF-beta1 increased smad-7 expression. Transfection with an IkappaBalpha plasmid enhanced basal smad-7 expression. CONCLUSION: Intestinal fibroblasts express TLR-4 and respond to LPS by activating NFkappaB and inducing collagen contraction. LPS acts in concert with TGF-beta1 to induce CTGF. LPS reduces the expression of the TGF-beta1 inhibitor, smad-7.

  14. Effects of polyhexamethylene guanidine phosphate on human gingival fibroblasts.

    Science.gov (United States)

    Vitt, Anton; Slizen, Veronica; Boström, Elisabeth A; Yucel-Lindberg, Tülay; Kats, Anna; Sugars, Rachael V; Gustafsson, Anders; Buhlin, Kåre

    2017-10-01

    Polyhexamethylene guanidine phosphate (PHMG-P) was compared to chlorhexidine (CHX) in order to determine potential cytotoxic and immune-modulatory effects on human gingival fibroblasts. Cytotoxic effects of PHMG-P and CHX on human gingival fibroblasts were assessed using cell viability assay at various time points and concentrations. The effects of PHMG-P and CHX on the secretion of prostaglandin (PG) E 2 , interleukin (IL)-6, IL-8 and matrix metalloproteinase (MMP)-1 by non-stimulated or IL-1β stimulated fibroblasts were evaluated by enzyme-linked immunosorbent assays. PHMG-P concentration 0.00009% led to the total loss of fibroblast viability within 24 h, whereas inhibition of fibroblast viability by CHX occurred at significantly higher concentrations of 0.0009% (p PHMG-P led to loss of fibroblast viability after 5 min, whilst cells exposed to 0.005% CHX survived 30 min of treatment (p PHMG-P or CHX at concentrations of 0.000045 or 0.0.00009% resulted in significantly decreased PGE 2 , IL-6, IL-8 and MMP-1 levels. PHMG-P or CHX alone did not affect the baseline secretion of PGE 2 , IL-6, IL-8 or MMP-1 by gingival fibroblasts. Cytotoxic effects on gingival fibroblasts were triggered by both PHMG-P and CHX at concentrations below those used in clinical practice. The tested antiseptics did not cause inflammation and reduced IL-1β-induced secretion of inflammatory mediators and collagenase by gingival fibroblasts, which suggests anti-inflammatory properties.

  15. Ets2 in tumor fibroblasts promotes angiogenesis in breast cancer.

    Directory of Open Access Journals (Sweden)

    Julie A Wallace

    Full Text Available Tumor fibroblasts are active partners in tumor progression, but the genes and pathways that mediate this collaboration are ill-defined. Previous work demonstrates that Ets2 function in stromal cells significantly contributes to breast tumor progression. Conditional mouse models were used to study the function of Ets2 in both mammary stromal fibroblasts and epithelial cells. Conditional inactivation of Ets2 in stromal fibroblasts in PyMT and ErbB2 driven tumors significantly reduced tumor growth, however deletion of Ets2 in epithelial cells in the PyMT model had no significant effect. Analysis of gene expression in fibroblasts revealed a tumor- and Ets2-dependent gene signature that was enriched in genes important for ECM remodeling, cell migration, and angiogenesis in both PyMT and ErbB2 driven-tumors. Consistent with these results, PyMT and ErbB2 tumors lacking Ets2 in fibroblasts had fewer functional blood vessels, and Ets2 in fibroblasts elicited changes in gene expression in tumor endothelial cells consistent with this phenotype. An in vivo angiogenesis assay revealed the ability of Ets2 in fibroblasts to promote blood vessel formation in the absence of tumor cells. Importantly, the Ets2-dependent gene expression signatures from both mouse models were able to distinguish human breast tumor stroma from normal stroma, and correlated with patient outcomes in two whole tumor breast cancer data sets. The data reveals a key function for Ets2 in tumor fibroblasts in signaling to endothelial cells to promote tumor angiogenesis. The results highlight the collaborative networks that orchestrate communication between stromal cells and tumor cells, and suggest that targeting tumor fibroblasts may be an effective strategy for developing novel anti-angiogenic therapies.

  16. Matrix metalloproteinase inhibition reduces contraction by dupuytren fibroblasts.

    Science.gov (United States)

    Townley, William A; Cambrey, Alison D; Khaw, Peng T; Grobbelaar, Adriaan O

    2008-11-01

    Dupuytren's disease is a common fibroproliferative condition of the hand characterized by fibrotic lesions (nodules and cords), leading to disability through progressive digital contracture. Although the etiology of the disease is poorly understood, recent evidence suggests that abnormal matrix metalloproteinase (MMP) activity may play a role in cell-mediated collagen contraction and tissue scarring. The aim of this study was to investigate the efficacy of ilomastat, a broad-spectrum MMP inhibitor, in an in vitro model of Dupuytren fibroblast-mediated contraction. Nodule-derived and cord-derived fibroblasts were isolated from Dupuytren patients; carpal ligament-derived fibroblasts acted as control. Stress-release fibroblast-populated collagen lattices (FPCLs) were used as a model of contraction. FPCLs were allowed to develop mechanical stress (48 hours) during treatment with ilomastat (0-100 micromol/L), released, and allowed to contract over a 48-hour period. Contraction was estimated by measuring lattice area compared with untreated cells or treatment with a control peptide. MMP-1, MMP-2, and MT1-MMP levels were assessed by zymography, Western blotting, and enzyme-linked immunosorbent assay. Nodule-derived fibroblasts contracted lattices (69% +/- 2) to a greater extent than did cord-derived (55% +/- 3) or carpal ligament-derived (55% +/- 1) fibroblasts. Exposure to ilomastat led to significant inhibition of lattice contraction by all fibroblasts, although a reduction in lattice contraction by nodule-derived fibroblasts was most prominent (84% +/- 8). In addition, treatment with ilomastat led to a concomitant suppression of MMP-1 and MMP-2 activity, whereas MT1-MMP activity was found to be upregulated. Our results demonstrate that inhibition of MMP activity results in a reduction in extracellular matrix contraction by Dupuytren fibroblasts and suggest that MMP activity may be a critical target in preventing recurrent contracture caused by this disease.

  17. Extracellular matrix organization modulates fibroblast growth and growth factor responsiveness.

    Science.gov (United States)

    Nakagawa, S; Pawelek, P; Grinnell, F

    1989-06-01

    To learn more about the relationship between extracellular matrix organization, cell shape, and cell growth control, we studied DNA synthesis by fibroblasts in collagen gels that were either attached to culture dishes or floating in culture medium during gel contraction. After 4 days of contraction, the collagen density (initially 1.5 mg/ml) reached 22 mg/ml in attached gels and 55 mg/ml in floating gels. After contraction, attached collagen gels were well organized; collagen fibrils were aligned in the plane of cell spreading; and fibroblasts had an elongated, bipolar morphology. Floating collagen gels, however, were unorganized; collagen fibrils were arranged randomly; and fibroblasts had a stellate morphology. DNA synthesis by fibroblasts in contracted collagen gels was suppressed if the gels were floating in medium but not if the gels were attached, and inhibition was independent of the extent of gel contraction. Therefore, growth of fibroblasts in contracted collagen gels could be regulated by differences in extracellular matrix organization and cell shape independently of extracellular matrix density. We also compared the responses of fibroblasts in contracted collagen gels and monolayer culture to peptide growth factors including fibroblast growth factor, platelet-derived growth factor, transforming growth factor-beta, and interleukin 1. Cells in floating collagen gels were generally unresponsive to any of the growth factors. Cells in attached collagen gels and monolayer culture were affected similarly by fibroblast growth factor but not by the others. Our results indicate that extracellular matrix organization influenced not only cell growth, but also fibroblast responsiveness to peptide growth factors.

  18. 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.

  19. 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

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

    Directory of Open Access Journals (Sweden)

    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. 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.

  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. Alzheimer skin fibroblasts show increased susceptibility to free radicals.

    Science.gov (United States)

    Tesco, G; Latorraca, S; Piersanti, P; Piacentini, S; Amaducci, L; Sorbi, S

    1992-11-01

    We have studied the response to toxic oxygen metabolites of fibroblasts derived from skin biopsies of 5 patients with familial (FAD) and 4 with sporadic (AD) Alzheimer's disease compared with those derived from 4 normal controls. Fibroblasts were damaged by the generation of oxygen metabolites during the enzymatic oxidation of acetaldehyde by 50 munits of xanthine-oxidase (Xo). To quantify cell damage we measured lactate dehydrogenase (LDH) activity in the culture medium and cell viability in fibroblast cultures. We found a significant increase in LDH activity in the FAD vs. controls and also in the AD vs. controls.

  4. Uptake of intact TPGS (d-alpha-tocopheryl polyethylene glycol 1000 succinate) a water-miscible form of vitamin E by human cells in vitro

    International Nuclear Information System (INIS)

    Traber, M.G.; Thellman, C.A.; Rindler, M.J.; Kayden, H.J.

    1988-01-01

    The mechanism by which TPGS (alpha-tocopheryl succinate esterified to polyethylene glycol 1000 [PEG 1000]) delivers tocopherol (vitamin E) was studied in human fibroblasts and erythrocytes and a human intestinal cell line, Caco-2. The total cellular tocopherol content of saponified samples of fibroblasts or Caco-2 incubated for 4 h with TPGS (4 mumol/L) increased 10-fold without an increase in the free tocopherol content of nonsaponified samples. A 24-h incubation resulted in a free tocopherol content of approximately 20%, suggesting that intracellular hydrolysis of ester bonds had occurred. The increase in total tocopherol content after a 4-h incubation with TPGS was temperature dependent; no change was measurable at 4 degrees C. Addition of metabolic inhibitors during incubation with TPGS at 37 degrees C did not prevent the increase. [ 14 C]TPGS (synthesized from [ 14 C]PEG 1000) was taken up by Caco-2 cells but [ 14 C]PEG 1000 was not. The intracellular total tocopherol (pmol) equaled the [ 14 C]TPGS (pmol), unequivocally demonstrating uptake of the intact TPGS molecule

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

    Directory of Open Access Journals (Sweden)

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

    Directory of Open Access Journals (Sweden)

    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.

  7. Cultured human foreskin fibroblasts produce a factor that stimulates their growth with properties similar to basic fibroblast growth factor

    International Nuclear Information System (INIS)

    Story, M.T.

    1989-01-01

    To determine if fibroblasts could be a source of fibroblast growth factor (FGF) in tissue, cells were initiated in culture from newborn human foreskin. Fibroblast cell lysates promoted radiolabeled thymidine uptake by cultured quiescent fibroblasts. Seventy-nine percent of the growth-promoting activity of lysates was recovered from heparin-Sepharose. The heparin-binding growth factor reacted on immunoblots with antiserum to human placenta-derived basic FGF and competed with iodinated basic FGF for binding to antiserum to (1-24)bFGF synthetic peptide. To confirm that fibroblasts were the source of the growth factor, cell lysates were prepared from cells incubated with radiolabeled methionine. Heparin affinity purified material was immunoprecipitated with basic FGF antiserum and electrophoresed. Radiolabeled material was detected on gel autoradiographs in the same molecular weight region as authentic iodinated basic FGF. The findings are consistant with the notion that cultured fibroblasts express basic FGF. As these cells also respond to the mitogen, it is possible that the regulation of their growth is under autocrine control. Fibroblasts may be an important source of the growth factor in tissue

  8. Phosphatidylinositol 3,4,5-trisphosphate modulation in SHIP2-deficient mouse embryonic fibroblasts.

    Science.gov (United States)

    Blero, Daniel; Zhang, Jing; Pesesse, Xavier; Payrastre, Bernard; Dumont, Jacques E; Schurmans, Stéphane; Erneux, Christophe

    2005-05-01

    SHIP2, the ubiquitous SH2 domain containing inositol 5-phosphatase, includes a series of protein interacting domains and has the ability to dephosphorylate phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]in vitro. The present study, which was undertaken to evaluate the impact of SHIP2 on PtdIns(3,4,5)P(3) levels, was performed in a mouse embryonic fibroblast (MEF) model using SHIP2 deficient (-/-) MEF cells derived from knockout mice. PtdIns(3,4,5)P(3) was upregulated in serum stimulated -/- MEF cells as compared to +/+ MEF cells. Although the absence of SHIP2 had no effect on basal PtdIns(3,4,5)P(3) levels, we show here that this lipid was significantly upregulated in SHIP2 -/- cells but only after short-term (i.e. 5-10 min) incubation with serum. The difference in PtdIns(3,4,5)P(3) levels in heterozygous fibroblast cells was intermediate between the +/+ and the -/- cells. In our model, insulin-like growth factor-1 stimulation did not show this upregulation. Serum stimulated phosphoinositide 3-kinase (PI 3-kinase) activity appeared to be comparable between +/+ and -/- cells. Moreover, protein kinase B, but not mitogen activated protein kinase activity, was also potentiated in SHIP2 deficient cells stimulated by serum. The upregulation of protein kinase B activity in serum stimulated cells was totally reversed in the presence of the PI 3-kinase inhibitor LY-294002, in both +/+ and -/- cells. Altogether, these data establish a link between SHIP2 and the acute control of PtdIns(3,4,5)P(3) levels in intact cells.

  9. The capsule of Porphyromonas gingivalis reduces the immune response of human gingival fibroblasts

    Directory of Open Access Journals (Sweden)

    van Winkelhoff Arie J

    2010-01-01

    Full Text Available Abstract Background Periodontitis is a bacterial infection of the periodontal tissues. The Gram-negative anaerobic bacterium Porphyromonas gingivalis is considered a major causative agent. One of the virulence factors of P. gingivalis is capsular polysaccharide (CPS. Non-encapsulated strains have been shown to be less virulent in mouse models than encapsulated strains. Results To examine the role of the CPS in host-pathogen interactions we constructed an insertional isogenic P. gingivalis knockout in the epimerase-coding gene epsC that is located at the end of the CPS biosynthesis locus. This mutant was subsequently shown to be non-encapsulated. K1 capsule biosynthesis could be restored by in trans expression of an intact epsC gene. We used the epsC mutant, the W83 wild type strain and the complemented mutant to challenge human gingival fibroblasts to examine the immune response by quantification of IL-1β, IL-6 and IL-8 transcription levels. For each of the cytokines significantly higher expression levels were found when fibroblasts were challenged with the epsC mutant compared to those challenged with the W83 wild type, ranging from two times higher for IL-1β to five times higher for IL-8. Conclusions These experiments provide the first evidence that P. gingivalis CPS acts as an interface between the pathogen and the host that may reduce the host's pro-inflammatory immune response. The higher virulence of encapsulated strains may be caused by this phenomenon which enables the bacteria to evade the immune system.

  10. Fibroblast growth factor receptor mediates fibroblast-dependent growth in EMMPRIN-depleted head and neck cancer tumor cells.

    Science.gov (United States)

    Liu, Zhiyong; Hartman, Yolanda E; Warram, Jason M; Knowles, Joseph A; Sweeny, Larissa; Zhou, Tong; Rosenthal, Eben L

    2011-08-01

    Head and neck squamous cell carcinoma tumors (HNSCC) contain a dense fibrous stroma which is known to promote tumor growth, although the mechanism of stroma-mediated growth remains unclear. As dysplastic mucosal epithelium progresses to cancer, there is incremental overexpression of extracellular matrix metalloprotease inducer (EMMPRIN) which is associated with tumor growth and metastasis. Here, we present evidence that gain of EMMPRIN expression allows tumor growth to be less dependent on fibroblasts by modulating fibroblast growth factor receptor-2 (FGFR2) signaling. We show that silencing EMMPRIN in FaDu and SCC-5 HNSCC cell lines inhibits cell growth, but when EMMPRIN-silenced tumor cells were cocultured with fibroblasts or inoculated with fibroblasts into severe combined immunodeficient mice, the growth inhibition by silencing EMMPRIN was blunted by the presence of fibroblasts. Coculture experiments showed fibroblast-dependent tumor cell growth occurred via a paracrine signaling. Analysis of tumor gene expression revealed expression of FGFR2 was inversely related to EMMPRIN expression. To determine the role of FGFR2 signaling in EMMPRIN-silenced tumor cells, ligands and inhibitors of FGFR2 were assessed. Both FGF1 and FGF2 enhanced tumor growth in EMMPRIN-silenced cells compared with control vector-transfected cells, whereas inhibition of FGFR2 with blocking antibody or with a synthetic inhibitor (PD173074) inhibited tumor cell growth in fibroblast coculture, suggesting the importance of FGFR2 signaling in fibroblast-mediated tumor growth. Analysis of xenografted tumors revealed that EMMPRIN-silenced tumors had a larger stromal compartment compared with control. Taken together, these results suggest that EMMPRIN acquired during tumor progression promotes fibroblast-independent tumor growth.

  11. Fibroblast growth factor receptor mediates fibroblast-dependent growth in EMMPRIN depleted head and neck cancer tumor cells

    Science.gov (United States)

    Liu, Zhiyong; Hartman, Yolanda E.; Warram, Jason M.; Knowles, Joseph A.; Sweeny, Larrisa; Zhou, Tong; Rosenthal, Eben L.

    2011-01-01

    Head and neck squamous cell carcinoma tumors (HNSCC) contain a dense fibrous stroma which is known to promote tumor growth, although the mechanism of stroma mediated growth remains unclear. As dysplastic mucosal epithelium progresses to cancer there is incremental overexpression of extracellular matrix metalloprotease inducer (EMMPRIN) which is associated with tumor growth and metastasis. Here we present evidence that gain of EMMPRIN expression allows tumor growth to be less dependent on fibroblasts by modulating fibroblast growth factor receptor-2 (FGFR2) signaling. We show that silencing EMMPRIN in FaDu and SCC-5 HNSCC cell lines inhibits cell growth, but when EMMPRIN-silenced tumor cells were co-cultured with fibroblasts or inoculated with fibroblasts into SCID mice, the growth inhibition by silencing EMMPRIN was blunted by the presence of fibroblasts. Co-culture experiments demonstrated fibroblast-dependent tumor cell growth occurred via a paracrine signaling. Analysis of tumor gene expression revealed expression of FGFR2 was inversely related to EMMPRIN expression. To determine the role of FGFR2 signaling in EMMPRIN silenced tumor cells, ligands and inhibitors of FGFR2 were assessed. Both FGF1 and FGF2 enhanced tumor growth in EMMPRIN silenced cells compared to control vector transfected cells, while inhibition of FGFR2 with blocking antibody or with a synthetic inhibitor (PD173074) inhibited tumor cell growth in fibroblast co-culture, suggesting the importance of FGFR2 signaling in fibroblast mediated tumor growth. Analysis of xenografted tumors revealed EMMPRIN silenced tumors had a larger stromal compartment compared to control. Taken together, these results suggest that EMMPRIN acquired during tumor progression promotes fibroblast independent tumor growth. PMID:21665938

  12. Interspecific variation of intracellular localization and postirradiation movement of Ku70-protein in fibroblastic cells

    International Nuclear Information System (INIS)

    Endoh, Daiji; Hayashi, Masanobu; Okui, Toyo; Kawase, Shiro; Kon, Yasushiro

    2003-01-01

    Ku (Ku70 and Ku80) Proteins are known as components of DNA-dependent protein kinase (DNA-PK) and play an important role for DNA repair. We previously reported that more than 70% of Ku proteins were located in cytoplasm of rat cells, the Ku proteins moved into nuclei of normal rat cells after X-irradiation, Ku proteins also moved into nuclei after X-irradiation but were not retained in nucleus of radiosensitive LEC rat cells. While reports have been shown about mechanisms on nuclear localization of Ku proteins, how Ku proteins export from nucleus is poorly understood. Here we show that C-terminal region of Ku70 protein is important for its cytoplasmic localization. When transfected into LEC rat cells, exogenous intact Ku70 (1-609) tagged with enhanced green fluorescent protein (EGFP-Ku70) localized mainly in the cytoplasm, whereas C-terminal-deletion mutant of Ku70 (1-593) tagged with EGFP (EGFP-Ku70D) was mainly localized in the nucleus. After X-irradiation, the endogenous intact EGFP-Ku70 once moved into nucleus, but returned into the cytoplasm. On the other hand, EGFP-Ku70D was retained in nucleus for two hours after X-irradiation. These results suggest that C-terminal region of Ku70 is included in the postirradiation nuclear export. Next, we investigated the intracellular localization of Ku70 proteins and the movement after X-irradiation of fibroblastic cells prepared from some mammalian species. Ku70 proteins were localized in nucleus and the postirradiation-extranuclear transport was not observed in human and African green monkey cells. On the other hand, Ku70 proteins were mainly localized in cytoplasm and moved into nucleus in mouse, Chinese hamster, Golden hamster, cotton rat, squirrel, cat and dog cells. These results may show that alternatively Ku70 protein is localized in the cytoplasm or nucleus depends on species and translocation of cytoplasmic Ku70 into nucleus is a response against low dose irradiation in fibroblasts of rodents, cats and dogs

  13. The radiation response of human dermal fibroblasts

    Science.gov (United States)

    Mitchell, Stephen Andrew

    A clinically reliable predictive assay based on normal-tissue radiosensitivity may lead to improved tumour control through individualised dose prescriptions. In-vitro fibroblast radiosensitivity has been shown, in several studies, to correlate with late radiation morbidity. The aim of this study was to investigate some of the cellular mechanisms underlying the normal-tissue response. In this study, seventeen primary fibroblast strains were established by enzymatic disaggregation of skin biopsies obtained from patients. These comprised seven who experienced acute tissue reactions to radiotherapy, four patients with a normal response and six non-cancer volunteers. An AT cell line was included as a positive control for radiosensitivity. In-vitro radiosensitivity was measured using a clonogenic assay at both high (HDR: 1.6 Gymin-1) and low dose rate (LDR: 0.01 Gymin-1). The radiation parameter HDR SF2 was the most sensitive in discriminating the seven sensitive patients from the remaining ten normal patients (range 0.11-0.19 sensitive patients compared with 0.17-0.34 control patients: puse of an internal control or LDR radiation protocol increased this discrimination. Pulsed-field gel electrophoresis (PFGE) was used to measure the level of initial and residual double-strand breaks following irradiation. No correlation was found between HDR SF2 and initial DNA damage. However, a strong correlation was found between clonogenic survival and both residual DNA damage (measured over 10-70 Gy, allowing 4 h repair, correlation coefficient: 0.90, <0.0001) and the ratio of residual/initial DNA damage, with the sensitive cell lines generally showing a higher level of residual DNA damage. Cell-cycle delays were found in all 18 cell strains in response to 2 Gy irradiation, but were not found to discriminate between sensitive and normal patients. Associated studies found no mutations of the ATM gene in the five radiosensitive patients studied. However, a coding sequence alteration

  14. Improved Fibroblast Functionalities by Microporous Pattern Fabricated by Microelectromechanical Systems

    Science.gov (United States)

    Wei, Hongbo; Zhao, Lingzhou; Chen, Bangdao; Bai, Shizhu; Zhao, Yimin

    2014-01-01

    Fibroblasts, which play an important role in biological seal formation and maintenance, determine the long-term success of percutaneous implants. In this study, well-defined microporous structures with micropore diameters of 10–60 µm were fabricated by microelectromechanical systems and their influence on the fibroblast functionalities was observed. The results show that the microporous structures with micropore diameters of 10–60 µm did not influence the initial adherent fibroblast number; however, those with diameters of 40 and 50 µm improved the spread, actin stress fiber organization, proliferation and fibronectin secretion of the fibroblasts. The microporous structures with micropore diameters of 40–50 µm may be promising for application in the percutaneous part of an implant. PMID:25054322

  15. Improved Fibroblast Functionalities by Microporous Pattern Fabricated by Microelectromechanical Systems

    Directory of Open Access Journals (Sweden)

    Hongbo Wei

    2014-07-01

    Full Text Available Fibroblasts, which play an important role in biological seal formation and maintenance, determine the long-term success of percutaneous implants. In this study, well-defined microporous structures with micropore diameters of 10–60 µm were fabricated by microelectromechanical systems and their influence on the fibroblast functionalities was observed. The results show that the microporous structures with micropore diameters of 10–60 µm did not influence the initial adherent fibroblast number; however, those with diameters of 40 and 50 µm improved the spread, actin stress fiber organization, proliferation and fibronectin secretion of the fibroblasts. The microporous structures with micropore diameters of 40–50 µm may be promising for application in the percutaneous part of an implant.

  16. Relationship among expression of basic-fibroblast growth factor ...

    African Journals Online (AJOL)

    Relationship among expression of basic-fibroblast growth factor, MTDH/Astrocyte elevated gene-1, adenomatous polyposis coli, matrix metalloproteinase 9,and COX-2 markers with prognostic factors in prostate carcinomas.

  17. Growth of fibroblasts and endothelial cells on wettability gradient surfaces

    NARCIS (Netherlands)

    Ruardy, TG; Moorlag, HE; Schakenraad, JM; VanderMei, HC; Busscher, HJ

    1997-01-01

    The growth, spreading, and shape of human skin fibroblasts (PK 84) and human umbilical cord endothelial cells on dichlorodimethylsilane (DDS) and dimethyloctadecylchlorosilane (DOGS) gradient surfaces were investigated in the presence of serum proteins. Gradient surfaces were prepared on glass using

  18. Factor XIIIa is expressed by fibroblasts in fibrovascular tumors.

    Science.gov (United States)

    Nemeth, A J; Penneys, N S

    1989-10-01

    Factor XIIIa (FXIIIa), a blood and intracellularly produced coagulation factor, has been found in a variety of cell types including fibroblast-like mesenchymal cells, and has been shown to stimulate the proliferation of fibroblasts and some neoplastic cells in vitro. We have already shown that the dendritic fibroblasts composing the fibrous papule contain this factor. We hypothesized that histopathologically similar fibrovascular tumors may also express FXIIIa and, in this report, show that the large stellate fibroblasts found in acquired digital fibrokeratomas, angiofibromas (adenoma sebaceum of Pringle), and oral fibroma (oral fibrous hyperplasia) also express FXIIIa. We postulate that FXIIIa, possibly acting as a growth factor, may be a common denominator in the pathogenesis of these tumors. Another possibility is that these tumors may be the consequence of a local overproduction of FXIIIa in response to an, as yet, unidentified stimulus.

  19. HGF is released from buccal fibroblasts after smokeless tobacco stimulation

    DEFF Research Database (Denmark)

    Dabelsteen, S; Christensen, S; Gron, B

    2005-01-01

    on exposure time and on concentration of the tobacco extract. High concentration increased production of HGF 4-fold. KGF production was doubled when high concentration of tobacco was used, low concentration did not stimulate cells. GM-CSF production was low in both stimulated and non-stimulated cells......To investigate the effect of smokeless tobacco (ST) on (1) HGF, KGF and GM-CSF expression by buccal fibroblasts and (2) on keratinocyte and fibroblast proliferation. Buccal fibroblasts were stimulated with different concentrations of ST extracts in a double dilution from 0.50% w/v to 0.03% w....... Keratinocytes and fibroblasts showed no increase in proliferation after stimulation with increased concentrations of ST. The results suggest that HGF and KGF may play an important role as a paracrine growth factor in epithelial hyperplasia in ST lesions....

  20. Transient expression of acidic fibroblast growth factor in pea (Pisum ...

    African Journals Online (AJOL)

    Yomi

    2012-03-15

    Mar 15, 2012 ... a 100 W, long-wave UV lamp (Black Ray model B-100 AP; Ultra- ... frequency) was used to estimate the treatment efficiency during 15 days post .... Crystal structure of fibroblast growth factor receptor ectodomain bound to.

  1. Generation of Five Human Lactoferrin Transgenic Cloned Goats Using Fibroblast Cells and Their Methylation Status of Putative Differential Methylation Regions of IGF2R and H19 Imprinted Genes

    Science.gov (United States)

    Sun, Yanyan; Zhang, Yanli; Wang, Ziyu; Song, Yang; Wang, Feng

    2013-01-01

    Background Somatic cell nuclear transfer (SCNT) is a promising technique to produce transgenic cloned mammalian, including transgenic goats which may produce Human Lactoferrin (hLF). However, success percentage of SCNT is low, because of gestational and neonatal failure of transgenic embryos. According to the studies on cattle and mice, DNA methylation of some imprinted genes, which plays a vital role in the reprogramming of embryo in NT maybe an underlying mechanism. Methodology/Principal Findings Fibroblast cells were derived from the ear of a two-month-old goat. The vector expressing hLF was constructed and transfected into fibroblasts. G418 selection, EGFP expression, PCR, and cell cycle distribution were applied sequentially to select transgenic cells clones. After NT and embryo transfer, five transgenic cloned goats were obtained from 240 cloned transgenic embryos. These transgenic goats were identified by 8 microsatellites genotyping and southern blot. Of the five transgenic goats, 3 were lived after birth, while 2 were dead during gestation. We compared differential methylation regions (DMR) pattern of two paternally imprinted genes (H19 and IGF2R) of the ear tissues from the lived transgenic goats, dead transgenic goats, and control goats from natural reproduction. Hyper-methylation pattern appeared in cloned aborted goats, while methylation status was relatively normal in cloned lived goats compared with normal goats. Conclusions/Significance In this study, we generated five hLF transgenic cloned goats by SCNT. This is the first time the DNA methylation of lived and dead transgenic cloned goats was compared. The results demonstrated that the methylation status of DMRs of H19 and IGF2R were different in lived and dead transgenic goats and therefore this may be potentially used to assess the reprogramming status of transgenic cloned goats. Understanding the pattern of gene imprinting may be useful to improve cloning techniques in future. PMID:24204972

  2. 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...

  3. 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.

  4. 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.

  5. 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.

  6. 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...

  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