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Sample records for cell nuclear reprogramming

  1. 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.

  2. Nuclear reprogramming by nuclear transplantation and defined transcription factors

    Institute of Scientific and Technical Information of China (English)

    WANG YiXuan; LIU Sheng; LAI LiangXue; GAO ShaoRong

    2009-01-01

    In the past ten years,great breakthroughs have been achieved in the nuclear reprogramming area.It has been demonstrated that highly differentiated somatic cell genome could be reprogrammed to a pluripotent state,which indicates that differentiated cell fate is not irreversible.Nuclear transplantation and induced pluripotent stem (iPS) cell generation are the two major approaches to inducing repro-gramming of differentiated somatic cell genome.In the present review,we will summarize the recent progress of nuclear reprogramming and further discuss the potential to generate patient specific pluripotent stem cells from differentiated somatic cells for therapeutic purpose.

  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

    , 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...

  4. Oncometabolic Nuclear Reprogramming of Cancer Stemness

    Science.gov (United States)

    Menendez, Javier A.; Corominas-Faja, Bruna; Cuyàs, Elisabet; García, María G.; Fernández-Arroyo, Salvador; Fernández, Agustín F.; Joven, Jorge; Fraga, Mario F.; Alarcón, Tomás

    2016-01-01

    Summary By impairing histone demethylation and locking cells into a reprogramming-prone state, oncometabolites can partially mimic the process of induced pluripotent stem cell generation. Using a systems biology approach, combining mathematical modeling, computation, and proof-of-concept studies with live cells, we found that an oncometabolite-driven pathological version of nuclear reprogramming increases the speed and efficiency of dedifferentiating committed epithelial cells into stem-like states with only a minimal core of stemness transcription factors. Our biomathematical model, which introduces nucleosome modification and epigenetic regulation of cell differentiation genes to account for the direct effects of oncometabolites on nuclear reprogramming, demonstrates that oncometabolites markedly lower the “energy barriers” separating non-stem and stem cell attractors, diminishes the average time of nuclear reprogramming, and increases the size of the basin of attraction of the macrostate occupied by stem cells. These findings establish the concept of oncometabolic nuclear reprogramming of stemness as a bona fide metabolo-epigenetic mechanism for generation of cancer stem-like cells. PMID:26876667

  5. Oncometabolic Nuclear Reprogramming of Cancer Stemness

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    Javier A. Menendez

    2016-03-01

    Full Text Available By impairing histone demethylation and locking cells into a reprogramming-prone state, oncometabolites can partially mimic the process of induced pluripotent stem cell generation. Using a systems biology approach, combining mathematical modeling, computation, and proof-of-concept studies with live cells, we found that an oncometabolite-driven pathological version of nuclear reprogramming increases the speed and efficiency of dedifferentiating committed epithelial cells into stem-like states with only a minimal core of stemness transcription factors. Our biomathematical model, which introduces nucleosome modification and epigenetic regulation of cell differentiation genes to account for the direct effects of oncometabolites on nuclear reprogramming, demonstrates that oncometabolites markedly lower the “energy barriers” separating non-stem and stem cell attractors, diminishes the average time of nuclear reprogramming, and increases the size of the basin of attraction of the macrostate occupied by stem cells. These findings establish the concept of oncometabolic nuclear reprogramming of stemness as a bona fide metabolo-epigenetic mechanism for generation of cancer stem-like cells.

  6. Epigenetic reprogramming in mammalian nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    LI Shijie; DU Weihua; LI Ning

    2004-01-01

    Somatic cloning has been succeeded in some species, but the cloning efficiency is very low, which limits the application of the technique in many areas of research and biotechnology. The cloning of mammals by somatic cell nuclear transfer (NT) requires epigenetic reprogramming of the differentiated state of donor cell to a totipotent, embryonic ground state. Accumulating evidence indicates that incomplete or inappropriate epigenetic reprogramming of donor nuclei is likely to be the primary cause of failures in nuclear transfer. This review summarizes the roles of various epigenetic mechanisms, including DNA methylation, histone acetylation, imprinting, X-chromosome inactivation, telomere maintenance and expressions of development-related genes on somatic nuclear transfer.

  7. Reprogrammed pluripotent stem cells from somatic cells.

    Science.gov (United States)

    Kim, Jong Soo; Choi, Hyun Woo; Choi, Sol; Do, Jeong Tae

    2011-06-01

    Pluripotent stem cells, such as embryonic stem (ES) cells, can differentiate into all cell types. So, these cells can be a biological resource for regenerative medicine. However, ES cells known as standard pluripotent cells have problem to be used for cell therapy because of ethical issue of the origin and immune response on the graft. Hence, recently reprogrammed pluripotent cells have been suggested as an alternative source for regenerative medicine. Somatic cells can acquire the ES cell-like pluripotency by transferring somatic cell nuclei into oocytes, by cell fusion with pluripotent cells. Retroviral-mediated introduction of four factors, Oct4, Sox2, Klf4 and c-Myc can successfully reprogram somatic cells into ES cell-like pluripotent stem cells, known as induced pluripotent stem (iPS) cells. These cells closely resemble ES cells in gene expression pattern, cell biologic and phenotypic characteristics. However, to reach the eventual goal of clinical application, it is necessary to overcome the major drawbacks such as low reprogramming efficiency and genomic alterations due to viral integration. In this review, we discuss the current reprogramming techniques and mechanisms of nuclear reprogramming induced by transcription factor transduction. PMID:24298328

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

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    Sebastian Thomas Balbach

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

  9. Nuclear Reprogramming and Mitosis--how does mitosis enhance changes in gene expression?

    Science.gov (United States)

    Halley-Stott, Richard P

    2015-01-01

    Nuclear reprogramming changes the identity of cells by changing gene expression programmes. Two recent pieces of work have highlighted the role that mitosis plays in enhancing the success of nuclear reprogramming. This Point of View article examines this work in the context of nuclear reprogramming.

  10. Optimal ROS Signaling Is Critical for Nuclear Reprogramming

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    Gang Zhou

    2016-05-01

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

  11. Reprogramming cells with synthetic proteins

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    Xiaoxiao Yang

    2015-06-01

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

  12. MicroRNA-mediated somatic cell reprogramming.

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    Kuo, Chih-Hao; Ying, Shao-Yao

    2013-02-01

    Since the first report of induced pluripotent stem cells (iPSCs) using somatic cell nuclear transfer (SCNT), much focus has been placed on iPSCs due to their great therapeutic potential for diseases such as abnormal development, degenerative disorders, and even cancers. Subsequently, Takahashi and Yamanaka took a novel approach by using four defined transcription factors to generate iPSCs in mice and human fibroblast cells. Scientists have since been trying to refine or develop better approaches to reprogramming, either by using different combinations of transcription factors or delivery methods. However, recent reports showed that the microRNA expression pattern plays a crucial role in somatic cell reprogramming and ectopic introduction of embryonic stem cell-specific microRNAs revert cells back to an ESC-like state, although, the exact mechanism underlying this effect remains unclear. This review describes recent work that has focused on microRNA-mediated approaches to somatic cell reprogramming as well as some of the pros and cons to these approaches and a possible mechanism of action. Based on the pivotal role of microRNAs in embryogenesis and somatic cell reprogramming, studies in this area must continue in order to gain a better understanding of the role of microRNAs in stem cells regulation and activity. PMID:22961769

  13. Reprogramming Cells for Brain Repair

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    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. The acetyllysine reader BRD3R promotes human nuclear reprogramming and regulates mitosis.

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    Shao, Zhicheng; Zhang, Ruowen; Khodadadi-Jamayran, Alireza; Chen, Bo; Crowley, Michael R; Festok, Muhamad A; Crossman, David K; Townes, Tim M; Hu, Kejin

    2016-01-01

    It is well known that both recipient cells and donor nuclei demonstrate a mitotic advantage as observed in the traditional reprogramming with somatic cell nuclear transfer (SCNT). However, it is not known whether a specific mitotic factor plays a critical role in reprogramming. Here we identify an isoform of human bromodomain-containing 3 (BRD3), BRD3R (BRD3 with Reprogramming activity), as a reprogramming factor. BRD3R positively regulates mitosis during reprogramming, upregulates a large set of mitotic genes at early stages of reprogramming, and associates with mitotic chromatin. Interestingly, a set of the mitotic genes upregulated by BRD3R constitutes a pluripotent molecular signature. The two BRD3 isoforms display differential binding to acetylated histones. Our results suggest a molecular interpretation for the mitotic advantage in reprogramming and show that mitosis may be a driving force of reprogramming.

  15. DNA methylation patterns in tissues from mid-gestation bovine foetuses produced by somatic cell nuclear transfer show subtle abnormalities in nuclear reprogramming

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    Lee Rita SF

    2010-03-01

    Full Text Available Abstract Background Cloning of cattle by somatic cell nuclear transfer (SCNT is associated with a high incidence of pregnancy failure characterized by abnormal placental and foetal development. These abnormalities are thought to be due, in part, to incomplete re-setting of the epigenetic state of DNA in the donor somatic cell nucleus to a state that is capable of driving embryonic and foetal development to completion. Here, we tested the hypothesis that DNA methylation patterns were not appropriately established during nuclear reprogramming following SCNT. A panel of imprinted, non-imprinted genes and satellite repeat sequences was examined in tissues collected from viable and failing mid-gestation SCNT foetuses and compared with similar tissues from gestation-matched normal foetuses generated by artificial insemination (AI. Results Most of the genomic regions examined in tissues from viable and failing SCNT foetuses had DNA methylation patterns similar to those in comparable tissues from AI controls. However, statistically significant differences were found between SCNT and AI at specific CpG sites in some regions of the genome, particularly those associated with SNRPN and KCNQ1OT1, which tended to be hypomethylated in SCNT tissues. There was a high degree of variation between individuals in methylation levels at almost every CpG site in these two regions, even in AI controls. In other genomic regions, methylation levels at specific CpG sites were tightly controlled with little variation between individuals. Only one site (HAND1 showed a tissue-specific pattern of DNA methylation. Overall, DNA methylation patterns in tissues of failing foetuses were similar to apparently viable SCNT foetuses, although there were individuals showing extreme deviant patterns. Conclusion These results show that SCNT foetuses that had developed to mid-gestation had largely undergone nuclear reprogramming and that the epigenetic signature at this stage was not a

  16. Reprogrammed Pluripotent Stem Cells from Somatic Cells

    OpenAIRE

    Kim, Jong Soo; Choi, Hyun Woo; Choi, Sol; Do, Jeong Tae

    2011-01-01

    Pluripotent stem cells, such as embryonic stem (ES) cells, can differentiate into all cell types. So, these cells can be a biological resource for regenerative medicine. However, ES cells known as standard pluripotent cells have problem to be used for cell therapy because of ethical issue of the origin and immune response on the graft. Hence, recently reprogrammed pluripotent cells have been suggested as an alternative source for regenerative medicine. Somatic cells can acquire the ES cell-li...

  17. Dynamic culture improves cell reprogramming efficiency.

    Science.gov (United States)

    Sia, Junren; Sun, Raymond; Chu, Julia; Li, Song

    2016-06-01

    Cell reprogramming to pluripotency is an inefficient process and various approaches have been devised to improve the yield of induced pluripotent stem cells. However, the effect of biophysical factors on cell reprogramming is not well understood. Here we showed that, for the first time, dynamic culture with orbital shaking significantly improved the reprogramming efficiency in adherent cells. Manipulating the viscosity of the culture medium suggested that the improved efficiency is mainly attributed to convective mixing rather than hydrodynamic shear stress. Temporal studies demonstrated that the enhancement of reprogramming efficiency required the dynamic culture in the middle but not early phase. In the early phase, fibroblasts had a high proliferation rate, but as the culture became over-confluent in the middle phase, expression of p57 was upregulated to inhibit cell proliferation and consequently, cell reprogramming. Subjecting the over confluent culture to orbital shaking prevented the upregulation of p57, thus improving reprogramming efficiency. Seeding cells at low densities to avoid over-confluency resulted in a lower efficiency, and optimal reprogramming efficiency was attained at a high seeding density with dynamic culture. Our findings provide insight into the underlying mechanisms of how dynamic culture condition regulate cell reprogramming, and will have broad impact on cell engineering for regenerative medicine and disease modeling.

  18. 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

    to cloning by somatic cell nuclear transfer. However, when cells are reprogrammed by less fundamental means, as for example treatment by Xenopus extract or expression of pluripotency genes, more subtle nucleolar modulations can also be noted. The monitoring and understanding of the reprogramming...

  19. Generation of Induced Pluripotent Stem (iPS) Cells by Nuclear Reprogramming

    OpenAIRE

    Evans, Gregory R.D.; Dilip Dey

    2011-01-01

    During embryonic development pluripotency is progressively lost irreversibly by cell division, differentiation, migration and organ formation. Terminally differentiated cells do not generate other kinds of cells. Pluripotent stem cells are a great source of varying cell types that are used for tissue regeneration or repair of damaged tissue. The pluripotent stem cells can be derived from inner cell mass of blastocyte but its application is limited due to ethical concerns. The recent discovery...

  20. Stem cell reprogramming: A 3D boost

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    Abilez, Oscar J.; Wu, Joseph C.

    2016-03-01

    Biophysical factors in an optimized three-dimensional microenvironment enhance the reprogramming efficiency of human somatic cells into pluripotent stem cells when compared to traditional cell-culture substrates.

  1. The labyrinth of nuclear reprogramming

    Institute of Scientific and Technical Information of China (English)

    Ignacio Sancho-Martinez; Emmanuel Nivet; Juan Carlos Izpisua Belmonte

    2011-01-01

    Human embryonic stem cells (ESCs) have the capability to differentiate into all somatic cell types,a process that recapitulates the early stages of human development.However,the use of ESCs raises several controversies,particularly concerning the ethical dilemma regarding the use of human embryonic material and the need for embryo destruction.

  2. Direct reprogramming of somatic cells: an update

    Directory of Open Access Journals (Sweden)

    Phuc Van Pham

    2015-03-01

    Full Text Available Direct epigenetic reprogramming is a technique that converts a differentiated adult cell into another differentiated cell and mdash;such fibroblasts to cardiomyocytes and mdash;without passage through an undifferentiated pluripotent stage. This novel technology is opening doors in biological research and regenerative medicine. Some preliminary studies about direct reprogramming started in the 1980s when differentiated adult cells could be converted into other differentiated cells by overexpressing transcription-factor genes. These studies also showed that differentiated cells have plasticity. Direct reprogramming can be a powerful tool in biological research and regenerative medicine, especially the new frontier of personalized medicine. This review aims to summarize all direct reprogramming studies of somatic cells by master control genes as well as potential applications of these techniques in research and treatment of selected human diseases. [Biomed Res Ther 2015; 2(3.000: 231-240

  3. Directed Dedifferentiation Using Partial Reprogramming Induces Invasive Phenotype in Melanoma Cells.

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    Knappe, Nathalie; Novak, Daniel; Weina, Kasia; Bernhardt, Mathias; Reith, Maike; Larribere, Lionel; Hölzel, Michael; Tüting, Thomas; Gebhardt, Christoffer; Umansky, Viktor; Utikal, Jochen

    2016-04-01

    The combination of cancer-focused studies and research related to nuclear reprogramming has gained increasing importance since both processes-reprogramming towards pluripotency and malignant transformation-share essential features. Studies have revealed that incomplete reprogramming of somatic cells leads to malignant transformation indicating that epigenetic regulation associated with iPSC generation can drive cancer development [J Mol Cell Biol 2011;341-350; Cell 2012;151:1617-1632; Cell 2014;156:663-677]. However, so far it is unclear whether incomplete reprogramming also affects cancer cells and their function. In the context of melanoma, dedifferentiation correlates to therapy resistance in mouse studies and has been documented in melanoma patients [Nature 2012;490:412-416; Clin Cancer Res 2014;20:2498-2499]. Therefore, we sought to investigate directed dedifferentiation using incomplete reprogramming of melanoma cells. Using a murine model we investigated the effects of partial reprogramming on the cellular plasticity of melanoma cells. We demonstrate for the first time that induced partial reprogramming results in a reversible phenotype switch in melanoma cells. Partially reprogrammed cells at day 12 after transgene induction display elevated invasive potential in vitro and increased lung colonization in vivo. Additionally, using global gene expression analysis of partially reprogrammed cells, we identified SNAI3 as a novel invasion-related marker in human melanoma. SNAI3 expression correlates with tumor thickness in primary melanomas and thus, may be of prognostic value. In summary, we show that investigating intermediate states during the process of reprogramming melanoma cells can reveal novel insights into the pathogenesis of melanoma progression. We propose that deeper analysis of partially reprogrammed melanoma cells may contribute to identification of yet unknown signaling pathways that can drive melanoma progression. Stem Cells 2016;34:832-846. PMID

  4. Reprogramming stem cells is a microenvironmental task

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    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

  5. Somatic cell reprogramming-free generation of genetically modified pigs.

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    Tanihara, Fuminori; Takemoto, Tatsuya; Kitagawa, Eri; Rao, Shengbin; Do, Lanh Thi Kim; Onishi, Akira; Yamashita, Yukiko; Kosugi, Chisato; Suzuki, Hitomi; Sembon, Shoichiro; Suzuki, Shunichi; Nakai, Michiko; Hashimoto, Masakazu; Yasue, Akihiro; Matsuhisa, Munehide; Noji, Sumihare; Fujimura, Tatsuya; Fuchimoto, Dai-Ichiro; Otoi, Takeshige

    2016-09-01

    Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs. PMID:27652340

  6. Somatic cell reprogramming-free generation of genetically modified pigs

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    Tanihara, Fuminori; Takemoto, Tatsuya; Kitagawa, Eri; Rao, Shengbin; Do, Lanh Thi Kim; Onishi, Akira; Yamashita, Yukiko; Kosugi, Chisato; Suzuki, Hitomi; Sembon, Shoichiro; Suzuki, Shunichi; Nakai, Michiko; Hashimoto, Masakazu; Yasue, Akihiro; Matsuhisa, Munehide; Noji, Sumihare; Fujimura, Tatsuya; Fuchimoto, Dai-ichiro; Otoi, Takeshige

    2016-01-01

    Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs. PMID:27652340

  7. Nuclear reprogramming of luminal-like breast cancer cells generates Sox2-overexpressing cancer stem-like cellular states harboring transcriptional activation of the mTOR pathway

    Science.gov (United States)

    Corominas-Faja, Bruna; Cufí, Sílvia; Oliveras-Ferraros, Cristina; Cuyàs, Elisabet; López-Bonet, Eugeni; Lupu, Ruth; Alarcón, Tomás; Vellon, Luciano; Iglesias, Juan Manuel; Leis, Olatz; Martín, Ángel G; Vazquez-Martin, Alejandro; Menendez, Javier A

    2013-01-01

    cells. Consistent with the downregulation of AMPK expression, immunoblotting procedures confirmed upregulation of p70S6K and increased phosphorylation of mTOR in Sox2-overexpressing CSC-like cell populations. Using an in vitro model of the de novo generation of CSC-like states through the nuclear reprogramming of an established breast cancer cell line, we reveal that the transcriptional suppression of mTOR repressors is an intrinsic process occurring during the acquisition of CSC-like properties by differentiated populations of luminal-like breast cancer cells. This approach may provide a new path for obtaining information about preventing the appearance of CSCs through the modulation of the AMPK/mTOR pathway. PMID:23974095

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

    Institute of Scientific and Technical Information of China (English)

    LiuWang; AihuaZheng; LingYi; ChongrenXu; MingxiaoDing; HongkuiDeng

    2005-01-01

    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.

  9. Metaplasia and somatic cell reprogramming.

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    Slack, J M W

    2009-01-01

    The nature and occurrence of metaplasia is briefly reviewed. A theory of how metaplasia is initiated is presented, depending on the idea that it represents an alteration in the combination of developmental transcription factors that are expressed. Two examples of experimental metaplasia, provoked by over-expression of specific transcription factors, are presented: the transformation of B lymphocytes to macrophages, and of pancreatic exocrine cells to hepatocytes. The formation of induced pluripotential stem cells (iPS cells) is considered an example of the same process, in which the destination state is the embryonic stem cell. It is noted that iPS cell production is a stochastic process, depending on selection to obtain the desired cell type. It is proposed that analogous technology, using the appropriate transcription factors, could be used to bring about transformation to cell types other than embryonic stem cells. PMID:18855879

  10. Reprogramming within hours following nuclear transfer into mouse but not human zygotes.

    Science.gov (United States)

    Egli, Dieter; Chen, Alice E; Saphier, Genevieve; Ichida, Justin; Fitzgerald, Claire; Go, Kathryn J; Acevedo, Nicole; Patel, Jay; Baetscher, Manfred; Kearns, William G; Goland, Robin; Leibel, Rudolph L; Melton, Douglas A; Eggan, Kevin

    2011-01-01

    Fertilized mouse zygotes can reprogram somatic cells to a pluripotent state. Human zygotes might therefore be useful for producing patient-derived pluripotent stem cells. However, logistical, legal and social considerations have limited the availability of human eggs for research. Here we show that a significant number of normal fertilized eggs (zygotes) can be obtained for reprogramming studies. Using these zygotes, we found that when the zygotic genome was replaced with that of a somatic cell, development progressed normally throughout the cleavage stages, but then arrested before the morula stage. This arrest was associated with a failure to activate transcription in the transferred somatic genome. In contrast to human zygotes, mouse zygotes reprogrammed the somatic cell genome to a pluripotent state within hours after transfer. Our results suggest that there may be a previously unappreciated barrier to successful human nuclear transfer, and that future studies could focus on the requirements for genome activation. PMID:21971503

  11. Metabostemness: Metaboloepigenetic reprogramming of cancer stem-cell functions

    Science.gov (United States)

    Menendez, Javier A.; Corominas-Faja, Bruna; Cuyàs, Elisabet; Alarcón, Tomás

    2014-01-01

    Cancer researchers are currently embarking on one of their field's biggest challenges, namely the understanding of how cellular metabolism or certain classes of elite metabolites (e.g., oncometabolites) can directly influence chromatin structure and the functioning of epi-transcriptional circuits to causally drive tumour formation. We here propose that refining the inherent cell attractor nature of nuclear reprogramming phenomena by adding the under-appreciated capacity of metabolism to naturally reshape the Waddingtonian landscape's topography provides a new integrative metabolo-epigenetic model of the cancer stem cell (CSC) theory. PMID:25621295

  12. Reprogramming Cells for Brain Repair

    OpenAIRE

    McKinnon, Randall D.; Alyx T. Guarino

    2013-01-01

    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 c...

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

    Science.gov (United States)

    Brouwer, Marinka; Zhou, Huiqing; Nadif Kasri, Nael

    2016-02-01

    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 concerns in the use of iPSCs, especially in clinical settings. Therefore, reprogramming methods have been under development to generate safer iPSCs with higher efficiency and better quality. Developments have mainly focused on the somatic cell source, the cocktail of reprogramming factors, the delivery method used to introduce reprogramming factors and culture conditions to maintain the generated iPSCs. This review discusses the developments on these topics and briefly discusses pros and cons of iPSCs in comparison with human embryonic stem cells generated from somatic cell nuclear transfer. PMID:26424535

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

    OpenAIRE

    Page Grier P; Kasinathan Poothappillai; Wang Zhongde; Rodriguez-Osorio Nelida; Robl James M; Memili Erdogan

    2009-01-01

    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 clo...

  15. Transcriptional Reprogramming of Gene Expression in Bovine Somatic Cell Chromatin Transfer Embryos

    OpenAIRE

    Rodriguez-Osorio, N.; Wang, Zhongde; Page, G. P.; Robl, J M; Memili, E.

    2009-01-01

    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 ...

  16. Translational research on reprogramming of somatic cells

    Institute of Scientific and Technical Information of China (English)

    Yanhua Li; Jiahui Yin; Bingbing Zhang; Ping Zhou; Bin Feng; Fangyi Zhang; Yongzhong Lin; Zhanhua Liang; Jianling Du; Minghui Lü; Tiezheng Zheng; Jie Lin; Siyu Liu; Hao Hong; Xing Meng; Dandan Xia; Yang Sun; Pan Wei; Nan Cai; Hongye Li; Shuang Wu; Hui Zhao; Changkai Sun; Yuyuan Li; Changyu Gao; Wei Li; Ye Dai; Junde Wang; Hui Zhao; Xiaoxin Tan; Lili Men; Hui Ma; Jun Xu; Xiaohan Yang; Zengchun Hu; Ling Wang; Hong Wang; Pin Sun; Huifang Guo; Guirong Song; Hui Liu1; Baoshuai Shan; Lu Han; Linlang Liang; Min Wang; Xiaochen Wang; Dan Wang; Guihua Chen; Jianting Chen; Xiangyou Sun; Jun Xue; Zhiqi Wang; Jing Wang; Yongqing Zhang; Dongfeng Cai; Mozhen Liu; Guiping Zhang; Guoming Luan; Jianli Wang; Ming Fan; Xuetao Cao; Chao Wan; Qigui Liu; Anchun Yin

    2014-01-01

    Cerebrovascular diseases,dementia,diabetes,malignant tumors and degenerative bone diseases remain high prevalence,incidence,disability and mortality rates.One important reason might be the slow or stagnated progress in translating and applying cytoprotection and cellular repair researches into clinical practice.Based on collaboration among biomedical re-searchers,database experts,computer programmers,statisticians and management engineers,this is the first study to apply quanti-tative comparison on the overall characteristics and partial correlation analysis on the large-scale complex information and data regarding the topic“mature cells can be reprogrammed to become pluripotent”proposed by Sir John B.Gurdon and Shinya Yamanaka who were jointly awarded with 2012 Nobel Prize in Physiology or Medicine,as well as articles that cited publications of the two Nobel Laureates to discuss the prospects of translating somatic cell reprogramming researches into clinical practice and cor-responding implementation strategies.The study found that there was statistically significant difference between the two Nobel Laureates with regard to the number,publication date,subject categories and scientific and technological focuses of their origi-nal researches.The study revealed the importance,objectives,approaches and research trends of translational medicine,especially translational neuroscience.The study also identified the challenges that China should overcome to improve its medical research management scheme.

  17. Signaling involved in stem cell reprogramming and differentiation

    Institute of Scientific and Technical Information of China (English)

    Shihori; Tanabe

    2015-01-01

    Stem cell differentiation is regulated by multiple signaling events. Recent technical advances have reve-aled that differentiated cells can be reprogrammed into stem cells. The signals involved in stem cell pro-gramming are of major interest in stem cell research. The signaling mechanisms involved in regulating stem cell reprogramming and differentiation are the subject of intense study in the field of life sciences. In this review,the molecular interactions and signaling pathways related to stem cell differentiation are discussed.

  18. Cell signalling pathways underlying induced pluripotent stem cell reprogramming

    Institute of Scientific and Technical Information of China (English)

    Kate; Hawkins; Shona; Joy; Tristan; Mc; Kay

    2014-01-01

    Induced pluripotent stem(i PS) cells, somatic cells reprogrammed to the pluripotent state by forced expression of defined factors, represent a uniquely valuable resource for research and regenerative medicine. However, this methodology remains inefficient due to incomplete mechanistic understanding of the reprogramming process. In recent years, various groups have endeavoured to interrogate the cell signalling that governs the reprogramming process, including LIF/STAT3, BMP, PI3 K, FGF2, Wnt, TGFβ and MAPK pathways, with the aim of increasing our understanding and identifying new mechanisms of improving safety, reproducibility and efficiency. This has led to a unified model of reprogramming that consists of 3 stages: initiation, maturation and stabilisation. Initiation of reprogramming occurs in almost all cells that receive the reprogramming transgenes; most commonly Oct4, Sox2, Klf4 and c Myc, and involves a phenotypic mesenchymal-to-epithelial transition. The initiation stage is also characterised by increased proliferation and a metabolic switch from oxidative phosphorylation to glycolysis. The maturation stage is considered the major bottleneck within the process, resulting in very few "stabilisation competent" cells progressing to the final stabilisation phase. To reach this stage in both mouse and human cells, pre-i PS cells must activate endogenous expression of the core circuitry of pluripotency, comprising Oct4, Sox2, and Nanog, and thus reach a state of transgene independence. By the stabilisation stage, i PS cells generally use the same signalling networks that govern pluripotency in embryonic stem cells. These pathways differ between mouse and human cells although recent work has demonstrated that this is context dependent. As i PS cell generation technologies move forward, tools are being developed to interrogate the process in more detail, thus allowing a greater understanding of this intriguing biological phenomenon.

  19. Wound signaling of regenerative cell reprogramming.

    Science.gov (United States)

    Lup, Samuel Daniel; Tian, Xin; Xu, Jian; Pérez-Pérez, José Manuel

    2016-09-01

    Plants are sessile organisms that must deal with various threats resulting in tissue damage, such as herbivore feeding, and physical wounding by wind, snow or crushing by animals. During wound healing, phytohormone crosstalk orchestrates cellular regeneration through the establishment of tissue-specific asymmetries. In turn, hormone-regulated transcription factors and their downstream targets coordinate cellular responses, including dedifferentiation, cell cycle reactivation and vascular regeneration. By comparing different examples of wound-induced tissue regeneration in the model plant Arabidopsis thaliana, a number of key regulators of developmental plasticity of plant cells have been identified. We present the relevance of these findings and of the dynamic establishment of differential auxin gradients for cell reprogramming after wounding. PMID:27457994

  20. Signaling involved in stem cell reprogramming and differentiation

    OpenAIRE

    Tanabe, Shihori

    2015-01-01

    Stem cell differentiation is regulated by multiple signaling events. Recent technical advances have revealed that differentiated cells can be reprogrammed into stem cells. The signals involved in stem cell programming are of major interest in stem cell research. The signaling mechanisms involved in regulating stem cell reprogramming and differentiation are the subject of intense study in the field of life sciences. In this review, the molecular interactions and signaling pathways related to s...

  1. Reprogrammed cell delivery for personalized medicine.

    Science.gov (United States)

    Wieland, Markus; Fussenegger, Martin

    2012-10-01

    In most approaches, personalized medicine requires time- and cost-intensive characterization of an individual's genetic background in order to achieve the best-adapted therapy. For this purpose, cell-based drug delivery offers a promising alternative. In particular, synthetic biology has introduced the vision of cells being programmable therapeutic production facilities that can be introduced into patients. This review highlights the progress made in synthetic biology-based cell engineering toward advanced drug delivery entities. Starting from basic one-input responsive transcriptional or post-transcriptional gene control systems, the field has reached a level on which cells can be engineered to detect cancer cells, to obtain control over T-cell proliferation, and to restore blood glucose homeostasis upon blue light illumination. Furthermore, a cellular implant was developed that detects blood urate level disorders and acts accordingly to restore homeostasis while another cellular implant was engineered as an artificial insemination device that releases bull sperm into bovine ovarian only during ovulation time by recording endogenous luteinizing hormone levels. Soon, the field will reach a stage at which cells can be reprogrammed to detect multiple metabolic parameters and self-sufficiently treat any disorder connected to them. PMID:22721864

  2. Cell reprogramming for the creation of patient-specific pluripotent stem cells by defined factors

    Institute of Scientific and Technical Information of China (English)

    Huiqun YIN; Heng WANG; Hongguo CAO; Yunhai ZHANG; Yong TAO; Xiaorong ZHANG

    2009-01-01

    Pluripotent stem cells (PSCs), characterized by being able to differentiate into various types of cells, are generally regarded as the most promising sources for cell replacement therapies. However, as typical PSCs, embryonic stem cells (ESCs) are still far away from human clinics so far due to ethical issues and immune rejection response. One way to avoid such problems is to use stem cells derived from autologous somatic cells. Up to date, PSCs could be obtained by reprogramming somatic cells to pluripotent state with approaches including somatic cell nuclear transfer (SCNT), fusion with stem cells, coculture with cells' extracts, and induction with defined factors. Among these, through reprogramming somatic cells directly by retroviral transduction of transcription factors, induced pluripotent stem (iPS) cells have been successfully generated in both mouse and human recently. These iPS cells shared similar morphology and growth properties to ESCs, could express ESCs marker genes, and could produce adult or germline-competent chimaeras and differentiate into a variety of cell types, including germ cells. Moreover, with iPS technique, patient specific PSCs could be derived more easily from handful somatic cells in human without immune rejection responses innately connected to ESCs. Consequently, generation of iPS cells would be of great help to further understand disease mechanisms, drug screening, and cell transplantation therapies as well.In summary,the recent progress in the study of cell reprogramming for the creation of patientspecific pluripotent stem cells, some existing problems, and research perspectives were suggested.

  3. 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

    2013-10-01

    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. PMID:24048479

  4. 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

    2013-10-01

    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.

  5. Molecular insights into the heterogeneity of telomere reprogramming in induced pluripotent stem cells

    Institute of Scientific and Technical Information of China (English)

    Fang Wang; Jiameng Dan; Bingfeng Zuo; Minshu Li; Qian Zhang; Na Liu; Lingyi Chen; Xinghua Pan,; Sarantis Gagos; David L Keefe; Lin Liu; Yu Yin; Xiaoying Ye; Kai Liu; Haiying Zhu; Lingling Wang; Maria Chiourea; Maja Okuka; Guangzhen Ji

    2012-01-01

    Rejuvenation of telomeres with various lengths has been found in induced pluripotent stem cells (iPSCs).Mechanisms of telomere length regulation during induction and proliferation of iPSCs remain elusive.We show that telomere dynamics are variable in mouse iPSCs during reprogramming and passage,and suggest that these differences likely result from multiple potential factors,including the telomerase machinery,teiomerase-independent mechanisms and clonal influences including reexpression of exogenous reprogramming factors.Using a genetic model of telomerase-deficient (Terc-/- and Terc+/-) cells for derivation and passages of iPSCs,we found that telomerase plays a critical role in reprogramming and self-renewal of iPSCs.Further,telomerase maintenance of telomeres is necessary for induction of true pluripotency while the alternative pathway of elongation and maintenance by recombination is also required,but not sufficient.Together,several aspects of telomere biology may account for the variable telomere dynamics in iPSCs.Notably,the mechanisms employed to maintain telomeres during iPSC reprogramming are very similar to those of embryonic stem cells.These findings may also relate to the cloning field where these mechanisms could be responsible for telomere heterogeneity after nuclear reprogramming by somatic cell nuclear transfer.

  6. Cell Reprogramming, IPS Limitations, and Overcoming Strategies in Dental Bioengineering

    Directory of Open Access Journals (Sweden)

    Gaskon Ibarretxe

    2012-01-01

    Full Text Available The procurement of induced pluripotent stem cells, or IPS cells, from adult differentiated animal cells has the potential to revolutionize future medicine, where reprogrammed IPS cells may be used to repair disease-affected tissues on demand. The potential of IPS cell technology is tremendous, but it will be essential to improve the methodologies for IPS cell generation and to precisely evaluate each clone and subclone of IPS cells for their safety and efficacy. Additionally, the current state of knowledge on IPS cells advises that research on their regenerative properties is carried out in appropriate tissue and organ systems that permit a safe assessment of the long-term behavior of these reprogrammed cells. In the present paper, we discuss the mechanisms of cell reprogramming, current technical limitations of IPS cells for their use in human tissue engineering, and possibilities to overcome them in the particular case of dental regeneration.

  7. Experimental Limitations Using Reprogrammed Cells for Hematopoietic Differentiation

    Directory of Open Access Journals (Sweden)

    Katharina Seiler

    2011-01-01

    Full Text Available We review here our experiences with the in vitro reprogramming of somatic cells to induced pluripotent stem cells (iPSC and subsequent in vitro development of hematopoietic cells from these iPSC and from embryonic stem cells (ESC. While, in principle, the in vitro reprogramming and subsequent differentiation can generate hematopoietic cell from any somatic cells, it is evident that many of the steps in this process need to be significantly improved before it can be applied to human cells and used in clinical settings of hematopoietic stem cell (HSC transplantations.

  8. Reprogramming fibroblasts into induced pluripotent stem cells with Bmi

    Institute of Scientific and Technical Information of China (English)

    Jai-Hee Moon; June Seok Heo; Jun Sung Kim; Eun Kyoung Jun; Jung Han Lee; Aeree Kim; Jonggun Kim

    2011-01-01

    Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by the transcription factors Oct4,Sox2,and Klf4 in combination with c-Myc.Recently,Sox2 plus Oct4 was shown to reprogram fibroblasts and Oct4 alone was able to reprogram mouse and human neural stem cells (NSCs) into iPS cells.Here,we report that Bmi1 leads to the transdifferentiation of mouse fibroblasts into NSC-like cells,and,in combination with Oct4,can replace Sox2,Klf4 and c-Myc during the reprogramming of fibroblasts into iPS cells.Furthermore,activation of sonic hedgehog signaling (by Shh,purmorphamine,or oxysterol) compensates for the effects of Bmil,and,in combination with Oct4,reprograms mouse embryonic and adult fibroblasts into iPS cells.One- and two-factor iPS cells are similar to mouse embryonic stem cells in their global gene expression profile,epigenetic status,and in vitro and in vivo differentiation into all three germ layers,as well as teratoma formation and germline transmission in vivo.These data support that converting fibroblasts with Bmi1 or activation of the sonic hedgehog pathway to an intermediate cell type that expresses Sox2,KIf4,and N-Myc allows iPS generation via the addition of Oct4.

  9. Mouse cloning and somatic cell reprogramming using electrofused blastomeres

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

    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.

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

    OpenAIRE

    Ana Belen Alvarez Palomo; Michaela Lucas; Dilley, Rodney J.; Samuel McLenachan; Fred Kuanfu Chen; Jordi Requena; Marti Farrera Sal; Andrew Lucas; Inaki Alvarez; Dolores Jaraquemada; Michael J. Edel

    2014-01-01

    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 rege...

  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. Cell Reprogramming, IPS Limitations, and Overcoming Strategies in Dental Bioengineering

    OpenAIRE

    Gaskon Ibarretxe; Antonia Alvarez; Maria-Luz Cañavate; Enrique Hilario; Maitane Aurrekoetxea; Fernando Unda

    2012-01-01

    The procurement of induced pluripotent stem cells, or IPS cells, from adult differentiated animal cells has the potential to revolutionize future medicine, where reprogrammed IPS cells may be used to repair disease-affected tissues on demand. The potential of IPS cell technology is tremendous, but it will be essential to improve the methodologies for IPS cell generation and to precisely evaluate each clone and subclone of IPS cells for their safety and efficacy. Additionally, the current stat...

  13. Telomere reprogramming and maintenance in porcine iPS cells.

    Directory of Open Access Journals (Sweden)

    Guangzhen Ji

    Full Text Available Telomere reprogramming and silencing of exogenous genes have been demonstrated in mouse and human induced pluripotent stem cells (iPS cells. Pigs have the potential to provide xenotransplant for humans, and to model and test human diseases. We investigated the telomere length and maintenance in porcine iPS cells generated and cultured under various conditions. Telomere lengths vary among different porcine iPS cell lines, some with telomere elongation and maintenance, and others telomere shortening. Porcine iPS cells with sufficient telomere length maintenance show the ability to differentiate in vivo by teratoma formation test. IPS cells with short or dysfunctional telomeres exhibit reduced ability to form teratomas. Moreover, insufficient telomerase and incomplete telomere reprogramming and/or maintenance link to sustained activation of exogenous genes in porcine iPS cells. In contrast, porcine iPS cells with reduced expression of exogenous genes or partial exogene silencing exhibit insufficient activation of endogenous pluripotent genes and telomerase genes, accompanied by telomere shortening with increasing passages. Moreover, telomere doublets, telomere sister chromatid exchanges and t-circles that presumably are involved in telomere lengthening by recombination also are found in porcine iPS cells. These data suggest that both telomerase-dependent and telomerase-independent mechanisms are involved in telomere reprogramming during induction and passages of porcine iPS cells, but these are insufficient, resulting in increased telomere damage and shortening, and chromosomal instability. Active exogenes might compensate for insufficient activation of endogenous genes and incomplete telomere reprogramming and maintenance of porcine iPS cells. Further understanding of telomere reprogramming and maintenance may help improve the quality of porcine iPS cells.

  14. Renal stem cell reprogramming: Prospects in regenerative medicine

    Institute of Scientific and Technical Information of China (English)

    Elvin; E; Morales; Rebecca; A; Wingert

    2014-01-01

    Stem cell therapy is a promising future enterprise for renal replacement in patients with acute and chronic kidney disease, conditions which affect millions worldwide and currently require patients to undergo lifelong medical treatments through dialysis and/or organ transplant. Reprogramming differentiated renal cells harvested from the patient back into a pluripotent state would decrease the risk of tissue rejection and provide a virtually unlimited supply of cells for regenerative medicine treatments, making it an exciting area of current research in nephrology. Among the major hurdles that need to be overcome before stem cell therapy for the kidney can be applied in a clinical setting are ensuring the fidelity and relative safety of the reprogrammed cells, as well as achieving feasible efficiency in the reprogramming processes that are utilized. Further, improved knowledge about the genetic control of renal lineage development is vital to identifying predictable and efficient reprogramming approaches, such as the expression of key modulators or the regulation of geneactivity through small molecule mimetics. Here, we discuss several recent advances in induced pluripotent stem cell technologies. We also explore strategies that have been successful in renal progenitor generation, and explore what these methods might mean for the development of cell-based regenerative therapies for kidney disease.

  15. Reprogramming T cell Lymphocytes to Induced Pluripotent Stem Cells

    Science.gov (United States)

    Bared, Kalia

    The discovery of induced pluripotent stem cells (iPSC) provided a novel technology for the study of development and pharmacology and complement embryonic stem cells (ES) for cell therapy applications. Though iPSC are derived from adult tissue they are comparable to ES cells in their behavior; multi-lineage differentiation and self-renewal. This makes iPSC research appealing because they can be studied in great detail and expanded in culture broadly. Fibroblasts were the first cell type reprogrammed to an iPSC using a retrovirus vector, since then alternative cell types including lymphocytes have been used to generate iPSC. Different types of vectors have also been developed to enhance iPSC formation and quality. However, specific T lymphocyte subsets have not been shown to reprogram to a pluripotent state to date. Here, we proposed to derive iPSC from peripheral blood effector and central memory T cells, reasoning that the resultant iPSC will maintain the epigenetic memory of a T lymphocyte, including the T cell receptor (TCR) gene rearrangement. This epigenetic memory will enable the differentiation and expansion of T cell iPSC into professional T cells containing a specific TCR. These could then be used for cell therapy to target specific antigens, as well as to improve culture techniques to expand T cells in vitro. We studied different gene delivery methods to derive iPSC from different types of T lymphocytes. We assessed the viability of viral transduction using flow cytometry to detect green fluorescent marker contained in the viral construct and quantitative real time polymerase chain reaction (qRT-PCR) to detect Oct4, Klf4, Sox2, and c-Myc gene expression. Our results demonstrate that the Sendai virus construct is the most feasible platform to reprogram T lymphocytes. We anticipate that this platform will provide an efficient and safe approach to derive iPSC from different T cell subsets, including memory T cells.

  16. Suv4-20h abrogation enhances telomere elongation during reprogramming and confers a higher tumorigenic potential to iPS cells

    OpenAIRE

    Rosa M Marión; Gunnar Schotta; Sagrario Ortega; Blasco, Maria A.

    2011-01-01

    Reprogramming of adult differentiated cells to induced pluripotent stem cells (iPS) cells has been achieved by over-expression of specific transcription factors. Nuclear reprogramming induces a series of profound changes at the telomeres of the parental differentiated cells, including a telomerase-dependent telomere elongation and the remodeling of telomeric chromatin. In particular, iPS cells show a decreased density of H4K20me3 heterochromatic mark at telomeres compared to the parental cell...

  17. 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.

  18. 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 doma

  19. Mitochondrial physiology and gene expression analyses reveal metabolic and translational dysregulation in oocyte-induced somatic nuclear reprogramming.

    Directory of Open Access Journals (Sweden)

    Telma C Esteves

    Full Text Available While reprogramming a foreign nucleus after somatic cell nuclear transfer (SCNT, the enucleated oocyte (ooplasm must signal that biomass and cellular requirements changed compared to the nucleus donor cell. Using cells expressing nuclear-encoded but mitochondria-targeted EGFP, a strategy was developed to directly distinguish maternal and embryonic products, testing ooplasm demands on transcriptional and post-transcriptional activity during reprogramming. Specifically, we compared transcript and protein levels for EGFP and other products in pre-implantation SCNT embryos, side-by-side to fertilized controls (embryos produced from the same oocyte pool, by intracytoplasmic injection of sperm containing the EGFP transgene. We observed that while EGFP transcript abundance is not different, protein levels are significantly lower in SCNT compared to fertilized blastocysts. This was not observed for Gapdh and Actb, whose protein reflected mRNA. This transcript-protein relationship indicates that the somatic nucleus can keep up with ooplasm transcript demands, whilst transcription and translation mismatch occurs after SCNT for certain mRNAs. We further detected metabolic disturbances after SCNT, suggesting a place among forces regulating post-transcriptional changes during reprogramming. Our observations ascribe oocyte-induced reprogramming with previously unsuspected regulatory dimensions, in that presence of functional proteins may no longer be inferred from mRNA, but rather depend on post-transcriptional regulation possibly modulated through metabolism.

  20. 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.

  1. 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...

  2. 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.

  3. Stress-mediated p38 activation promotes somatic cell reprogramming

    Institute of Scientific and Technical Information of China (English)

    Xinxiu Xu; Quan Wang; Yuan Long; Ru Zhang; Xiaoyuan Wei; Mingzhe Xing; Haifeng Gu

    2013-01-01

    Environmental stress-mediated adaptation plays essential roles in the evolution of life.Cellular adaptation mechanisms usually involve the regulation of chromatin structure,transcription,mRNA stability and translation,which eventually lead to efficient changes in gene expression.Global epigenetic change is also involved in the reprogramming of somatic cells into induced pluripotent stem (iPS) cells by defined factors.Here we report that environmental stress such as hyperosmosis not only facilitates four factor-mediated reprogramming,but also enhances two or one factor-induced iPS cell generation.Hyperosmosis-induced p38 activation plays a critical role in this process.Constitutive active p38 mimics the positive effect of hyperosmosis,while dominant negative p38 and p38 inhibitor block the effect of hyperosmosis.Further study indicates stress-mediated p38 activation may promote reprogramming by reducing the global DNA methylation level and enhancing the expression of pluripotency genes.Our results demonstrate how simple environmental stress like hyperosmosis helps to alter the fate of cells via intracellular signaling and epigenetic modulation.

  4. Clinical significance of T cell metabolic reprogramming in cancer.

    Science.gov (United States)

    Herbel, Christoph; Patsoukis, Nikolaos; Bardhan, Kankana; Seth, Pankaj; Weaver, Jessica D; Boussiotis, Vassiliki A

    2016-12-01

    Conversion of normal cells to cancer is accompanied with changes in their metabolism. During this conversion, cell metabolism undergoes a shift from oxidative phosphorylation to aerobic glycolysis, also known as Warburg effect, which is a hallmark for cancer cell metabolism. In cancer cells, glycolysis functions in parallel with the TCA cycle and other metabolic pathways to enhance biosynthetic processes and thus support proliferation and growth. Similar metabolic features are observed in T cells during activation but, in contrast to cancer, metabolic transitions in T cells are part of a physiological process. Currently, there is intense interest in understanding the cause and effect relationship between metabolic reprogramming and T cell differentiation. After the recent success of cancer immunotherapy, the crosstalk between immune system and cancer has come to the forefront of clinical and basic research. One of the key goals is to delineate how metabolic alterations of cancer influence metabolism-regulated function and differentiation of tumor resident T cells and how such effects might be altered by immunotherapy. Here, we review the unique metabolic features of cancer, the implications of cancer metabolism on T cell metabolic reprogramming during antigen encounters, and the translational prospective of harnessing metabolism in cancer and T cells for cancer therapy. PMID:27510264

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-03-26

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

  6. Lineage-specific reprogramming as a strategy for cell therapy.

    Science.gov (United States)

    Darabi, Radbod; Perlingeiro, Rita C R

    2008-06-15

    Embryonic stem (ES) cells are endowed with extensive ability for self renewal and differentiation. These features make them a promising candidate for cell therapy. However, despite the enthusiasm and hype surrounding the potential therapeutic use of human ES cells and more recently induced pluripotent stem (iPS) cells, to date few reports have documented successful therapeutic outcome with ES-derived cell populations. This is probably due to two main caveats associated with ES cells, their capacity to form teratomas and the challenge of isolating the appropriate therapeutic cell population from differentiating ES cells. We have focused our efforts on the derivation of skeletal muscle progenitors from ES cells and here we will discuss the strategy of reprogramming lineage choices by overexpression of a master regulator, which has proven successful for the generation of the skeletal myogenic lineage from mouse ES cells.

  7. Roles of small molecules in somatic cell reprogramming

    Institute of Scientific and Technical Information of China (English)

    Jian-bin SU; Duan-qing PEI; Bao-ming QIN

    2013-01-01

    The Nobel Prize in Physiology and Medicine 2012 was awarded to Sir John B GURDON and Shinya YAMANAKA for their discovery that mature cells can be reprogrammed to become pluripotent.This event reaffirms the importance of research on cell fate plasticity and the technology progress in the stem cell field and regenerative medicine.Indeed,reprogramming technology has developed at a dazzling speed within the past 6 years,yet we are still at the early stages of understanding the mechanisms of cell fate identity.This is particularly true in the case of human induced pluripotent stem ceils (iPSCs),which lack reliable standards in the evaluation of their fidelity and safety prior to their application.Along with the genetic approaches,small molecules nowadays become convenient tools for modulating endogenous protein functions and regulating key cellular processes,including the mesenchymal-to-epithelial transition,metabolism,signal transduction and epigenetics.Moreover,small molecules may affect not only the efficiency of clone formation but also the quality of the resulting cells.With increasing availability of such chemicals,we can better understand the biology of stems cells and further improve the technology of generation of stem cells.

  8. Dendritic cell reprogramming by the hypoxic environment.

    Science.gov (United States)

    Bosco, Maria Carla; Varesio, Luigi

    2012-12-01

    Myeloid dendritic cells (DCs) are professional antigen-presenting cells central to the orchestration of innate and acquired immunity and the maintenance of self-tolerance. The local microenvironment contributes to the regulation of DC development and functions, and deregulated DC responses may result in amplification of inflammation, loss of tolerance, or establishment of immune escape mechanisms. DC generation from monocytic precursors recruited at sites of inflammation, tissue damage, or neoplasia occurs under condition of low partial oxygen pressure (pO(2), hypoxia). We reviewed the literature addressing the phenotypic and functional changes triggered by hypoxia in monocyte-derived immature (i) and mature (m) DCs. The discussion will revolve around in vitro studies of gene expression profile, which give a comprehensive representation of the complexity of response of these cells to low pO(2). The gene expression pattern of hypoxic DC will be discussed to address the question of the relationship with a specific maturation stage. We will summarize data relative to the regulation of the chemotactic network, which points to a role for hypoxia in promoting a migratory phenotype in iDCs and a highly proinflammatory state in mDCs. Current knowledge of the strict regulatory control exerted by hypoxia on the expression of immune-related cell surface receptors will also be addressed, with a particular focus on a newly identified marker of hypoxic DCs endowed with proinflammatory properties. Furthermore, we discuss the literature on the transcription mechanisms underlying hypoxia-regulated gene expression in DCs, which support a major role for the HIF/HRE pathway. Finally, recent advances shedding light on the in vivo influence of the local hypoxic microenvironment on DCs infiltrating the inflamed joints of juvenile idiopathic arthritis patients are outlined. PMID:22901977

  9. Embryonic Development following Somatic Cell Nuclear Transfer Impeded by Persisting Histone Methylation

    OpenAIRE

    Matoba, Shogo; Liu, Yuting; Lu, Falong; Iwabuchi, Kumiko A.; Inoue, Azusa; Zhang, Yi

    2014-01-01

    Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major epigenetic barrier for efficient reprogramming by SCNT. Comparative transcriptome analysis identified reprogramming resistant regions (RRRs) that are expressed n...

  10. Privileged Communication Embryonic Development Following Somatic Cell Nuclear Transfer Impeded by Persisting Histone Methylation

    OpenAIRE

    Matoba, Shogo; Liu, Yuting; Lu, Falong; Iwabuchi, Kumiko A.; Shen, Li; Inoue, Azusa; Zhang, Yi

    2014-01-01

    Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major epigenetic barrier for efficient reprogramming by SCNT. Comparative transcriptome analysis identified reprogramming resistant regions (RRRs) that are expressed n...

  11. 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.

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

    DEFF Research Database (Denmark)

    Swales, Nathalie; Martens, Geert A; Bonné, Stefan;

    2012-01-01

    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....

  13. 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...

  14. A New Avenue to Cure Cancer by Turning Adaptive Immune T Cells to Innate Immune NK Cells via Reprogramming

    Institute of Scientific and Technical Information of China (English)

    Dong-Ming Su; Ramakrishna Vankayalapati

    2010-01-01

    Thymocytes after T-lineage commitment develop in the T-cell pathway. However, in a recent study, Li et al. (2010) demonstrated that inducing to delete Bcl11b gene in these thymocytes, even in mature T cells turns these cells into natural killer (NK) cells during the culture. They called this conversion 'reprogramming', and the reprogrammed killer cells 'ITNK cells'.

  15. Reprogramming human B cells into induced pluripotent stem cells and its enhancement by C/EBPα.

    Science.gov (United States)

    Bueno, C; Sardina, J L; Di Stefano, B; Romero-Moya, D; Muñoz-López, A; Ariza, L; Chillón, M C; Balanzategui, A; Castaño, J; Herreros, A; Fraga, M F; Fernández, A; Granada, I; Quintana-Bustamante, O; Segovia, J C; Nishimura, K; Ohtaka, M; Nakanishi, M; Graf, T; Menendez, P

    2016-03-01

    B cells have been shown to be refractory to reprogramming and B-cell-derived induced pluripotent stem cells (iPSC) have only been generated from murine B cells engineered to carry doxycycline-inducible Oct4, Sox2, Klf4 and Myc (OSKM) cassette in every tissue and from EBV/SV40LT-immortalized lymphoblastoid cell lines. Here, we show for the first time that freshly isolated non-cultured human cord blood (CB)- and peripheral blood (PB)-derived CD19+CD20+ B cells can be reprogrammed to iPSCs carrying complete VDJH immunoglobulin (Ig) gene monoclonal rearrangements using non-integrative tetracistronic, but not monocistronic, OSKM-expressing Sendai Virus. Co-expression of C/EBPα with OSKM facilitates iPSC generation from both CB- and PB-derived B cells. We also demonstrate that myeloid cells are much easier to reprogram than B and T lymphocytes. Differentiation potential back into the cell type of their origin of B-cell-, T-cell-, myeloid- and fibroblast-iPSCs is not skewed, suggesting that their differentiation does not seem influenced by 'epigenetic memory'. Our data reflect the actual cell-autonomous reprogramming capacity of human primary B cells because biased reprogramming was avoided by using freshly isolated primary cells, not exposed to cytokine cocktails favoring proliferation, differentiation or survival. The ability to reprogram CB/PB-derived primary human B cells offers an unprecedented opportunity for studying developmental B lymphopoiesis and modeling B-cell malignancies. PMID:26500142

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

    Science.gov (United States)

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

    2016-01-01

    Summary 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 domain (TAD) repositioning and alterations of tissue-restricted genomic neighborhoods and chromatin loops, effectively erasing the somatic-cell-specific genome structures while establishing an embryonic stem-cell-like 3D genome. Yet, early passage iPSCs carry topological hallmarks that enable recognition of their cell of origin. These hallmarks are not remnants of somatic chromosome topologies. Instead, the distinguishing topological features are acquired during reprogramming, as we also find for cell-of-origin-dependent gene expression patterns. PMID:26971819

  17. Global epigenetic changes during somatic cell reprogramming to iPS cells

    Institute of Scientific and Technical Information of China (English)

    Anna Mattout; Alva Biran; Eran Meshorer

    2011-01-01

    Embryonic stem cells (ESCs) exhibit unique chromatin features,including a permissive transcriptional program and an open,decondensed chromatin state.Induced pluripotent stem cells (iPSCs),which are very similar to ESCs,hold great promise for therapy and basic research.However,the mechanisms by which reprogramming occurs and the chromatin organization that underlies the reprogramming process are largely unknown.Here we characterize and compare the epigenetic landscapes of partially and fully reprogrammed iPSCs to mouse embryonic fibroblasts (MEFs) and ESCs,which serves as a standard for pluripotency.Using immunofluorescence and biochemical fractionations,we analyzed the levels and distribution of a battery of histone modifications (H3ac,H4ac,H4KSac,H3Kgac,H3K27ac,H3K4me3,H3K36me2,H3K9me3,H3K27me3,and yH2AX),as well as HP1α and lamin A.We find that fully reprogrammed iPSCs are epigenetically identical to ESCs,and that partially reprogrammed iPSCs are closer to MEFs.Intriguingly,combining both time-course reprogramming experiments and data from the partially reprogrammed iPSCs,we find that heterochromatin reorganization precedes Nanog expression and active histone marking.Together,these data delineate the global epigenetic state of iPSCs in conjunction with their pluripotent state,and demonstrate that heterochromatin precedes euchromatin in reorganization during reprogramming.

  18. Brains in metamorphosis: reprogramming cell identity within the central nervous system

    OpenAIRE

    Arlotta, Paola; Berninger, Benedikt

    2014-01-01

    During embryonic development, uncommitted pluripotent cells undergo progressive epigenetic changes that lock them into a final differentiated state. Can mammalian cells change identity within the living organism? Direct lineage reprogramming of cells has attracted attention as a means to achieve organ regeneration. However, it is unclear whether cells in the CNS are endowed with the plasticity to reprogram. Neurons in particular are considered among the most immutable cell types, able to reta...

  19. 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.

  20. Study of mitochondrial respiratory defects on reprogramming to human induced pluripotent stem cells

    Science.gov (United States)

    Hung, Sandy S.C.; Van Bergen, Nicole J.; Jackson, Stacey; Liang, Helena; Mackey, David A.; Hernández, Damián; Lim, Shiang Y.; Hewitt, Alex W.; Trounce, Ian; Pébay, Alice; Wong, Raymond C.B.

    2016-01-01

    Reprogramming of somatic cells into a pluripotent state is known to be accompanied by extensive restructuring of mitochondria and switch in metabolic requirements. Here we utilized Leber's hereditary optic neuropathy (LHON) as a mitochondrial disease model to study the effects of homoplasmic mtDNA mutations and subsequent oxidative phosphorylation (OXPHOS) defects in reprogramming. We obtained fibroblasts from a total of 6 LHON patients and control subjects, and showed a significant defect in complex I respiration in LHON fibroblasts by high-resolution respiratory analysis. Using episomal vector reprogramming, our results indicated that human induced pluripotent stem cell (hiPSC) generation is feasible in LHON fibroblasts. In particular, LHON-specific OXPHOS defects in fibroblasts only caused a mild reduction and did not significantly affect reprogramming efficiency, suggesting that hiPSC reprogramming can tolerate a certain degree of OXPHOS defects. Our results highlighted the induction of genes involved in mitochondrial biogenesis (TFAM, NRF1), mitochondrial fusion (MFN1, MFN2) and glycine production (GCAT) during reprogramming. However, LHON-associated OXPHOS defects did not alter the kinetics or expression levels of these genes during reprogramming. Together, our study provides new insights into the effects of mtDNA mutation and OXPHOS defects in reprogramming and genes associated with various aspects of mitochondrial biology. PMID:27127184

  1. 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.

  2. 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

  3. Genomic imprinting is variably lost during reprogramming of mouse iPS cells

    OpenAIRE

    Takikawa, Sachiko; Ray, Chelsea; Wang, Xin; Shamis, Yulia; Wu, Tien-Yuan; Li, Xiajun

    2013-01-01

    Derivation of induced pluripotent stem (iPS) cells is mainly an epigenetic reprogramming process. It is still quite controversial how genomic imprinting is reprogrammed in iPS cells. Thus, we derived multiple iPS clones from genetically identical mouse somatic cells. We found that parentally inherited imprint was variably lost among these iPS clones. Concurrent with the loss of DNA methylation imprint at the corresponding Snrpn and Peg3 imprinted regions, parental origin-specific expression o...

  4. Early ERK1/2 activation promotes DRP1-dependent mitochondrial fission necessary for cell reprogramming.

    Science.gov (United States)

    Prieto, Javier; León, Marian; Ponsoda, Xavier; Sendra, Ramón; Bort, Roque; Ferrer-Lorente, Raquel; Raya, Angel; López-García, Carlos; Torres, Josema

    2016-01-01

    During the process of reprogramming to induced pluripotent stem (iPS) cells, somatic cells switch from oxidative to glycolytic metabolism, a transition associated with profound mitochondrial reorganization. Neither the importance of mitochondrial remodelling for cell reprogramming, nor the molecular mechanisms controlling this process are well understood. Here, we show that an early wave of mitochondrial fragmentation occurs upon expression of reprogramming factors. Reprogramming-induced mitochondrial fission is associated with a minor decrease in mitochondrial mass but not with mitophagy. The pro-fission factor Drp1 is phosphorylated early in reprogramming, and its knockdown and inhibition impairs both mitochondrial fragmentation and generation of iPS cell colonies. Drp1 phosphorylation depends on Erk activation in early reprogramming, which occurs, at least in part, due to downregulation of the MAP kinase phosphatase Dusp6. Taken together, our data indicate that mitochondrial fission controlled by an Erk-Drp1 axis constitutes an early and necessary step in the reprogramming process to pluripotency. PMID:27030341

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

    OpenAIRE

    Brouwer, Marinka; Zhou, Huiqing; Nadif Kasri, Nael

    2015-01-01

    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 concerns in the use of iPSCs, especially in clinical settings. Therefore, reprogramming methods have been under development to generate safer iPSCs with higher efficiency and better quality. Develo...

  6. Cell reprogramming: a new chemical approach to stem cell biology and tissue regeneration.

    Science.gov (United States)

    Anastasia, L; Piccoli, M; Garatti, A; Conforti, E; Scaringi, R; Bergante, S; Castelvecchio, S; Venerando, B; Menicanti, L; Tettamanti, G

    2011-02-01

    Generation of pluripotent stem cells (iPSCs) from adult fibroblasts starts a "new era" in stem cell biology, as it overcomes several key issues associated with previous approaches, including the ethical concerns associated with human embryonic stem cells. However, as the genetic approach for cell reprogramming has already shown potential safety issues, a chemical approach may be a safer and easier alternative. Moreover, a chemical approach could be advantageous not only for the de-differentiation phase, but also for inducing reprogrammed cells into the desired cell type with higher efficiency than current methodologies. Finally, a chemical approach may be envisioned to activate resident adult stem cells to proliferate and regenerate damaged tissues in situ, without the need for exogenous cell injections.

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

    Institute of Scientific and Technical Information of China (English)

    Shaoping Hou; Paul Lu

    2016-01-01

    Direct reprogramming of somatic cells into neurons or neural stem cells is one of the most important fron-tier ifelds 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 cellsin vitro andin vivo and their potential treatments of neurological disorders.

  8. Plant Hormones Increase Efficiency of Reprogramming Mouse Somatic Cells to Induced Pluripotent Stem Cells and Reduce Tumorigenicity

    OpenAIRE

    Alvarez Palomo, Ana Belén; McLenachan, Samuel; Requena Osete, Jordi; Menchón, Cristina; Barrot, Carme; Chen, Fred; Munné-Bosch, Sergi; Michael J. Edel

    2013-01-01

    Reprogramming of somatic cells into induced pluripotent stem (iPS) cells by defined pluripotency and self-renewal factors has taken stem cell technology to the forefront of regenerative medicine. However, a number of challenges remain in the field including efficient protocols and the threat of cancer. Reprogramming of plant somatic cells to plant embryonic stem cells using a combination of two plant hormones was discovered in 1957 and has been a routine university laboratory practical for ov...

  9. 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. PMID:26098448

  10. 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.

  11. Limiting replication stress during somatic cell reprogramming reduces genomic instability in induced pluripotent stem cells

    OpenAIRE

    Ruiz, Sergio; Lopez Contreras, Andres J.; Gabut, Mathieu; Marion, Rosa M.; Guti??rrez Mart??nez, Paula; Bua, Sabela; Ram??rez, Oscar; Olalde, I??igo; Rodrigo Perez, Sara; Li, Han; Marqu??s i Bonet, Tom??s, 1975-; Serrano, Manuel; Blasco, Maria A; Batada, Nizar N; Fern??ndez Capetillo, Oscar

    2015-01-01

    The generation of induced pluripotent stem cells (iPSC) from adult somatic cells is one of the most remarkable discoveries in recent decades. However, several works have reported evidence of genomic instability in iPSC, raising concerns on their biomedical use. The reasons behind the genomic instability observed in iPSC remain mostly unknown. Here we show that, similar to the phenomenon of oncogene-induced replication stress, the expression of reprogramming factors induces replication stress....

  12. Critical POU domain residues confer Oct4 uniqueness in somatic cell reprogramming.

    Science.gov (United States)

    Jin, Wensong; Wang, Lei; Zhu, Fei; Tan, Weiqi; Lin, Wei; Chen, Dahua; Sun, Qinmiao; Xia, Zongping

    2016-01-01

    The POU domain transcription factor Oct4 plays critical roles in self-renewal and pluripotency of embryonic stem cells (ESCs). Together with Sox2, Klf4 and c-Myc, Oct4 can reprogram any other cell types to pluripotency, in which Oct4 is the only factor that cannot be functionally replaced by other POU family members. To investigate the determinant elements of Oct4 uniqueness, we performed Ala scan on all Ser, Thr, Tyr, Lys and Arg of murine Oct4 by testing their capability in somatic cell reprogramming. We uncovered a series of residues that are important for Oct4 functionality, in which almost all of these key residues are within the POU domains making direct interaction with DNA. The Oct4 N- and C-terminal transactivation domains (TADs) are not unique and could be replaced by the Yes-associated protein (YAP) TAD domain to support reprogramming. More importantly, we uncovered two important residues that confer Oct4 uniqueness in somatic cell reprogramming. Our systematic structure-function analyses bring novel mechanistic insight into the molecular basis of how critical residues function together to confer Oct4 uniqueness among POU family for somatic cell reprogramming. PMID:26877091

  13. Interpretation of reprogramming to predict the success of somatic cell cloning.

    Science.gov (United States)

    Eckardt, Sigrid; McLaughlin, K John

    2004-07-01

    In the context of mammalian somatic cell cloning, the term reprogramming refers to the processes that enable a somatic cell nucleus to adopt the role of a zygotic nucleus. Gene re-expression is one measure of reprogramming if correlated with subsequent developmental potential. This paper describes several experiments utilizing pre-implantation gene expression to evaluate reprogramming and clone viability. We have established a direct correlation between Oct4 expression in mouse clones at the blastocyst stage and their potential to maintain pluripotent embryonic cells essential for post-implantation development. Furthermore, the quality of gene expression in clones dramatically improves when genetically identical clones are combined in clone-clone aggregate chimeras. Clone--clone aggregates exhibit a higher developmental potential than single clones both in vitro and in vivo. This could be mediated by complementation between blastomeres from epigenetically different clones within the aggregate rather than by the increase in cell number resulting from aggregation. We also discuss the use of tetraploid embryos as a model to evaluate reprogramming using gene expression and demonstrate that somatic cell nuclei can be reprogrammed by blastomeres to re-express embryonic specific genes but not to contribute to post-implantation development.

  14. Reprogramming Müller glia via in vivo cell fusion regenerates murine photoreceptors.

    Science.gov (United States)

    Sanges, Daniela; Simonte, Giacoma; Di Vicino, Umberto; Romo, Neus; Pinilla, Isabel; Nicolás, Marta; Cosma, Maria Pia

    2016-08-01

    Vision impairments and blindness caused by retinitis pigmentosa result from severe neurodegeneration that leads to a loss of photoreceptors, the specialized light-sensitive neurons that enable vision. Although the mammalian nervous system is unable to replace neurons lost due to degeneration, therapeutic approaches to reprogram resident glial cells to replace retinal neurons have been proposed. Here, we demonstrate that retinal Müller glia can be reprogrammed in vivo into retinal precursors that then differentiate into photoreceptors. We transplanted hematopoietic stem and progenitor cells (HSPCs) into retinas affected by photoreceptor degeneration and observed spontaneous cell fusion events between Müller glia and the transplanted cells. Activation of Wnt signaling in the transplanted HSPCs enhanced survival and proliferation of Müller-HSPC hybrids as well as their reprogramming into intermediate photoreceptor precursors. This suggests that Wnt signaling drives the reprogrammed cells toward a photoreceptor progenitor fate. Finally, Müller-HSPC hybrids differentiated into photoreceptors. Transplantation of HSPCs with activated Wnt functionally rescued the retinal degeneration phenotype in rd10 mice, a model for inherited retinitis pigmentosa. Together, these results suggest that photoreceptors can be generated by reprogramming Müller glia and that this approach may have potential as a strategy for reversing retinal degeneration. PMID:27427986

  15. PXD101 significantly improves nuclear reprogramming and the in vitro developmental competence of porcine SCNT embryos

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Jun-Xue; Kang, Jin-Dan; Li, Suo; Jin, Long; Zhu, Hai-Ying; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun, E-mail: yinxj33@msn.com

    2015-01-02

    Highlights: • First explored that the effects of PXD101 on the development of SCNT embryos in vitro. • 0.5 μM PXD101 treated for 24 h improved the development of porcine SCNT embryos. • Level of AcH3K9 was significantly higher than control group at early stages. - Abstract: In this study, we investigated the effects of the histone deacetylase inhibitor PXD101 (belinostat) on the preimplantation development of porcine somatic cell nuclear transfer (SCNT) embryos and their expression of the epigenetic markers histone H3 acetylated at lysine 9 (AcH3K9). We compared the in vitro developmental competence of SCNT embryos treated with various concentrations of PXD101 for 24 h. Treatment with 0.5 μM PXD101 significantly increased the proportion of SCNT embryos that reached the blastocyst stage, in comparison to the control group (23.3% vs. 11.5%, P < 0.05). We tested the in vitro developmental competence of SCNT embryos treated with 0.5 μM PXD101 for various amounts of times following activation. Treatment for 24 h significantly improved the development of porcine SCNT embryos, with a significantly higher proportion of embryos reaching the blastocyst stage in comparison to the control group (25.7% vs. 10.6%, P < 0.05). PXD101-treated SCNT embryos were transferred into two surrogate sows, one of whom became pregnant and four fetuses developed. PXD101 treatment significantly increased the fluorescence intensity of immunostaining for AcH3K9 in embryos at the pseudo-pronuclear and 2-cell stages. At these stages, the fluorescence intensities of immunostaining for AcH3K9 were significantly higher in PXD101-treated embryos than in control untreated embryos. In conclusion, this study demonstrates that PXD101 can significantly improve the in vitro and in vivo developmental competence of porcine SCNT embryos and can enhance their nuclear reprogramming.

  16. PXD101 significantly improves nuclear reprogramming and the in vitro developmental competence of porcine SCNT embryos

    International Nuclear Information System (INIS)

    Highlights: • First explored that the effects of PXD101 on the development of SCNT embryos in vitro. • 0.5 μM PXD101 treated for 24 h improved the development of porcine SCNT embryos. • Level of AcH3K9 was significantly higher than control group at early stages. - Abstract: In this study, we investigated the effects of the histone deacetylase inhibitor PXD101 (belinostat) on the preimplantation development of porcine somatic cell nuclear transfer (SCNT) embryos and their expression of the epigenetic markers histone H3 acetylated at lysine 9 (AcH3K9). We compared the in vitro developmental competence of SCNT embryos treated with various concentrations of PXD101 for 24 h. Treatment with 0.5 μM PXD101 significantly increased the proportion of SCNT embryos that reached the blastocyst stage, in comparison to the control group (23.3% vs. 11.5%, P < 0.05). We tested the in vitro developmental competence of SCNT embryos treated with 0.5 μM PXD101 for various amounts of times following activation. Treatment for 24 h significantly improved the development of porcine SCNT embryos, with a significantly higher proportion of embryos reaching the blastocyst stage in comparison to the control group (25.7% vs. 10.6%, P < 0.05). PXD101-treated SCNT embryos were transferred into two surrogate sows, one of whom became pregnant and four fetuses developed. PXD101 treatment significantly increased the fluorescence intensity of immunostaining for AcH3K9 in embryos at the pseudo-pronuclear and 2-cell stages. At these stages, the fluorescence intensities of immunostaining for AcH3K9 were significantly higher in PXD101-treated embryos than in control untreated embryos. In conclusion, this study demonstrates that PXD101 can significantly improve the in vitro and in vivo developmental competence of porcine SCNT embryos and can enhance their nuclear reprogramming

  17. Direct reprogramming of human fibroblasts into dopaminergic neuron-like cells

    Institute of Scientific and Technical Information of China (English)

    Xinjian Liu; Dabing Zhang; Timothy A Benke; John R Sladek; Nancy R Zahniser; Chuan-Yuan Li; Fang Li; Elizabeth A Stubblefield; Barbara Blanchard; Toni L Richards; Gaynor A Larson; Yujun He; Qian Huang; Aik-Choon Tan

    2012-01-01

    Transplantation of exogenous dopaminergic neuron (DA neurons) is a promising approach for treating Parkinson's disease (PD).However,a major stumbling block has been the lack of a reliable source of donor DA neurons.Here we show that a combination of five transcriptional factors Mash1,Ngn2,Sox2,Nurr1,and Pitx3 can directly and effectively reprogram human fibroblasts into DA neuron-like cells.The reprogrammed cells stained positive for various markers for DA neurons.They also showed characteristic DA uptake and production properties.Moreover,they exhibited DA neuron-specific electrophysiological profiles.Finally,they provided symptomatic relief in a rat PD model.Therefore,our directly reprogrammed DA neuron-like cells are a promising source of cell-replacement therapy for PD.

  18. Quiescence Loosens Epigenetic Constraints in Bovine Somatic Cells and Improves Their Reprogramming into Totipotency.

    Science.gov (United States)

    Kallingappa, Prasanna K; Turner, Pavla M; Eichenlaub, Michael P; Green, Andria L; Oback, Fleur C; Chibnall, Alice M; Wells, David N; Oback, Björn

    2016-07-01

    Reprogramming by nuclear transfer (NT) cloning forces cells to lose their lineage-specific epigenetic marks and reacquire totipotency. This process often produces molecular anomalies that compromise clone development. We hypothesized that quiescence alters the epigenetic status of somatic NT donor cells and elevates their reprogrammability. To test this idea, we compared chromatin composition and cloning efficiency of serum-starved quiescent (G0) fibroblasts versus nonstarved mitotically selected (G1) controls. We show that G0 chromatin contains reduced levels of Polycomb group proteins EED, SUZ12, PHC1, and RING2, as well as histone variant H2A.Z. Using quantitative confocal immunofluorescence microscopy and fluorometric enzyme-linked immunosorbent assay, we further show that G0 induced DNA and histone hypomethylation, specifically at H3K4me3, H3K9me2/3 and H3K27me3, but not H3K9me1. Collectively, these changes resulted in a more relaxed G0 chromatin state. Following NT, G0 donors developed into blastocysts that retained H3K9me3 hypomethylation, both in the inner cell mass and trophectoderm. G0 blastocysts from different cell types and cell lines developed significantly better into adult offspring. In conclusion, serum starvation induced epigenetic changes, specifically hypotrimethylation, that provide a mechanistic correlate for increased somatic cell reprogrammability. PMID:27281704

  19. Reprogramming of Human Fibroblasts to Induced Pluripotent Stem Cells with Sleeping Beauty Transposon-Based Stable Gene Delivery.

    Science.gov (United States)

    Sebe, Attila; Ivics, Zoltán

    2016-01-01

    Human induced pluripotent stem (iPS) cells are a source of patient-specific pluripotent stem cells and resemble human embryonic stem (ES) cells in gene expression profiles, morphology, pluripotency, and in vitro differentiation potential. iPS cells are applied in disease modeling, drug screenings, toxicology screenings, and autologous cell therapy. In this protocol, we describe how to derive human iPS cells from fibroblasts by Sleeping Beauty (SB) transposon-mediated gene transfer of reprogramming factors. First, the components of the non-viral Sleeping Beauty transposon system, namely a transposon vector encoding reprogramming transcription factors and a helper plasmid expressing the SB transposase, are electroporated into human fibroblasts. The reprogramming cassette undergoes transposition from the transfected plasmids into the fibroblast genome, thereby resulting in stable delivery of the reprogramming factors. Reprogramming by using this protocol takes ~4 weeks, after which the iPS cells are isolated and clonally propagated.

  20. Reprogramming of Human Fibroblasts to Induced Pluripotent Stem Cells with Sleeping Beauty Transposon-Based Stable Gene Delivery.

    Science.gov (United States)

    Sebe, Attila; Ivics, Zoltán

    2016-01-01

    Human induced pluripotent stem (iPS) cells are a source of patient-specific pluripotent stem cells and resemble human embryonic stem (ES) cells in gene expression profiles, morphology, pluripotency, and in vitro differentiation potential. iPS cells are applied in disease modeling, drug screenings, toxicology screenings, and autologous cell therapy. In this protocol, we describe how to derive human iPS cells from fibroblasts by Sleeping Beauty (SB) transposon-mediated gene transfer of reprogramming factors. First, the components of the non-viral Sleeping Beauty transposon system, namely a transposon vector encoding reprogramming transcription factors and a helper plasmid expressing the SB transposase, are electroporated into human fibroblasts. The reprogramming cassette undergoes transposition from the transfected plasmids into the fibroblast genome, thereby resulting in stable delivery of the reprogramming factors. Reprogramming by using this protocol takes ~4 weeks, after which the iPS cells are isolated and clonally propagated. PMID:26895068

  1. In Vivo Reprogramming for CNS Repair: Regenerating Neurons from Endogenous Glial Cells.

    Science.gov (United States)

    Li, Hedong; Chen, Gong

    2016-08-17

    Neuroregeneration in the CNS has proven to be difficult despite decades of research. The old dogma that CNS neurons cannot be regenerated in the adult mammalian brain has been overturned; however, endogenous adult neurogenesis appears to be insufficient for brain repair. Stem cell therapy once held promise for generating large quantities of neurons in the CNS, but immunorejection and long-term functional integration remain major hurdles. In this Perspective, we discuss the use of in vivo reprogramming as an emerging technology to regenerate functional neurons from endogenous glial cells inside the brain and spinal cord. Besides the CNS, in vivo reprogramming has been demonstrated successfully in the pancreas, heart, and liver and may be adopted in other organs. Although challenges remain for translating this technology into clinical therapies, we anticipate that in vivo reprogramming may revolutionize regenerative medicine by using a patient's own internal cells for tissue repair. PMID:27537482

  2. Physiological, pathological, and engineered cell identity reprogramming in the central nervous system.

    Science.gov (United States)

    Smith, Derek K; Wang, Lei-Lei; Zhang, Chun-Li

    2016-07-01

    Multipotent neural stem cells persist in restricted regions of the adult mammalian central nervous system. These proliferative cells differentiate into diverse neuron subtypes to maintain neural homeostasis. This endogenous process can be reprogrammed as a compensatory response to physiological cues, traumatic injury, and neurodegeneration. In addition to innate neurogenesis, recent research has demonstrated that new neurons can be engineered via cell identity reprogramming in non-neurogenic regions of the adult central nervous system. A comprehensive understanding of these reprogramming mechanisms will be essential to the development of therapeutic neural regeneration strategies that aim to improve functional recovery after injury and neurodegeneration. WIREs Dev Biol 2016, 5:499-517. doi: 10.1002/wdev.234 For further resources related to this article, please visit the WIREs website. PMID:27258392

  3. Zscan4 promotes genomic stability during reprogramming and dramatically improves the quality of iPS cells as demonstrated by tetraploid complementation

    Institute of Scientific and Technical Information of China (English)

    Jing Jiang; Wenjian Lv; Xiaoying Ye; Lingbo Wang; Man Zhang; Hui Yang; Maja Okuka

    2013-01-01

    Induced pluripotent stem (iPS) cells generated using Yamanaka factors have great potential for use in autologous cell therapy.However,genomic abnormalities exist in human iPS cells,and most mouse iPS cells are not fully pluripotent,as evaluated by the tetraploid complementation assay (TCA); this is most likely associated with the DNA damage response (DDR) occurred in early reprogramming induced by Yamanaka factors.In contrast,nuclear transfer can faithfully reprogram somatic cells into embryonic stem (ES) cells that satisfy the TCA.We thus hypothesized that factors involved in oocyte-induced reprogramming may stabilize the somatic genome during reprogramming,and improve the quality of the resultant iPS cells.To test this hypothesis,we screened for factors that could decrease DDR signals during iPS cell induction.We determined that Zscan4,in combination with the Yamanaka factors,not only remarkably reduced the DDR but also markedly promoted the efficiency of iPS cell generation.The inclusion of Zscan4 stabilized the genomic DNA,resulting in p53 downregulation.Furthermore,Zscan4 also enhanced telomere lengthening as early as 3 days post-infection through a telomere recombination-based mechanism.As a result,iPS cells generated with addition of Zscan4 exhibited longer telomeres than classical iPS cells.Strikingly,more than 50%of iPS cell lines (11/19) produced via this "Zscan4 protocol" gave rise to live-borne all-iPS cell mice as determined by TCA,compared to 1/12 for lines produced using the classical Yamanaka factors.Our findings provide the first demonstration that maintaining genomic stability during reprogramming promotes the generation of high quality iPS cells.

  4. Effects of Collective Histone State Dynamics on Epigenetic Landscape and Kinetics of Cell Reprogramming

    Science.gov (United States)

    Ashwin, S. S.; Sasai, Masaki

    2015-11-01

    Cell reprogramming is a process of transitions from differentiated to pluripotent cell states via transient intermediate states. Within the epigenetic landscape framework, such a process is regarded as a sequence of transitions among basins on the landscape; therefore, theoretical construction of a model landscape which exhibits experimentally consistent dynamics can provide clues to understanding epigenetic mechanism of reprogramming. We propose a minimal gene-network model of the landscape, in which each gene is regulated by an integrated mechanism of transcription-factor binding/unbinding and the collective chemical modification of histones. We show that the slow collective variation of many histones around each gene locus alters topology of the landscape and significantly affects transition dynamics between basins. Differentiation and reprogramming follow different transition pathways on the calculated landscape, which should be verified experimentally via single-cell pursuit of the reprogramming process. Effects of modulation in collective histone state kinetics on transition dynamics and pathway are examined in search for an efficient protocol of reprogramming.

  5. 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.

  6. Make no bones about it: cells could soon be reprogrammed to grow replacement bones?

    Science.gov (United States)

    de Peppo, Giuseppe Maria; Marolt, Darja

    2014-01-01

    Recent developments in nuclear reprogramming allow the generation of patient-matched stem cells with broad potential for applications in cell therapies, disease modeling and drug discovery. An increasing body of work is reporting the derivation of lineage-specific progenitors from human-induced pluripotent stem cells (hiPSCs), which could in the near future be used to engineer personalized tissue substitutes, including those for reconstructive therapies of bone. Although the potential clinical impact of such technology is not arguable, significant challenges remain to be addressed before hiPSC-derived progenitors can be employed to engineer bone substitutes of clinical relevance. The most important challenge is indeed the construction of personalized multicellular bone substitutes for the treatment of complex skeletal defects that integrate fast, are immune tolerated and display biofunctionality and long-term safety. As recent studies suggest, the merging of iPSC technology with advanced biomaterials and bioreactor technologies offers a way to generate bone substitutes in a controllable, automated manner with potential to meet the needs for scale-up and requirements for translation into clinical practice. It is only via the use of state-of-the-art cell culture technologies, process automation under GMP-compliant conditions, application of appropriate engineering strategies and compliance with regulatory policies that personalized lab-made bone grafts can start being used to treat human patients. PMID:24053578

  7. 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.

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

    Science.gov (United States)

    Papadimou, Evangelia; Morigi, Marina; Iatropoulos, Paraskevas; Xinaris, Christodoulos; Tomasoni, Susanna; Benedetti, Valentina; Longaretti, Lorena; Rota, Cinzia; Todeschini, Marta; Rizzo, Paola; Introna, Martino; Grazia de Simoni, Maria; Remuzzi, Giuseppe; Goligorsky, Michael S.; Benigni, Ariela

    2015-01-01

    Summary 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. PMID:25754206

  9. Temporal Perturbation of the Wnt Signaling Pathway in the Control of Cell Reprogramming Is Modulated by TCF1

    Directory of Open Access Journals (Sweden)

    Francesco Aulicino

    2014-05-01

    Full Text Available Cyclic activation of the Wnt/β-catenin signaling pathway controls cell fusion-mediated somatic cell reprogramming. TCFs belong to a family of transcription factors that, in complex with β-catenin, bind and transcriptionally regulate Wnt target genes. Here, we show that Wnt/β-catenin signaling needs to be off during the early reprogramming phases of mouse embryonic fibroblasts (MEFs into iPSCs. In MEFs undergoing reprogramming, senescence genes are repressed and mesenchymal-to-epithelial transition is favored. This is correlated with a repressive activity of TCF1, which contributes to the silencing of Wnt/β-catenin signaling at the onset of reprogramming. In contrast, the Wnt pathway needs to be active in the late reprogramming phases to achieve successful reprogramming. In conclusion, continued activation or inhibition of the Wnt/β-catenin signaling pathway is detrimental to the reprogramming of MEFs; instead, temporal perturbation of the pathway is essential for efficient reprogramming, and the “Wnt-off” state can be considered an early reprogramming marker.

  10. Predicting pancreas cell fate decisions and reprogramming with a hierarchical multi-attractor model.

    Directory of Open Access Journals (Sweden)

    Joseph Xu Zhou

    Full Text Available Cell fate reprogramming, such as the generation of insulin-producing β cells from other pancreas cells, can be achieved by external modulation of key transcription factors. However, the known gene regulatory interactions that form a complex network with multiple feedback loops make it increasingly difficult to design the cell reprogramming scheme because the linear regulatory pathways as schemes of causal influences upon cell lineages are inadequate for predicting the effect of transcriptional perturbation. However, sufficient information on regulatory networks is usually not available for detailed formal models. Here we demonstrate that by using the qualitatively described regulatory interactions as the basis for a coarse-grained dynamical ODE (ordinary differential equation based model, it is possible to recapitulate the observed attractors of the exocrine and β, δ, α endocrine cells and to predict which gene perturbation can result in desired lineage reprogramming. Our model indicates that the constraints imposed by the incompletely elucidated regulatory network architecture suffice to build a predictive model for making informed decisions in choosing the set of transcription factors that need to be modulated for fate reprogramming.

  11. 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...

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

    International Nuclear Information System (INIS)

    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)

  13. Inducing pluripotency in vitro: recent advances and highlights in induced pluripotent stem cells generation and pluripotency reprogramming.

    Science.gov (United States)

    Rony, I K; Baten, A; Bloomfield, J A; Islam, M E; Billah, M M; Islam, K D

    2015-04-01

    Induced pluripotent stem cells (iPSCs) are considered patient-specific counterparts of embryonic stem cells as they originate from somatic cells after forced expression of pluripotency reprogramming factors Oct4, Sox2, Klf4 and c-Myc. iPSCs offer unprecedented opportunity for personalized cell therapies in regenerative medicine. In recent years, iPSC technology has undergone substantial improvement to overcome slow and inefficient reprogramming protocols, and to ensure clinical-grade iPSCs and their functional derivatives. Recent developments in iPSC technology include better reprogramming methods employing novel delivery systems such as non-integrating viral and non-viral vectors, and characterization of alternative reprogramming factors. Concurrently, small chemical molecules (inhibitors of specific signalling or epigenetic regulators) have become crucial to iPSC reprogramming; they have the ability to replace putative reprogramming factors and boost reprogramming processes. Moreover, common dietary supplements, such as vitamin C and antioxidants, when introduced into reprogramming media, have been found to improve genomic and epigenomic profiles of iPSCs. In this article, we review the most recent advances in the iPSC field and potent application of iPSCs, in terms of cell therapy and tissue engineering.

  14. Temporal repression of endogenous pluripotency genes during reprogramming of porcine induced pluripotent stem cells

    DEFF Research Database (Denmark)

    Hall, Vanessa Jane; Christensen, Marianne; Rasmussen, Mikkel Aabech;

    2012-01-01

    Porcine induced pluripotent stem cells (piPSCs) have the capacity to differentiate in vitro and in vivo and form chimeras. However, the lack of transgene silencing of exogenous DNA integrated into the genome and the inability of cells to proliferate in the absence of transgene expression are unde......Porcine induced pluripotent stem cells (piPSCs) have the capacity to differentiate in vitro and in vivo and form chimeras. However, the lack of transgene silencing of exogenous DNA integrated into the genome and the inability of cells to proliferate in the absence of transgene expression...... are underlying reported problems, suggesting that reprogramming is not complete. The aim of the present study was to evaluate reprogramming events using a partially reprogrammed piPSC-like line expressing hOCT4, hNANOG, and hcMYC under tetracycline-regulated control to investigate the effects of these particular....... Despite the ability for some endogenous genes to be expressed in these lines, the piPSC-like cells still cannot be maintained without doxycycline, indicating that the culture system of piPSCs may not be optimal or that the reprogramming factor combination used may not currently be optimal for maintaining...

  15. 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.

  16. The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming

    Institute of Scientific and Technical Information of China (English)

    Athanasia D Panopoulos; Margaret Lutz; W Travis Berggren; Kun Zhang; Ronald M Evans; Gary Siuzdak; Juan Carlos Izpisua Belmonte; Oscar Yanes; SergioRuiz; Yasuyuki S Kida; Dinh Diep; Ralf Tautenhahn; Aida Herrerias; Erika M Batchelder; Nongluk Plongthongkum

    2012-01-01

    Metabolism is vital to every aspect of cell function,yet the metabolome of induced pluripotent stem cells (iPSCs)remains largely unexplored.Here we report,using an untargeted metabolomics approach,that human iPSCs share a pluripotent metabolomic signature with embryonic stem cells (ESCs) that is distinct from their parental cells,and that is characterized by changes in metabolites involved in cellular respiration.Examination of cellular bioenergetics corroborated with our metabolomic analysis,and demonstrated that somatic cells convert from an oxidative state to a glycolytic state in pluripotency.Interestingly,the bioenergetics of various somatic cells correlated with their reprogramming efficiencies.We further identified metabolites that differ between iPSCs and ESCs,which revealed novel metabolic pathways that play a critical role in regulating somatic cell reprogramming.Our findings are the first to globally analyze the metabolome of iPSCs,and provide mechanistic insight into a new layer of regulation involved in inducing pluripotency,and in evaluating iPSC and ESC equivalence.

  17. JNK/SAPK Signaling Is Essential for Efficient Reprogramming of Human Fibroblasts to Induced Pluripotent Stem Cells

    Science.gov (United States)

    Neganova, Irina; Shmeleva, Evgenija; Munkley, Jennifer; Chichagova, Valeria; Anyfantis, George; Anderson, Rhys; Passos, Joao; Elliott, David J.; Armstrong, Lyle

    2016-01-01

    Abstract Reprogramming of somatic cells to the phenotypic state termed “induced pluripotency” is thought to occur through three consecutive stages: initiation, maturation, and stabilisation. The initiation phase is stochastic but nevertheless very important as it sets the gene expression pattern that permits completion of reprogramming; hence a better understanding of this phase and how this is regulated may provide the molecular cues for improving the reprogramming process. c‐Jun N‐terminal kinase (JNK)/stress‐activated protein kinase (SAPKs) are stress activated MAPK kinases that play an essential role in several processes known to be important for successful completion of the initiation phase such as cellular proliferation, mesenchymal to epithelial transition (MET) and cell cycle regulation. In view of this, we postulated that manipulation of this pathway would have significant impacts on reprogramming of human fibroblasts to induced pluripotent stem cells. Accordingly, we found that key components of the JNK/SAPK signaling pathway increase expression as early as day 3 of the reprogramming process and continue to rise in reprogrammed cells throughout the initiation and maturation stages. Using both chemical inhibitors and RNA interference of MKK4, MKK7 and JNK1, we tested the role of JNK/SAPK signaling during the initiation stage of neonatal and adult fibroblast reprogramming. These resulted in complete abrogation of fully reprogrammed colonies and the emergence of partially reprogrammed colonies which disaggregated and were lost from culture during the maturation stage. Inhibition of JNK/SAPK signaling resulted in reduced cell proliferation, disruption of MET and loss of the pluripotent phenotype, which either singly or in combination prevented establishment of pluripotent colonies. Together these data provide new evidence for an indispensable role for JNK/SAPK signaling to overcome the well‐established molecular barriers in human somatic cell

  18. Ordered chromatin changes and human X chromosome reactivation by cell fusion-mediated pluripotent reprogramming

    Science.gov (United States)

    Cantone, Irene; Bagci, Hakan; Dormann, Dirk; Dharmalingam, Gopuraja; Nesterova, Tatyana; Brockdorff, Neil; Rougeulle, Claire; Vallot, Celine; Heard, Edith; Chaligne, Ronan; Merkenschlager, Matthias; Fisher, Amanda G.

    2016-01-01

    Erasure of epigenetic memory is required to convert somatic cells towards pluripotency. Reactivation of the inactive X chromosome (Xi) has been used to model epigenetic reprogramming in mouse, but human studies are hampered by Xi epigenetic instability and difficulties in tracking partially reprogrammed iPSCs. Here we use cell fusion to examine the earliest events in the reprogramming-induced Xi reactivation of human female fibroblasts. We show that a rapid and widespread loss of Xi-associated H3K27me3 and XIST occurs in fused cells and precedes the bi-allelic expression of selected Xi-genes by many heterokaryons (30–50%). After cell division, RNA-FISH and RNA-seq analyses confirm that Xi reactivation remains partial and that induction of human pluripotency-specific XACT transcripts is rare (1%). These data effectively separate pre- and post-mitotic events in reprogramming-induced Xi reactivation and reveal a complex hierarchy of epigenetic changes that are required to reactivate the genes on the human Xi chromosome. PMID:27507283

  19. Experimental Advances Towards Neural Regeneration from Induced Stem Cells to Direct In Vivo Reprogramming.

    Science.gov (United States)

    Dametti, Sara; Faravelli, Irene; Ruggieri, Margherita; Ramirez, Agnese; Nizzardo, Monica; Corti, Stefania

    2016-05-01

    Neuronal loss is a common substrate of many neurological diseases that still lack effective treatments and highly burden lives of affected individuals. The discovery of self-renewing stem cells within the central nervous system (CNS) has opened the doors to the possibility of using the plasticity of CNS as a potential strategy for the development of regenerative therapies after injuries. The role of neural progenitor cells appears to be crucial, but insufficient in reparative processes after damage. In addition, the mechanisms that regulate these events are still largely unknown. Stem cell-based therapeutic approaches have primarily focused on the use of either induced pluripotent stem cells or induced neural stem cells as sources for cell transplantation. More recently, in vivo direct reprogramming of endogenous CNS cells into multipotent neural stem/progenitor cells has been proposed as an alternative strategy that could overcome the limits connected with both the invasiveness of exogenous cell transplantation and the technical issues of in vitro reprogramming (i.e., the time requested and the limited available amount of directly induced neuronal cells). In this review, we aim to highlight the recent studies on in vivo direct reprogramming, focusing on astrocytes conversion to neurons or to neural stem/precursors cells, in the perspective of future therapeutic purposes for neurological disorders.

  20. 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.

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

    Science.gov (United States)

    Zhao, Chunli; Farruggio, Alfonso P; Bjornson, Christopher R R; Chavez, Christopher L; Geisinger, Jonathan M; Neal, Tawny L; Karow, Marisa; Calos, Michele P

    2014-01-01

    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. PMID:24781921

  2. 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.

  3. 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. PMID:24086768

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

    Directory of Open Access Journals (Sweden)

    Catherine D McCusker

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

  5. 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.

  6. A blueprint for engineering cell fate: current technologies to reprogram cell identity

    Institute of Scientific and Technical Information of China (English)

    Samantha A Morris; George Q Daley

    2013-01-01

    Human diseases such as heart failure,diabetes,neurodegenerative disorders,and many others result from the deficiency or dysfunction of critical cell types.Strategies for therapeutic tissue repair or regeneration require the in vitro manufacture of clinically relevant quantities of defined cell types.In addition to transplantation therapy,the generation of otherwise inaccessible cells also permits disease modeling,toxicology testing and drug discovery in vitro.In this review,we discuss current strategies to manipulate the identity of abundant and accessible cells by differentiation from an induced pluripotent state or direct conversion between differentiated states.We contrast these approaches with recent advances employing partial reprogramming to facilitate lineage switching,and discuss the mechanisms underlying the engineering of cell fate.Finally,we address the current limitations of the field and how the resulting cell types can be assessed to ensure the production of medically relevant populations.

  7. 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.

  8. Bmi1 reprograms CML B-lymphoid progenitors to become B-ALL–initiating cells

    OpenAIRE

    Sengupta, Amitava; Ficker, Ashley M.; Dunn, Susan K.; Madhu, Malav; Cancelas, Jose A.

    2012-01-01

    The characterization and targeting of Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL)–initiating cells remains unresolved. Expression of the polycomb protein Bmi1 is up-regulated in patients with advanced stages of chronic myelogenous leukemia (CML). We report that Bmi1 transforms and reprograms CML B-lymphoid progenitors into stem cell leukemia (Scl) promoter-driven, self-renewing, leukemia-initiating cells to result in B-lymphoid leukemia (B-ALL) in vivo. In vitro,...

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

    Science.gov (United States)

    Hermann, Andreas; Kim, Jeong Beom; Srimasorn, Sumitra; Zaehres, Holm; Reinhardt, Peter; Schöler, Hans R; Storch, Alexander

    2016-01-01

    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.

  10. 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.

  11. Targeted alternative splicing of TAF4: a new strategy for cell reprogramming.

    Science.gov (United States)

    Kazantseva, Jekaterina; Sadam, Helle; Neuman, Toomas; Palm, Kaia

    2016-01-01

    Reprogramming of somatic cells has become a versatile tool for biomedical research and for regenerative medicine. In the current study, we show that manipulating alternative splicing (AS) is a highly potent strategy to produce cells for therapeutic applications. We demonstrate that silencing of hTAF4-TAFH activity of TAF4 converts human facial dermal fibroblasts to melanocyte-like (iMel) cells. iMel cells produce melanin and express microphthalmia-associated transcription factor (MITF) and its target genes at levels comparable to normal melanocytes. Reprogramming of melanoma cells by manipulation with hTAF4-TAFH activity upon TAFH RNAi enforces cell differentiation towards chondrogenic pathway, whereas ectoptic expression of TAF4 results in enhanced multipotency and neural crest-like features in melanoma cells. In both cell states, iMels and cancer cells, hTAF4-TAFH activity controls migration by supporting E- to N-cadherin switches. From our data, we conclude that targeted splicing of hTAF4-TAFH coordinates AS of other TFIID subunits, underscoring the role of TAF4 in synchronised changes of Pol II complex composition essential for efficient cellular reprogramming. Taken together, targeted AS of TAF4 provides a unique strategy for generation of iMels and recapitulating stages of melanoma progression. PMID:27499390

  12. Targeted alternative splicing of TAF4: a new strategy for cell reprogramming

    Science.gov (United States)

    Kazantseva, Jekaterina; Sadam, Helle; Neuman, Toomas; Palm, Kaia

    2016-01-01

    Reprogramming of somatic cells has become a versatile tool for biomedical research and for regenerative medicine. In the current study, we show that manipulating alternative splicing (AS) is a highly potent strategy to produce cells for therapeutic applications. We demonstrate that silencing of hTAF4-TAFH activity of TAF4 converts human facial dermal fibroblasts to melanocyte-like (iMel) cells. iMel cells produce melanin and express microphthalmia-associated transcription factor (MITF) and its target genes at levels comparable to normal melanocytes. Reprogramming of melanoma cells by manipulation with hTAF4-TAFH activity upon TAFH RNAi enforces cell differentiation towards chondrogenic pathway, whereas ectoptic expression of TAF4 results in enhanced multipotency and neural crest-like features in melanoma cells. In both cell states, iMels and cancer cells, hTAF4-TAFH activity controls migration by supporting E- to N-cadherin switches. From our data, we conclude that targeted splicing of hTAF4-TAFH coordinates AS of other TFIID subunits, underscoring the role of TAF4 in synchronised changes of Pol II complex composition essential for efficient cellular reprogramming. Taken together, targeted AS of TAF4 provides a unique strategy for generation of iMels and recapitulating stages of melanoma progression. PMID:27499390

  13. Reprogramming resistant genes: in-depth comparison of gene expressions among iPS, ES and somatic cells.

    Directory of Open Access Journals (Sweden)

    Natalia ePolouliakh

    2013-01-01

    Full Text Available Transcription factor based reprogramming reverts adult cells to an embryonic state, yielding potential for generating different tissue types. However, recent reports indicated the substantial differences in pattern of gene expression between induced pluripotent stem (iPS cells and embryonic stem (ES cells. In this study we compare gene expression signatures of different iPS and ES cell lines and relate expression profiles of differently expressed genes to their expression status in somatic cells. As a result, we discovered that genes resistant to reprogramming comprise two major clusters, which are reprogramming dependent ‘Induced Genes’ and somatic origin ‘Inherited Genes’, both exhibiting preferences in methylation marks. Closer look into the Induced Genes by means of the transcription regulation analysis predicted several groups of genes with various roles in reprogramming and transgene DNA binding model. We believe that our results are a helpful source for biologists for further improvement of iPS cell technology.

  14. HIV infection of naturally occurring and genetically reprogrammed human regulatory T-cells.

    Directory of Open Access Journals (Sweden)

    Kyra Oswald-Richter

    2004-07-01

    Full Text Available A T-cell subset, defined as CD4(+CD25(hi (regulatory T-cells [Treg cells], was recently shown to suppress T-cell activation. We demonstrate that human Treg cells isolated from healthy donors express the HIV-coreceptor CCR5 and are highly susceptible to HIV infection and replication. Because Treg cells are present in very few numbers and are difficult to expand in vitro, we genetically modified conventional human T-cells to generate Treg cells in vitro by ectopic expression of FoxP3, a transcription factor associated with reprogramming T-cells into a Treg subset. Overexpression of FoxP3 in naïve human CD4(+ T-cells recapitulated the hyporesponsiveness and suppressive function of naturally occurring Treg cells. However, FoxP3 was less efficient in reprogramming memory T-cell subset into regulatory cells. In addition, FoxP3-transduced T-cells also became more susceptible to HIV infection. Remarkably, a portion of HIV-positive individuals with a low percentage of CD4(+ and higher levels of activated T-cells have greatly reduced levels of FoxP3(+CD4(+CD25(hi T-cells, suggesting disruption of the Treg cells during HIV infection. Targeting and disruption of the T-cell regulatory system by HIV may contribute to hyperactivation of conventional T-cells, a characteristic of HIV disease progression. Moreover, the ability to reprogram human T-cells into Treg cells in vitro will greatly aid in decoding their mechanism of suppression, their enhanced susceptibility to HIV infection, and the unique markers expressed by this subset.

  15. microRNA-29b is a novel mediator of Sox2 function in the regulation of somatic cell reprogramming

    Institute of Scientific and Technical Information of China (English)

    Xudong Guo; Qidong Liu; Guiying Wang; Songcheng Zhu; Longfei Gao; Wujun Hong; Yafang Chen

    2013-01-01

    Fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSCs) by the application of Yamanaka factors (OSKM),but the mechanisms underlying this reprogramming remain poorly understood.Here,we report that Sox2 directly regulates endogenous microRNA-29b (miR-29b) expression during iPSC generation and that miR-29b expression is required for OSKM-and OSK-mediated reprogramming.Mechanistic studies show that Dnmt3a and Dnmt3b are in vivo targets of miR-29b and that Dnmt3a and Dnmt3b expression is inversely correlated with miR-29b expression during reprogramming.Moreover,the effect of miR-29b on reprogramming can be blocked by Dnmt3a or Dnmt3b overexpression.Further experiments indicate that miR-29b-DNMT signaling is significantly involved in the regulation of DNA methylation-related reprogramming events,such as mesenchymal-to-epithelial transition (MET)and Dlk1-Dio3 region transcription.Thus,our studies not only reveal that miR-29b is a novel mediator of reprogramming factor Sox2 but also provide evidence for a muitistep mechanism in which Sox2 drives a miR-29b-DNMT signaling axis that regulates DNA methylation-related events during reprogramming.

  16. 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.

    2015-01-01

    Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors1,2. 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 lineage differentiation3–6. 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 different methods. PMID:26098448

  17. In vivo reprogrammed pluripotent stem cells from teratomas share analogous properties with their in vitro counterparts

    OpenAIRE

    Hyun Woo Choi; Jong Soo Kim; Yean Ju Hong; Hyuk Song; Han Geuk Seo; Jeong Tae Do

    2015-01-01

    Recently, induced pluripotent stem cells (iPSCs) have been generated in vivo from reprogrammable mice. These in vivo iPSCs display features of totipotency, i.e., they differentiate into the trophoblast lineage, as well as all 3 germ layers. Here, we developed a new reprogrammable mouse model carrying an Oct4-GFP reporter gene to facilitate the detection of reprogrammed pluripotent stem cells. Without doxycycline administration, some of the reprogrammable mice developed aggressively growing te...

  18. Turning the fate of reprogramming cells from retinal disorder to regeneration by Pax6 in newts.

    Science.gov (United States)

    Casco-Robles, Martin Miguel; Islam, Md Rafiqul; Inami, Wataru; Tanaka, Hibiki Vincent; Kunahong, Ailidana; Yasumuro, Hirofumi; Hanzawa, Shiori; Casco-Robles, Roman Martin; Toyama, Fubito; Maruo, Fumiaki; Chiba, Chikafumi

    2016-01-01

    The newt, a urodele amphibian, has an outstanding ability- even as an adult -to regenerate a functional retina through reprogramming and proliferation of the retinal pigment epithelium (RPE) cells, even though the neural retina is completely removed from the eye by surgery. It remains unknown how the newt invented such a superior mechanism. Here we show that disability of RPE cells to regenerate the retina brings about a symptom of proliferative vitreoretinopathy (PVR), even in the newt. When Pax6, a transcription factor that is re-expressed in reprogramming RPE cells, is knocked down in transgenic juvenile newts, these cells proliferate but eventually give rise to cell aggregates that uniformly express alpha smooth muscle actin, Vimentin and N-cadherin, the markers of myofibroblasts which are a major component of the sub-/epi-retinal membranes in PVR. Our current study demonstrates that Pax6 is an essential factor that directs the fate of reprogramming RPE cells toward the retinal regeneration. The newt may have evolved the ability of retinal regeneration by modifying a mechanism that underlies the RPE-mediated retinal disorders. PMID:27640672

  19. Reprogramming of adult human neural stem cells into induced pluripotent stem cells

    Institute of Scientific and Technical Information of China (English)

    XIE Li-qian; SUN Hua-ping; WANG Tian; TANG Hai-liang; WANG Pu; ZHU Jian-hong; YAO Zheng-wei

    2013-01-01

    Background Since an effective method for generating induced pluripotent stem cells (iPSCs) from human neural stem cells (hNSCs) can offer us a promising tool for studying brain diseases,here we reported direct reprogramming of adult hNSCs into iPSCs by retroviral transduction of four defined factors.Methods NSCs were successfully isolated and cultured from the hippocampus tissue of epilepsy patients.When combined with four factors (OCT3/4,SOX2,KLF4,and c-MYC),iPSCs colonies were successfully obtained.Results Morphological characterization and specific genetic expression confirmed that these hNSCs-derived iPSCs showed embryonic stem cells-like properties,which include the ability to differentiate into all three germ layers both in vitro and in vivo.Conclusion Our method would be useful for generating human iPSCs from NSCs and provide an important tool for studying neurological diseases.

  20. A CRISPR/Cas9-Based System for Reprogramming Cell Lineage Specification

    Directory of Open Access Journals (Sweden)

    Syandan Chakraborty

    2014-12-01

    Full Text Available Gene activation by the CRISPR/Cas9 system has the potential to enable new approaches to science and medicine, but the technology must be enhanced to robustly control cell behavior. We show that the fusion of two transactivation domains to Cas9 dramatically enhances gene activation to a level that is necessary to reprogram cell phenotype. Targeted activation of the endogenous Myod1 gene locus with this system led to stable and sustained reprogramming of mouse embryonic fibroblasts into skeletal myocytes. The levels of myogenic marker expression obtained by the activation of endogenous Myod1 gene were comparable to that achieved by overexpression of lentivirally delivered MYOD1 transcription factor.

  1. Epiblastin A Induces Reprogramming of Epiblast Stem Cells Into Embryonic Stem Cells by Inhibition of Casein Kinase 1.

    Science.gov (United States)

    Ursu, Andrei; Illich, Damir J; Takemoto, Yasushi; Porfetye, Arthur T; Zhang, Miao; Brockmeyer, Andreas; Janning, Petra; Watanabe, Nobumoto; Osada, Hiroyuki; Vetter, Ingrid R; Ziegler, Slava; Schöler, Hans R; Waldmann, Herbert

    2016-04-21

    The discovery of novel small molecules that induce stem cell reprogramming and give efficient access to pluripotent stem cells is of major importance for potential therapeutic applications and may reveal novel insights into the factors controlling pluripotency. Chemical reprogramming of mouse epiblast stem cells (EpiSCs) into cells corresponding to embryonic stem cells (cESCs) is an inefficient process. In order to identify small molecules that promote this cellular transition, we analyzed the LOPAC library in a phenotypic screen monitoring Oct4-GFP expression and identified triamterene (TR) as initial hit. Synthesis of a TR-derived compound collection and investigation for reprogramming of EpiSCs into cESCs identified casein kinases 1 (CK1) α/δ/ɛ as responsible cellular targets of TR and unraveled the structural parameters that determine reprogramming. Delineation of a structure-activity relationship led to the development of Epiblastin A, which engages CK1 isoenzymes in cell lysate and induces efficient conversion of EpiSCs into cESCs. PMID:27049670

  2. Single-Cell XIST Expression in Human Preimplantation Embryos and Newly Reprogrammed Female Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Briggs, Sharon F; Dominguez, Antonia A; Chavez, Shawn L; Reijo Pera, Renee A

    2015-06-01

    The process of X chromosome inactivation (XCI) during reprogramming to produce human induced pluripotent stem cells (iPSCs), as well as during the extensive programming that occurs in human preimplantation development, is not well-understood. Indeed, studies of XCI during reprogramming to iPSCs report cells with two active X chromosomes and/or cells with one inactive X chromosome. Here, we examine expression of the long noncoding RNA, XIST, in single cells of human embryos through the oocyte-to-embryo transition and in new mRNA reprogrammed iPSCs. We show that XIST is first expressed beginning at the 4-cell stage, coincident with the onset of embryonic genome activation in an asynchronous manner. Additionally, we report that mRNA reprogramming produces iPSCs that initially express XIST transcript; however, expression is rapidly lost with culture. Loss of XIST and H3K27me3 enrichment at the inactive X chromosome at late passage results in X chromosome expression changes. Our data may contribute to applications in disease modeling and potential translational applications of female stem cells.

  3. Embryonic hybrid cells: a powerful tool for studying pluripotency and reprogramming of the differentiated cell chromosomes

    Directory of Open Access Journals (Sweden)

    SEROV OLEG

    2001-01-01

    Full Text Available The properties of embryonic hybrid cells obtained by fusion of embryonic stem (ES or teratocarcinoma (TC cells with differentiated cells are reviewed. Usually, ES-somatic or TC-somatic hybrids retain pluripotent capacity at high levels quite comparable or nearly identical with those of the pluripotent partner. When cultured in vitro, ES-somatic- and TC-somatic hybrid cell clones, as a rule, lose the chromosomes derived from the somatic partner; however, in some clones the autosomes from the ES cell partner were also eliminated, i.e. the parental chromosomes segregated bilaterally in the ES-somatic cell hybrids. This opens up ways for searching correlation between the pluripotent status of the hybrid cells and chromosome segregation patterns and therefore for identifying the particular chromosomes involved in the maintenance of pluripotency. Use of selective medium allows to isolate in vitro the clones of ES-somatic hybrid cells in which "the pluripotent" chromosome can be replaced by "the somatic" counterpart carrying the selectable gene. Unlike the TC-somatic cell hybrids, the ES-somatic hybrids with a near-diploid complement of chromosomes are able to contribute to various tissues of chimeric animals after injection into the blastocoel cavity. Analysis of the chimeric animals showed that the "somatic" chromosome undergoes reprogramming during development. The prospects for the identification of the chromosomes that are involved in the maintenance of pluripotency and its cis- and trans-regulation in the hybrid cell genome are discussed.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. 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.

  6. Regulatory T cell reprogramming toward a Th2-cell-like lineage impairs oral tolerance and promotes food allergy.

    Science.gov (United States)

    Noval Rivas, Magali; Burton, Oliver T; Wise, Petra; Charbonnier, Louis-Marie; Georgiev, Peter; Oettgen, Hans C; Rachid, Rima; Chatila, Talal A

    2015-03-17

    Oral immunotherapy has had limited success in establishing tolerance in food allergy, reflecting failure to elicit an effective regulatory T (Treg) cell response. We show that disease-susceptible (Il4ra(F709)) mice with enhanced interleukin-4 receptor (IL-4R) signaling exhibited STAT6-dependent impaired generation and function of mucosal allergen-specific Treg cells. This failure was associated with the acquisition by Treg cells of a T helper 2 (Th2)-cell-like phenotype, also found in peripheral-blood allergen-specific Treg cells of food-allergic children. Selective augmentation of IL-4R signaling in Treg cells induced their reprogramming into Th2-like cells and disease susceptibility, whereas Treg-cell-lineage-specific deletion of Il4 and Il13 was protective. IL-4R signaling impaired the capacity of Treg cells to suppress mast cell activation and expansion, which in turn drove Th2 cell reprogramming of Treg cells. Interruption of Th2 cell reprogramming of Treg cells might thus provide candidate therapeutic strategies in food allergy. PMID:25769611

  7. Non-genetic direct reprogramming and biomimetic platforms in a preliminary study for adipose-derived stem cells into corneal endothelia-like cells.

    Directory of Open Access Journals (Sweden)

    Ying Dai

    Full Text Available Cell fate and function can be regulated and reprogrammed by intrinsic genetic program, extrinsic factors and niche microenvironment. Direct reprogramming has shown many advantages in the field of cellular reprogramming. Here we tried the possibility to generate corneal endothelia (CE -like cells from human adipose-derived stem cells (ADSCs by the non-genetic direct reprogramming of recombinant cell-penetrating proteins Oct4/Klf4/Sox2 (PTD-OKS and small molecules (purmorphamine, RG108 and other reprogramming chemical reagents, as well as biomimetic platforms of simulate microgravity (SMG bioreactor. Co-cultured with corneal cells and decellularized corneal ECM, Reprogrammed ADSCs revealed spherical growth and positively expressing Nanog for RT-PCR analysis and CD34 for immunofluorescence staining after 7 days-treatment of both purmorphamine and PTD-OKS (P-OKS and in SMG culture. ADSCs changed to CEC polygonal morphology from spindle shape after the sequential non-genetic direct reprogramming and biomimetic platforms. At the same time, induced cells converted to weakly express CD31, AQP-1 and ZO-1. These findings demonstrated that the treatments were able to promote the stem-cell reprogramming for human ADSCs. Our study also indicates for the first time that SMG rotary cell culture system can be used as a non-genetic means to promote direct reprogramming. Our methods of reprogramming provide an alternative strategy for engineering patient-specific multipotent cells for cellular plasticity research and future autologous CEC replacement therapy that avoids complications associated with the use of human pluripotent stem cells.

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

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    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.

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

    Science.gov (United States)

    Trokovic, Ras; Weltner, Jere; Noisa, Parinya; Raivio, Taneli; Otonkoski, Timo

    2015-07-01

    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.

  10. Direct reprogramming of Sertoli cells into multipotent neural stem cells by defined factors

    Institute of Scientific and Technical Information of China (English)

    Chao Sheng; Ziwei Wang; Changlong Guo; Hua-Jun Wu; Zhonghua Liu; Liu Wang; Shigang He; Xiu-Jie Wang; Zhiguo Chen; Qi Zhou; Qinyuan Zheng; Jianyu Wu; Zhen Xu; Libin Wang; Wei Li; Haijiang Zhang; Xiao-YangZhao; Lei Liu

    2012-01-01

    Multipotent neural stem/progenitor cells hold great promise for cell therapy.The reprogramming of fibroblasts to induced pluripotent stem cells as well as mature neurons suggests a possibility to convert a terminally differentiated somatic cell into a muitipotent state without first establishing pluripotency.Here,we demonstrate that sertoli cells derived from mesoderm can be directly converted into a multipotent state that possesses neural stem/progenitor cell properties.The induced neural stem/progenitor cells (iNSCs) express multiple NSC-specific markers,exhibit a global gene-expression profile similar to normal NSCs,and are capable of self-renewal and differentiating into glia and electrophysiologically functional neurons,iNSC-derived neurons stain positive for tyrosine hydroxylase (TH),γ-aminobutyric acid,and choline acetyltransferase.In addition,iNSCs can survive and generate synapses following transplantation into the dentate gyrus.Generation of iNSCs may have important implications for disease modeling and regenerative medicine.

  11. The role of p53 in limiting somatic cell reprogramming

    Institute of Scientific and Technical Information of China (English)

    Yan Liu; Ruben Hoya-Arias; Stephen D Nimer

    2009-01-01

    @@ The first successful generation of induced pluripotent stem(iPS)cells from somatic cells was accomplished by introducing four genes into the cell,0ct3/4,Sox2,Klf4,and c-myc [1].While a tour-de-force,this approach to iPS cell generation is inefficient,and unlikely to be directly translated into therapeutic use since it involves the use of retroviruses to introduce these genes into the cell.Subsequent studies have used non-integrating genetic elements,chemical compounds,or proteins rather than DNA to bypass concerns about retroviral insertional mutagenesis [2-5].

  12. Re-programming tumour cell metabolism to treat cancer: no lone target for lonidamine

    Science.gov (United States)

    Bhutia, Yangzom D.; Babu, Ellappan; Ganapathy, Vadivel

    2016-01-01

    Tumour cell metabolism is very different from normal cell metabolism; cancer cells re-programme the metabolic pathways that occur in normal cells in such a manner that it optimizes their proliferation, growth and survival. Although this metabolic re-programming obviously operates to the advantage of the tumour, it also offers unique opportunities for effective cancer therapy. Molecules that target the tumour cell-specific metabolic pathways have potential as novel anti-cancer drugs. Lonidamine belongs to this group of molecules and is already in use in some countries for cancer treatment. It has been known for a long time that lonidamine interferes with energy production in tumour cells by inhibiting hexokinase II (HKII), a glycolytic enzyme. However, subsequent studies have uncovered additional pharmacological targets for the drug, which include the electron transport chain and the mitochondrial permeability transition pore, thus expanding the pharmacological effects of the drug on tumour cell metabolism. A study by Nancolas et al. in a recent issue of the Biochemical Journal identifies two additional new targets for lonidamine: the pyruvate transporter in the mitochondria and the H+-coupled monocarboxylate transporters in the plasma membrane (PM). It is thus becoming increasingly apparent that the anti-cancer effects of lonidamine do not occur through a single target; the drug works at multiple sites. Irrespective of the molecular targets, what lonidamine does in the end is to undo what the tumour cells have done in terms of re-programming cellular metabolism and mitochondrial function. PMID:27234586

  13. The effect of immunosuppressive molecules on T-cell metabolic reprogramming.

    Science.gov (United States)

    Fernández-Ramos, Ana A; Poindessous, Virginie; Marchetti-Laurent, Catherine; Pallet, Nicolas; Loriot, Marie-Anne

    2016-08-01

    T lymphocytes undergo metabolic reprogramming to adapt to extracellular and intracellular cues. Specifically, T-cell metabolism results into ATP production, anabolism and catabolism pathways that not only support rapid cell growth and proliferation, but also differentiation and effector functions, recently referred as "immunometabolism". Quiescent naïve T cells rely on oxidative phosphorylation whereas aerobic glycolysis (Warburg effect) occurs in activated T cells (effector CD4(+) and CD8(+)). The molecular mechanisms that sense metabolic status and influence T-cell function require metabolic checkpoints including sensors of metabolic signals and transducers (Myc, HIF-1α, AMPK and mTOR). These metabolic checkpoints represent a novel therapeutic strategy for immune modulation. Interestingly, many immunosuppressive drugs including mTOR inhibitors (rapamycin), calcineurin inhibitors (tacrolimus, cyclosporine A) and inhibitors of de novo purine synthesis (6-mercaptopurine, mycophenolic acid and methotrexate) provide examples into how modulating these metabolic checkpoints can regulate T-cell activation, differentiation and function. In this Review we highlight emerging concepts about metabolic reprogramming in T-cell responses and we discuss the potential therapeutic interventions to influence T-cell fate and effector function.

  14. Induced Pluripotent Stem Cells: Generation Strategy and Epigenetic Mystery behind Reprogramming

    Directory of Open Access Journals (Sweden)

    Pengfei Ji

    2016-01-01

    Full Text Available Possessing the ability of self-renewal with immortalization and potential for differentiation into different cell types, stem cells, particularly embryonic stem cells (ESC, have attracted significant attention since their discovery. As ESC research has played an essential role in developing our understanding of the mechanisms underlying reproduction, development, and cell (dedifferentiation, significant efforts have been made in the biomedical study of ESC in recent decades. However, such studies of ESC have been hampered by the ethical issues and technological challenges surrounding them, therefore dramatically inhibiting the potential applications of ESC in basic biomedical studies and clinical medicine. Induced pluripotent stem cells (iPSCs, generated from the reprogrammed somatic cells, share similar characteristics including but not limited to the morphology and growth of ESC, self-renewal, and potential differentiation into various cell types. The discovery of the iPSC, unhindered by the aforementioned limitations of ESC, introduces a viable alternative to ESC. More importantly, the applications of iPSC in the development of disease models such as neurodegenerative disorders greatly enhance our understanding of the pathogenesis of such diseases and also facilitate the development of clinical therapeutic strategies using iPSC generated from patient somatic cells to avoid an immune rejection. In this review, we highlight the advances in iPSCs generation methods as well as the mechanisms behind their reprogramming. We also discuss future perspectives for the development of iPSC generation methods with higher efficiency and safety.

  15. Induced Pluripotent Stem Cells: Generation Strategy and Epigenetic Mystery behind Reprogramming.

    Science.gov (United States)

    Ji, Pengfei; Manupipatpong, Sasicha; Xie, Nina; Li, Yujing

    2016-01-01

    Possessing the ability of self-renewal with immortalization and potential for differentiation into different cell types, stem cells, particularly embryonic stem cells (ESC), have attracted significant attention since their discovery. As ESC research has played an essential role in developing our understanding of the mechanisms underlying reproduction, development, and cell (de)differentiation, significant efforts have been made in the biomedical study of ESC in recent decades. However, such studies of ESC have been hampered by the ethical issues and technological challenges surrounding them, therefore dramatically inhibiting the potential applications of ESC in basic biomedical studies and clinical medicine. Induced pluripotent stem cells (iPSCs), generated from the reprogrammed somatic cells, share similar characteristics including but not limited to the morphology and growth of ESC, self-renewal, and potential differentiation into various cell types. The discovery of the iPSC, unhindered by the aforementioned limitations of ESC, introduces a viable alternative to ESC. More importantly, the applications of iPSC in the development of disease models such as neurodegenerative disorders greatly enhance our understanding of the pathogenesis of such diseases and also facilitate the development of clinical therapeutic strategies using iPSC generated from patient somatic cells to avoid an immune rejection. In this review, we highlight the advances in iPSCs generation methods as well as the mechanisms behind their reprogramming. We also discuss future perspectives for the development of iPSC generation methods with higher efficiency and safety.

  16. Mouse cloning and somatic cell reprogramming using electrofused blastomeres

    OpenAIRE

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

    2010-01-01

    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 s...

  17. An RNA editing fingerprint of cancer stem cell reprogramming

    OpenAIRE

    Crews, Leslie A; Jiang, Qingfei; Zipeto, Maria A; de Lazzari, Elisa; Court, Angela C.; Ali, Shawn; Barrett, Christian L.; Frazer, Kelly A; Jamieson, Catriona HM

    2015-01-01

    Background Deregulation of RNA editing by adenosine deaminases acting on dsRNA (ADARs) has been implicated in the progression of diverse human cancers including hematopoietic malignancies such as chronic myeloid leukemia (CML). Inflammation-associated activation of ADAR1 occurs in leukemia stem cells specifically in the advanced, often drug-resistant stage of CML known as blast crisis. However, detection of cancer stem cell-associated RNA editing by RNA sequencing in these rare cell populatio...

  18. Ustilago maydis reprograms cell proliferation in maize anthers.

    Science.gov (United States)

    Gao, Li; Kelliher, Timothy; Nguyen, Linda; Walbot, Virginia

    2013-09-01

    The basidiomycete Ustilago maydis is a ubiquitous pathogen of maize (Zea mays), one of the world's most important cereal crops. Infection by this smut fungus triggers tumor formation in aerial plant parts within which the fungus sporulates. Using confocal microscopy to track U. maydis infection on corn anthers for 7 days post-injection, we found that U. maydis is located on the epidermis during the first 2 days, and has reached all anther lobe cell types by 3 days post-injection. Fungal infection alters cell-fate specification events, cell division patterns, host cell expansion and host cell senescence, depending on the developmental stage and cell type. Fungal effects on tassel and plant growth were also quantified. Transcriptome profiling using a dual organism microarray identified thousands of anther genes affected by fungal infection at 3 days post-injection during the cell-fate specification and rapid cell proliferation phases of anther development. In total, 4147 (17%) of anther-expressed genes were altered by infection, 2018 fungal genes were expressed in anthers, and 206 fungal secretome genes may be anther-specific. The results confirm that U. maydis deploys distinct genes to cause disease in specific maize organs, and suggest mechanisms by which the host plant is manipulated to generate a tumor.

  19. [Competence factors of retinal pigment epithelium cells for reprogramming in the neuronal direction during retinal regeneration in newts].

    Science.gov (United States)

    Grigorian, E N

    2015-01-01

    Retinal pigment epithelium (RPE) cells that have the unique ability to reprogram retinal cells @in vivo@ were analyzed in the adult newt. Our own data and that available in the literature on the peculiarities of the biology of these cells (from morphology to molecular profile, which can be associated with the capability of phenotype change) were summarized: It was established that the molecular traits of specialized and poorly differentiated cells are combined in RPE of the adult newt. It was registered that persistent (at a low level) proliferation and rapid change of specific cytoskeleton proteins can contribute to the success of RPE cell reprogramming in the neuronal direction. Each of the considered factors of competence for reprogramming can be found for animal RPE, whose cells are not able @in vivo@ to change the phenotype to a neuronal one; however, their totality (supported by the epigenetic state permissive for conversion) is probably an internal property of only newt RPE. PMID:25872395

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

    Directory of Open Access Journals (Sweden)

    Soham Chanda

    2014-08-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  2. Conversion of Prostate Adenocarcinoma to Small Cell Carcinoma-Like by Reprogramming.

    Science.gov (United States)

    Borges, Gisely T; Vêncio, Eneida F; Quek, Sue-Ing; Chen, Adeline; Salvanha, Diego M; Vêncio, Ricardo Z N; Nguyen, Holly M; Vessella, Robert L; Cavanaugh, Christopher; Ware, Carol B; Troisch, Pamela; Liu, Alvin Y

    2016-09-01

    The lineage relationship between prostate adenocarcinoma and small cell carcinoma was studied by using the LuCaP family of xenografts established from primary neoplasm to metastasis. Expression of four stem cell transcription factor (TF) genes, LIN28A, NANOG, POU5F1, SOX2, were analyzed in the LuCaP lines. These genes, when force expressed in differentiated cells, can reprogram the recipients into stem-like induced pluripotent stem (iPS) cells. Most LuCaP lines expressed POU5F1, while LuCaP 145.1, representative of small cell carcinoma, expressed all four. Through transcriptome database query, many small cell carcinoma genes were also found in stem cells. To test the hypothesis that prostate cancer progression from "differentiated" adenocarcinoma to "undifferentiated" small cell carcinoma could involve re-expression of stem cell genes, the four TF genes were transduced via lentiviral vectors into five adenocarcinoma LuCaP lines-70CR, 73CR, 86.2, 92, 105CR-as done in iPS cell reprogramming. The resultant cells from these five transductions displayed a morphology of small size and dark appearing unlike the parentals. Transcriptome analysis of LuCaP 70CR* ("*" to denote transfected progeny) revealed a unique gene expression close to that of LuCaP 145.1. In a prostate principal components analysis space based on cell-type transcriptomes, the different LuCaP transcriptome datapoints were aligned to suggest a possible ordered sequence of expression changes from the differentiated luminal-like adenocarcinoma cell types to the less differentiated, more stem-like small cell carcinoma types, and LuCaP 70CR*. Prostate cancer progression can thus be molecularly characterized by loss of differentiation with re-expression of stem cell genes. J. Cell. Physiol. 231: 2040-2047, 2016. © 2016 Wiley Periodicals, Inc.

  3. Conversion of Prostate Adenocarcinoma to Small Cell Carcinoma-Like by Reprogramming.

    Science.gov (United States)

    Borges, Gisely T; Vêncio, Eneida F; Quek, Sue-Ing; Chen, Adeline; Salvanha, Diego M; Vêncio, Ricardo Z N; Nguyen, Holly M; Vessella, Robert L; Cavanaugh, Christopher; Ware, Carol B; Troisch, Pamela; Liu, Alvin Y

    2016-09-01

    The lineage relationship between prostate adenocarcinoma and small cell carcinoma was studied by using the LuCaP family of xenografts established from primary neoplasm to metastasis. Expression of four stem cell transcription factor (TF) genes, LIN28A, NANOG, POU5F1, SOX2, were analyzed in the LuCaP lines. These genes, when force expressed in differentiated cells, can reprogram the recipients into stem-like induced pluripotent stem (iPS) cells. Most LuCaP lines expressed POU5F1, while LuCaP 145.1, representative of small cell carcinoma, expressed all four. Through transcriptome database query, many small cell carcinoma genes were also found in stem cells. To test the hypothesis that prostate cancer progression from "differentiated" adenocarcinoma to "undifferentiated" small cell carcinoma could involve re-expression of stem cell genes, the four TF genes were transduced via lentiviral vectors into five adenocarcinoma LuCaP lines-70CR, 73CR, 86.2, 92, 105CR-as done in iPS cell reprogramming. The resultant cells from these five transductions displayed a morphology of small size and dark appearing unlike the parentals. Transcriptome analysis of LuCaP 70CR* ("*" to denote transfected progeny) revealed a unique gene expression close to that of LuCaP 145.1. In a prostate principal components analysis space based on cell-type transcriptomes, the different LuCaP transcriptome datapoints were aligned to suggest a possible ordered sequence of expression changes from the differentiated luminal-like adenocarcinoma cell types to the less differentiated, more stem-like small cell carcinoma types, and LuCaP 70CR*. Prostate cancer progression can thus be molecularly characterized by loss of differentiation with re-expression of stem cell genes. J. Cell. Physiol. 231: 2040-2047, 2016. © 2016 Wiley Periodicals, Inc. PMID:26773436

  4. Metabolic reprogramming in mutant IDH1 glioma cells.

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    Jose L Izquierdo-Garcia

    Full Text Available Mutations in isocitrate dehydrogenase (IDH 1 have been reported in over 70% of low-grade gliomas and secondary glioblastomas. IDH1 is the enzyme that catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate while mutant IDH1 catalyzes the conversion of α-ketoglutarate into 2-hydroxyglutarate. These mutations are associated with the accumulation of 2-hydroxyglutarate within the tumor and are believed to be one of the earliest events in the development of low-grade gliomas. The goal of this work was to determine whether the IDH1 mutation leads to additional magnetic resonance spectroscopy (MRS-detectable changes in the cellular metabolome.Two genetically engineered cell models were investigated, a U87-based model and an E6/E7/hTERT immortalized normal human astrocyte (NHA-based model. For both models, wild-type IDH1 cells were generated by transduction with a lentiviral vector coding for the wild-type IDH1 gene while mutant IDH1 cells were generated by transduction with a lentiviral vector coding for the R132H IDH1 mutant gene. Metabolites were extracted from the cells using the dual-phase extraction method and analyzed by 1H-MRS. Principal Component Analysis was used to analyze the MRS data.Principal Component Analysis clearly discriminated between wild-type and mutant IDH1 cells. Analysis of the loading plots revealed significant metabolic changes associated with the IDH1 mutation. Specifically, a significant drop in the concentration of glutamate, lactate and phosphocholine as well as the expected elevation in 2-hydroxyglutarate were observed in mutant IDH1 cells when compared to their wild-type counterparts.The IDH1 mutation leads to several, potentially translatable MRS-detectable metabolic changes beyond the production of 2-hydroxyglutarate.

  5. Microspore embryogenesis: reprogramming cell fate from pollen to embryo development

    NARCIS (Netherlands)

    Hui Li,

    2014-01-01

    Microspore embryogenesis is an expression of plant cell totipotency that leads to the production of haploid embryos. Besides being a widely exploited plant breeding tool, microspore embryogenesis is also a fascinating system that can be used to obtain a deeper mechanistic understanding of plant toti

  6. Enhancing the efficiency of direct reprogramming of human mesenchymal stem cells into mature neuronal-like cells with the combination of small molecule modulators of chromatin modifying enzymes, SMAD signaling and cyclic adenosine monophosphate levels.

    Science.gov (United States)

    Alexanian, Arshak R; Liu, Qing-song; Zhang, Zhiying

    2013-08-01

    Advances in cell reprogramming technologies to generate patient-specific cells of a desired type will revolutionize the field of regenerative medicine. While several cell reprogramming methods have been developed over the last decades, the majority of these technologies require the exposure of cell nuclei to reprogramming large molecules via transfection, transduction, cell fusion or nuclear transfer. This raises several technical, safety and ethical issues. Chemical genetics is an alternative approach for cell reprogramming that uses small, cell membrane penetrable substances to regulate multiple cellular processes including cell plasticity. Recently, using the combination of small molecules that are involved in the regulation chromatin structure and function and agents that favor neural differentiation we have been able to generate neural-like cells from human mesenchymal stem cells. In this study, to improve the efficiency of neuronal differentiation and maturation, two specific inhibitors of SMAD signaling (SMAD1/3 and SMAD3/5/8) that play an important role in neuronal differentiation of embryonic stem cells, were added to our previous neural induction recipe. Results demonstrated that human mesenchymal stem cells grown in this culture conditions exhibited higher expression of several mature neuronal genes, formed synapse-like structures and exerted electrophysiological properties of differentiating neural stem cells. Thus, an efficient method for production of mature neuronal-like cells from human adult bone marrow derived mesenchymal stem cells has been developed. We concluded that specific combinations of small molecules that target specific cell signaling pathways and chromatin modifying enzymes could be a promising approach for manipulation of adult stem cell plasticity.

  7. The reprogrammed pancreatic progenitor-like intermediate state of hepatic cells is more susceptible to pancreatic beta cell differentiation.

    Science.gov (United States)

    Wang, Qiwei; Wang, Hai; Sun, Yu; Li, Shi-Wu; Donelan, William; Chang, Lung-Ji; Jin, Shouguang; Terada, Naohiro; Cheng, Henrique; Reeves, Westley H; Yang, Li-Jun

    2013-08-15

    Induced pluripotent stem cells (iPSCs) hold great promise for cell therapy. However, their low efficiency of lineage-specific differentiation and tumorigenesis severely hinder clinical translation. We hypothesized that reprogramming of somatic cells into lineage-specific progenitor cells might allow for large-scale expansion, avoiding the tumorigenesis inherent with iPSCs and simultaneously facilitating lineage-specific differentiation. Here we aimed at reprogramming rat hepatic WB cells, using four Yamanaka factors, into pancreatic progenitor cells (PPCs) or intermediate (IM) cells that have characteristics of PPCs. IM clones were selected based on their specific morphology and alkaline phosphatase activity and stably passaged under defined culture conditions. IM cells did not have iPSC properties, could be stably expanded in large quantity, and expressed all 14 genes that are used to define the PPC developmental stage. Directed differentiation of IM and WB cells by Pdx1-Ngn3-MafA (PNM) into pancreatic beta-like cells revealed that the IM cells are more susceptible to directed beta cell differentiation because of their open chromatin configuration, as demonstrated by expression of key pancreatic beta cell genes, secretion of insulin in response to glucose stimulation, and easy access to exogenous PNM proteins at the rat insulin 1 and Pdx1 promoters. This notion that IM cells are superior to their parental cells is further supported by the epigenetic demonstration of accessibility of Pdx1 and insulin 1 promoters. In conclusion, we have developed a strategy to derive and expand PPC cells from hepatic WB cells using conventional cell reprogramming. This proof-of-principal study may offer a novel, safe and effective way to generate autologous pancreatic beta cells for cell therapy of diabetes. PMID:23750005

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

    Directory of Open Access Journals (Sweden)

    Michael K Skinner

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

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

    Science.gov (United States)

    Skinner, Michael K; Guerrero-Bosagna, Carlos; Haque, M; Nilsson, Eric; Bhandari, Ramji; McCarrey, John R

    2013-01-01

    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.

  10. Melatonin improves reprogramming efficiency and proliferation of bovine-induced pluripotent stem cells.

    Science.gov (United States)

    Bai, Chunyu; Li, Xiangchen; Gao, Yuhua; Yuan, Ziao; Hu, Pengfei; Wang, Hui; Liu, Changqing; Guan, Weijun; Ma, Yuehui

    2016-09-01

    Melatonin can modulate neural stem cell (NSC) functions such as proliferation and differentiation into NSC-derived pluripotent stem cells (N-iPS) in brain tissue, but the effect and mechanism underlying this are unclear. Thus, we studied how primary cultured bovine NSCs isolated from the retinal neural layer could transform into N-iPS cell. NSCs were exposed to 0.01, 0.1, 1, 10, or 100 μm melatonin, and cell viability studies indicated that 10 μm melatonin can significantly increase cell viability and promote cell proliferation in NSCs in vitro. Thus, 10 μm melatonin was used to study miR-302/367-mediated cell reprogramming of NSCs. We noted that this concentration of melatonin increased reprogramming efficiency of N-iPS cell generation from primary cultured bovine NSCs and that this was mediated by downregulation of apoptosis-related genes p53 and p21. Then, N-iPS cells were treated with 1, 10, 100, or 500 μm melatonin, and N-iPS (M-N-iPS) cell proliferation was measured. We noted that 100 μm melatonin increased proliferation of N-iPS cells via increased phosphorylation of intracellular ERK1/2 via activation of its pathway in M-N-iPS via melatonin receptors 1 (MT1). Finally, we verified that N-iPS cells and M-N-iPS cells are similar to typical embryonic stem cells including the expression of pluripotency markers (Oct4 and Nanog), the ability to form teratomas in vivo, and the capacity to differentiate into all three embryonic germ layers. PMID:27090494

  11. Melatonin improves reprogramming efficiency and proliferation of bovine-induced pluripotent stem cells.

    Science.gov (United States)

    Bai, Chunyu; Li, Xiangchen; Gao, Yuhua; Yuan, Ziao; Hu, Pengfei; Wang, Hui; Liu, Changqing; Guan, Weijun; Ma, Yuehui

    2016-09-01

    Melatonin can modulate neural stem cell (NSC) functions such as proliferation and differentiation into NSC-derived pluripotent stem cells (N-iPS) in brain tissue, but the effect and mechanism underlying this are unclear. Thus, we studied how primary cultured bovine NSCs isolated from the retinal neural layer could transform into N-iPS cell. NSCs were exposed to 0.01, 0.1, 1, 10, or 100 μm melatonin, and cell viability studies indicated that 10 μm melatonin can significantly increase cell viability and promote cell proliferation in NSCs in vitro. Thus, 10 μm melatonin was used to study miR-302/367-mediated cell reprogramming of NSCs. We noted that this concentration of melatonin increased reprogramming efficiency of N-iPS cell generation from primary cultured bovine NSCs and that this was mediated by downregulation of apoptosis-related genes p53 and p21. Then, N-iPS cells were treated with 1, 10, 100, or 500 μm melatonin, and N-iPS (M-N-iPS) cell proliferation was measured. We noted that 100 μm melatonin increased proliferation of N-iPS cells via increased phosphorylation of intracellular ERK1/2 via activation of its pathway in M-N-iPS via melatonin receptors 1 (MT1). Finally, we verified that N-iPS cells and M-N-iPS cells are similar to typical embryonic stem cells including the expression of pluripotency markers (Oct4 and Nanog), the ability to form teratomas in vivo, and the capacity to differentiate into all three embryonic germ layers.

  12. Conditionally reprogrammed normal and transformed mouse mammary epithelial cells display a progenitor-cell-like phenotype.

    Directory of Open Access Journals (Sweden)

    Francisco R Saenz

    Full Text Available Mammary epithelial (ME cells cultured under conventional conditions senesce after several passages. Here, we demonstrate that mouse ME cells isolated from normal mammary glands or from mouse mammary tumor virus (MMTV-Neu-induced mammary tumors, can be cultured indefinitely as conditionally reprogrammed cells (CRCs on irradiated fibroblasts in the presence of the Rho kinase inhibitor Y-27632. Cell surface progenitor-associated markers are rapidly induced in normal mouse ME-CRCs relative to ME cells. However, the expression of certain mammary progenitor subpopulations, such as CD49f+ ESA+ CD44+, drops significantly in later passages. Nevertheless, mouse ME-CRCs grown in a three-dimensional extracellular matrix gave rise to mammary acinar structures. ME-CRCs isolated from MMTV-Neu transgenic mouse mammary tumors express high levels of HER2/neu, as well as tumor-initiating cell markers, such as CD44+, CD49f+, and ESA+ (EpCam. These patterns of expression are sustained in later CRC passages. Early and late passage ME-CRCs from MMTV-Neu tumors that were implanted in the mammary fat pads of syngeneic or nude mice developed vascular tumors that metastasized within 6 weeks of transplantation. Importantly, the histopathology of these tumors was indistinguishable from that of the parental tumors that develop in the MMTV-Neu mice. Application of the CRC system to mouse mammary epithelial cells provides an attractive model system to study the genetics and phenotype of normal and transformed mouse epithelium in a defined culture environment and in vivo transplant studies.

  13. Cell reprogramming and neuronal differentiation applied to neurodegenerative diseases: Focus on Parkinson's disease.

    Science.gov (United States)

    Wenker, Shirley D; Casalía, Mariana; Candedo, Verónica Cavaliere; Casabona, Juan Cruz; Pitossi, Fernando J

    2015-11-14

    Adult cells from patients can be reprogrammed to induced pluripotent stem cells (iPSCs) which successively can be used to obtain specific cells such as neurons. This remarkable breakthrough represents a new way of studying diseases and brought new therapeutic perspectives in the field of regenerative medicine. This is particular true in the neurology field, where few techniques are amenable to study the affected tissue of the patient during illness progression, in addition to the lack of neuroprotective therapies for many diseases. In this review we discuss the advantages and unresolved issues of cell reprogramming and neuronal differentiation. We reviewed evidence using iPSCs-derived neurons from neurological patients. Focusing on data obtained from Parkinson's disease (PD) patients, we show that iPSC-derived neurons possess morphological and functional characteristics of this disease and build a case for the use of this technology to study PD and other neuropathologies while disease is in progress. These data show the enormous impact that this new technology starts to have on different purposes such as the study and design of future therapies of neurological disease, especially PD.

  14. Hallmarks of progeroid syndromes: lessons from mice and reprogrammed cells

    Directory of Open Access Journals (Sweden)

    Dido Carrero

    2016-07-01

    Full Text Available Ageing is a process that inevitably affects most living organisms and involves the accumulation of macromolecular damage, genomic instability and loss of heterochromatin. Together, these alterations lead to a decline in stem cell function and to a reduced capability to regenerate tissue. In recent years, several genetic pathways and biochemical mechanisms that contribute to physiological ageing have been described, but further research is needed to better characterize this complex biological process. Because premature ageing (progeroid syndromes, including progeria, mimic many of the characteristics of human ageing, research into these conditions has proven to be very useful not only to identify the underlying causal mechanisms and identify treatments for these pathologies, but also for the study of physiological ageing. In this Review, we summarize the main cellular and animal models used in progeria research, with an emphasis on patient-derived induced pluripotent stem cell models, and define a series of molecular and cellular hallmarks that characterize progeroid syndromes and parallel physiological ageing. Finally, we describe the therapeutic strategies being investigated for the treatment of progeroid syndromes, and their main limitations.

  15. 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

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

    Science.gov (United States)

    Cerdá-Esteban, Nuria; Spagnoli, Francesca M

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    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. 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.

  19. Messenger RNA- versus retrovirus-based induced pluripotent stem cell reprogramming strategies: analysis of genomic integrity.

    Science.gov (United States)

    Steichen, Clara; Luce, Eléanor; Maluenda, Jérôme; Tosca, Lucie; Moreno-Gimeno, Inmaculada; Desterke, Christophe; Dianat, Noushin; Goulinet-Mainot, Sylvie; Awan-Toor, Sarah; Burks, Deborah; Marie, Joëlle; Weber, Anne; Tachdjian, Gérard; Melki, Judith; Dubart-Kupperschmitt, Anne

    2014-06-01

    The use of synthetic messenger RNAs to generate human induced pluripotent stem cells (iPSCs) is particularly appealing for potential regenerative medicine applications, because it overcomes the common drawbacks of DNA-based or virus-based reprogramming strategies, including transgene integration in particular. We compared the genomic integrity of mRNA-derived iPSCs with that of retrovirus-derived iPSCs generated in strictly comparable conditions, by single-nucleotide polymorphism (SNP) and copy number variation (CNV) analyses. We showed that mRNA-derived iPSCs do not differ significantly from the parental fibroblasts in SNP analysis, whereas retrovirus-derived iPSCs do. We found that the number of CNVs seemed independent of the reprogramming method, instead appearing to be clone-dependent. Furthermore, differentiation studies indicated that mRNA-derived iPSCs differentiated efficiently into hepatoblasts and that these cells did not load additional CNVs during differentiation. The integration-free hepatoblasts that were generated constitute a new tool for the study of diseased hepatocytes derived from patients' iPSCs and their use in the context of stem cell-derived hepatocyte transplantation. Our findings also highlight the need to conduct careful studies on genome integrity for the selection of iPSC lines before using them for further applications.

  20. Reversible Reprogramming of Circulating Memory T Follicular Helper Cell Function during Chronic HIV Infection.

    Science.gov (United States)

    Cubas, Rafael; van Grevenynghe, Julien; Wills, Saintedym; Kardava, Lela; Santich, Brian H; Buckner, Clarisa M; Muir, Roshell; Tardif, Virginie; Nichols, Carmen; Procopio, Francesco; He, Zhong; Metcalf, Talibah; Ghneim, Khader; Locci, Michela; Ancuta, Petronella; Routy, Jean-Pierre; Trautmann, Lydie; Li, Yuxing; McDermott, Adrian B; Koup, Rick A; Petrovas, Constantinos; Migueles, Steven A; Connors, Mark; Tomaras, Georgia D; Moir, Susan; Crotty, Shane; Haddad, Elias K

    2015-12-15

    Despite the overwhelming benefits of antiretroviral therapy (ART) in curtailing viral load in HIV-infected individuals, ART does not fully restore cellular and humoral immunity. HIV-infected individuals under ART show reduced responses to vaccination and infections and are unable to mount an effective antiviral immune response upon ART cessation. Many factors contribute to these defects, including persistent inflammation, especially in lymphoid tissues, where T follicular helper (Tfh) cells instruct and help B cells launch an effective humoral immune response. In this study we investigated the phenotype and function of circulating memory Tfh cells as a surrogate of Tfh cells in lymph nodes and found significant impairment of this cell population in chronically HIV-infected individuals, leading to reduced B cell responses. We further show that these aberrant memory Tfh cells exhibit an IL-2-responsive gene signature and are more polarized toward a Th1 phenotype. Treatment of functional memory Tfh cells with IL-2 was able to recapitulate the detrimental reprogramming. Importantly, this defect was reversible, as interfering with the IL-2 signaling pathway helped reverse the abnormal differentiation and improved Ab responses. Thus, reversible reprogramming of memory Tfh cells in HIV-infected individuals could be used to enhance Ab responses. Altered microenvironmental conditions in lymphoid tissues leading to altered Tfh cell differentiation could provide one explanation for the poor responsiveness of HIV-infected individuals to new Ags. This explanation has important implications for the development of therapeutic interventions to enhance HIV- and vaccine-mediated Ab responses in patients under ART.

  1. 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.

  2. Reversibility of cellular aging by reprogramming through an embryonic-like state : a new paradigm for human cell rejuvenation

    Directory of Open Access Journals (Sweden)

    Jean-Marc Lemaitre

    2014-01-01

    Full Text Available Direct reprogramming of somatic cells into induced pluripotent stem cells (iPSCs provides a unique opportunity to derive patient-specific stem cells with potential application in autologous tissue replacement therapies and without the ethical concerns of Embryonic Stem Cells (hESC. However, this strategy still suffers from several hurdles that need to be overcome before clinical applications. Among them, cellular senescence, which contributes to aging and restricted longevity, has been described as a barrier to the derivation of iPSCs. This suggests that aging might be an important limitation for therapeutic purposes for elderly individuals. Senescence is characterized by an irreversible cell cycle arrest in response to various forms of stress, including activation of oncogenes, shortened telomeres, DNA damage, oxidative stress, and mitochondrial dysfunction. To overcome this barrier, we developed an optimized 6-factor-based reprogramming protocol that is able to cause efficient reversing of cellular senescence and reprogramming into iPSCs. We demonstrated that iPSCs derived from senescent and centenarian fibroblasts have reset telomere size, gene expression profiles, oxidative stress, and mitochondrial metabolism, and are indistinguishable from hESC. Finally, we demonstrate that re-differentiation led to rejuvenated cells with a reset cellular physiology, defining a new paradigm for human cell rejuvenation. We discuss the molecular mechanisms involved in cell reprogramming of senescent cells

  3. PDX1- and NGN3-mediated in vitro reprogramming of human bone marrow-derived mesenchymal stromal cells into pancreatic endocrine lineages

    DEFF Research Database (Denmark)

    Limbert, Catarina; Päth, Günter; Ebert, Regina;

    2011-01-01

    Reprogramming of multipotent adult bone marrow (BM)-derived mesenchymal stromal/stem cells (MSC) (BM-MSC) represents one of several strategies for cell-based therapy of diabetes. However, reprogramming primary BM-MSC into pancreatic endocrine lineages has not yet been consistently demonstrated....

  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.

  5. 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. PMID:23828660

  6. Application of Induced Pluripotent Stem Cells Reprogrammed from Dental Pulp Cells: a Novel Approach for Tooth Regeneration

    Directory of Open Access Journals (Sweden)

    Xiaoyan Zhou

    2011-03-01

    Full Text Available Introduction: Candidate human dental stem/progenitor cells have been isolated and charac-terized from dental tissues and shown to hold the capability to differentiate into tooth-generating cells. However, ad-vances in engineering a whole tooth by these stem cells are hindered by various factors, such as the poor availability of human primitive tooth bud stem cells, difficulties in isolating and purifying dental mesenchymal stem cells and ethical controversies when using embryonic oral epithelium. As a result it is meaningful to find other autologous dental cells for the purpose of reconstructing a tooth.The hypothesis: Previous studies demonstrated that somatic cells can be reprogrammed into induced pluripotent stem cells by ex-ogenous expression Oct-4 and Sox-2. On the basis of these findings we can reasonably hypothesize that when transfected with specific transcription factors Oct-4 and Sox-2, dental pulp cells, the main cell in pulp, could also be reprogrammed into induced pluripotent stem cells, which are considered to be of best potential to regenerate a whole tooth. Evaluation of the hypothesis: After transfection with Oct-4 and Sox-2 into human dental pulp cells, the positive colonies are isolated and then identified according to the characteristics of iPS cells. These cells are further investigated the capability in differentiating into ameloblasts and odontoblasts and finally seeded onto the sur-face of a tooth-shaped biodegradable polymer scaffold to detect the ability of constructing a bioengineered tooth.

  7. 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. PMID:26618522

  8. 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.

  9. Protein kinase D1 drives pancreatic acinar cell reprogramming and progression to intraepithelial neoplasia

    Science.gov (United States)

    Liou, Geou-Yarh; Döppler, Heike; Braun, Ursula B.; Panayiotou, Richard; Scotti Buzhardt, Michele; Radisky, Derek C.; Crawford, Howard C.; Fields, Alan P.; Murray, Nicole R.; Wang, Q. Jane; Leitges, Michael; Storz, Peter

    2015-02-01

    The transdifferentiation of pancreatic acinar cells to a ductal phenotype (acinar-to-ductal metaplasia, ADM) occurs after injury or inflammation of the pancreas and is a reversible process. However, in the presence of activating Kras mutations or persistent epidermal growth factor receptor (EGF-R) signalling, cells that underwent ADM can progress to pancreatic intraepithelial neoplasia (PanIN) and eventually pancreatic cancer. In transgenic animal models, ADM and PanINs are initiated by high-affinity ligands for EGF-R or activating Kras mutations, but the underlying signalling mechanisms are not well understood. Here, using a conditional knockout approach, we show that protein kinase D1 (PKD1) is sufficient to drive the reprogramming process to a ductal phenotype and progression to PanINs. Moreover, using 3D explant culture of primary pancreatic acinar cells, we show that PKD1 acts downstream of TGFα and Kras, to mediate formation of ductal structures through activation of the Notch pathway.

  10. Reprogramming of Melanoma Tumor-Infiltrating Lymphocytes to Induced Pluripotent Stem Cells

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    Hidehito Saito

    2016-01-01

    Full Text Available Induced pluripotent stem cells (iPSCs derived from somatic cells of patients hold great promise for autologous cell therapies. One of the possible applications of iPSCs is to use them as a cell source for producing autologous lymphocytes for cell-based therapy against cancer. Tumor-infiltrating lymphocytes (TILs that express programmed cell death protein-1 (PD-1 are tumor-reactive T cells, and adoptive cell therapy with autologous TILs has been found to achieve durable complete response in selected patients with metastatic melanoma. Here, we describe the derivation of human iPSCs from melanoma TILs expressing high level of PD-1 by Sendai virus-mediated transduction of the four transcription factors, OCT3/4, SOX2, KLF4, and c-MYC. TIL-derived iPSCs display embryonic stem cell-like morphology, have normal karyotype, express stem cell-specific surface antigens and pluripotency-associated transcription factors, and have the capacity to differentiate in vitro and in vivo. A wide variety of T cell receptor gene rearrangement patterns in TIL-derived iPSCs confirmed the heterogeneity of T cells infiltrating melanomas. The ability to reprogram TILs containing patient-specific tumor-reactive repertoire might allow the generation of patient- and tumor-specific polyclonal T cells for cancer immunotherapy.

  11. Production of Induced Pluripotent Stem Cells by Reprogramming of Blood Cells

    Directory of Open Access Journals (Sweden)

    Sadia Zia

    2011-06-01

    Full Text Available Blood cells are the simple, efficient and economical source for the production of induced pluripotent cells. The discovery of induced pluripotent cells was not novel; it was pedestal on the scientific principals and technologies which have been developed over last six decades. These are nuclear transfer and the cloning of Animals, Pluripotent cell lines and fusion hybrids and Transcription Factors and lineage switching. The use of human embryonic stem cells in regenerative medicines was a breakthrough but make use of these cells arise ethical issues as they are obtained from human embryos. An alternative advancement using induced pluripotent stem cells, which mimics the embryonic stem cells has the significant gain that they replaced the embryonic stem cells. The pluripotent cells can be induced from terminally differentiated somatic cells by the Induction of only four defined factors including c-Myc, klf4, Oct4 and Sox2 which are enough to alter the fate of cell.

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

    Directory of Open Access Journals (Sweden)

    Stéphane Bellafiore

    2008-10-01

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

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

    Science.gov (United States)

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

    2015-09-01

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

  14. Using somatic-cell nuclear transfer to study aging.

    Science.gov (United States)

    Kishigami, Satoshi; Lee, Ah Reum; Wakayama, Teruhiko

    2013-01-01

    In mammals, a diploid genome following fertilization of haploid cells, an egg, and a spermatozoon is unique and irreproducible. This implies that the generated unique diploid genome is doomed with the individual's inevitable demise. Since it was first reported in 1997 that Dolly the sheep had been cloned, many mammalian species have been cloned successfully using somatic-cell nuclear transfer (SCNT). The success of SCNT in mammals enables us not only to reproduce offspring without germ cells, that is, to "passage" a unique diploid genome, but also to address valuable biological questions on development, nuclear reprogramming, and epigenetic memory. Successful cloning can also support epigenetic reprogramming where the aging clock is reset or reversed. Recent work using iPS cell technology has explored the practicality and led to the recapitulation of premature aging with iPSCs from progeroid laminopathies. As a result, reprogramming tools are also expected to contribute to studying biological age. However, the efficiency of animal cloning is still low in most cases and the mechanism of reprogramming in cloned embryos is still largely unclear. Here, based on recent advances, we describe an improved, more efficient mouse cloning protocol using histone deacetylase inhibitors (HDACis) and latrunculin A, which increases the success rates of producing cloned mice or establishing ES cells fivefold. This improved method of cloning will provide a strong tool to address many issues including biological aging more easily and with lower cost. PMID:23929101

  15. Reprogramming of non-genomic estrogen signaling by the stemness factor SOX2 enhances the tumor-initiating capacity of breast cancer cells

    Science.gov (United States)

    Vazquez-Martin, Alejandro; Cufí, Sílvia; López-Bonet, Eugeni; Corominas-Faja, Bruna; Cuyàs, Elisabet; Vellon, Luciano; Iglesias, Juan Manuel; Leis, Olatz; Martín, Ángel G; Menendez, Javier A

    2013-01-01

    The restoration of pluripotency circuits by the reactivation of endogenous stemness factors, such as SOX2, may provide a new paradigm in cancer development. The tumoral stem cell reprogramming hypothesis, i.e., the ability of stemness factors to redirect normal and differentiated tumor cells toward a less-differentiated and stem-like state, adds new layers of complexity to cancer biology, because the effects of such reprogramming may remain dormant until engaged later in response to (epi)genetic and/or (micro)environmental events. To test this hypothesis, we utilized an in vitro model of a SOX2-overexpressing cancer stem cell (CSC)-like cellular state that was recently developed in our laboratory by employing Yamanaka’s nuclear reprogramming technology in the estrogen receptor α (ERα)-positive MCF-7 breast cancer cell line. Despite the acquisition of distinct molecular features that were compatible with a breast CSC-like cellular state, such as strong aldehyde dehydrogenase activity, as detected by ALDEFLUOR, and overexpression of the SSEA-4 and CD44 breast CSC markers, the tumor growth-initiating ability of SOX2-overexpressing CSC-like MCF-7 cells solely occurred in female nude mice supplemented with estradiol when compared with MCF-7 parental cells. Ser118 phosphorylation of estrogen receptor α (ERα), which is a pivotal integrator of the genomic and nongenomic E2/ERα signaling pathways, drastically accumulated in nuclear speckles in the interphase nuclei of SOX2-driven CSC-like cell populations. Moreover, SOX2-positive CSC-like cells accumulated significantly higher numbers of actively dividing cells, and the highest levels of phospho-Ser118-ERα occurred when chromosomes lined up on a metaphase plate. The previously unrecognized link between E2/ERα signaling and SOX2-driven stem cell circuitry may significantly impact our current understanding of breast cancer initiation and progression, i.e., SOX2 can promote non-genomic E2 signaling that leads to

  16. A “Hit and Run” Approach to Inducible Direct Reprogramming of Astrocytes to Neural Stem Cells

    Science.gov (United States)

    Poulou, Maria; Mandalos, Nikolaos P.; Karnavas, Theodoros; Saridaki, Marannia; McKay, Ronald D. G.; Remboutsika, Eumorphia

    2016-01-01

    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, 2 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. PMID:27148066

  17. Transgenic expression of Telomerase reverse transcriptase (Tert) improves cell proliferation of primary cells and enhances reprogramming efficiency into the induced pluripotent stem cell.

    Science.gov (United States)

    Hidema, Shizu; Fukuda, Tomokazu; Date, Shiori; Tokitake, Yuko; Matsui, Yasuhisa; Sasaki, Hiroki; Nishimori, Katsuhiko

    2016-10-01

    The enzymatic activity of telomerase is important for the extension of the telomere repeat sequence and overcoming cellular senescence. We generated a conditional transgenic mouse line, carrying the telomerase reverse transcriptase (Tert) expression cassette, controlled by the Cre-loxP-mediated recombination. In our study, Cre recombinase expression efficiently activated Tert expression, resulting in its increased enzymatic activity, which extended the period of cellular proliferation until the keratinocytes entered senescence. This suggests that transgenic Tert expression is effective in enhancing primary cell proliferation. Notably, Tert expression increased colony formation of induced pluripotent stem (iPS) cells after the introduction of four reprogramming factors, Oct-4, klf4, SOX-2, and c-Myc into the transgenic fibroblasts. To the best of our knowledge, this is the first study to show that the transgenic Tert expression enhances reprogramming efficiency of iPS cells, which indicates a critical role for Tert in the reprogramming process. PMID:27297181

  18. Global indiscriminate methylation in cell-specific gene promoters following reprogramming into human induced pluripotent stem cells.

    Science.gov (United States)

    Nissenbaum, Jonathan; Bar-Nur, Ori; Ben-David, Eyal; Benvenisty, Nissim

    2013-01-01

    Molecular reprogramming of somatic cells into human induced pluripotent stem cells (iPSCs) is accompanied by extensive changes in gene expression patterns and epigenetic marks. To better understand the link between gene expression and DNA methylation, we have profiled human somatic cells from different embryonic cell types (endoderm, mesoderm, and parthenogenetic germ cells) and the iPSCs generated from them. We show that reprogramming is accompanied by extensive DNA methylation in CpG-poor promoters, sparing CpG-rich promoters. Intriguingly, methylation in CpG-poor promoters occurred not only in downregulated genes, but also in genes that are not expressed in the parental somatic cells or their respective iPSCs. These genes are predominantly tissue-specific genes of other cell types from different lineages. Our results suggest a role of DNA methylation in the silencing of the somatic cell identity by global nonspecific methylation of tissue-specific genes from all lineages, regardless of their expression in the parental somatic cells.

  19. Cell fusion of bone marrow cells and somatic cell reprogramming by embryonic stem cells

    OpenAIRE

    Bonde, Sabrina; Pedram, Mehrdad; Stultz, Ryan; Zavazava, Nicholas

    2010-01-01

    Bone marrow transplantation is a curative treatment for many diseases, including leukemia, autoimmune diseases, and a number of immunodeficiencies. Recently, it was claimed that bone marrow cells transdifferentiate, a much desired property as bone marrow cells are abundant and therefore could be used in regenerative medicine to treat incurable chronic diseases. Using a Cre/loxP system, we studied cell fusion after bone marrow transplantation. Fused cells were chiefly Gr-1+, a myeloid cell mar...

  20. Macrohistone Variants Preserve Cell Identity by Preventing the Gain of H3K4me2 during Reprogramming to Pluripotency

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    María J. Barrero

    2013-04-01

    Full Text Available Transcription-factor-induced reprogramming of somatic cells to pluripotency is a very inefficient process, probably due to the existence of important epigenetic barriers that are imposed during differentiation and that contribute to preserving cell identity. In an effort to decipher the molecular nature of these barriers, we followed a genome-wide approach, in which we identified macrohistone variants (macroH2A as highly expressed in human somatic cells but downregulated after reprogramming to pluripotency, as well as strongly induced during differentiation. Knockdown of macrohistone variants in human keratinocytes increased the efficiency of reprogramming to pluripotency, whereas overexpression had opposite effects. Genome-wide occupancy profiles show that in human keratinocytes, macroH2A.1 preferentially occupies genes that are expressed at low levels and are marked with H3K27me3, including pluripotency-related genes and bivalent developmental regulators. The presence of macroH2A.1 at these genes prevents the regain of H3K4me2 during reprogramming, imposing an additional layer of repression that preserves cell identity.

  1. Metformin-induced metabolic reprogramming of chemoresistant ALDHbright breast cancer cells.

    Science.gov (United States)

    Cioce, Mario; Valerio, MariaCristina; Casadei, Luca; Pulito, Claudio; Sacconi, Andrea; Mori, Federica; Biagioni, Francesca; Manetti, Cesare; Muti, Paola; Strano, Sabrina; Blandino, Giovanni

    2014-06-30

    Metabolic remodeling is a hallmark of cancer progression and may affect tumor chemoresistance. Here we investigated by 1H-NMR/PCA analysis the metabolic profile of chemoresistant breast cancer cell subpopulations (ALDHbright cells) and their response to metformin, a promising anticancer metabolic modulator. The purified ALDHbright cells exhibited a different metabolic profile as compared to their chemosensitive ALDHlow counterparts. Metformin treatment strongly affected the metabolism of the ALDHbright cells thereby affecting, among the others, the glutathione metabolism, whose upregulation is a feature of progenitor-like, chemoresistant cell subpopulations. Globally, metformin treatment reduced the differences between ALDHbright and ALDHlow cells, making the former more similar to the latter. Metformin broadly modulated microRNAs in the ALDHbright cells, with a large fraction of them predicted to target the same metabolic pathways experimentally identified by 1H-NMR. Additionally, metformin modulated the levels of c-MYC and IRS-2, and this correlated with changes of the microRNA-33a levels. In summary, we observed, both by 1H-NMR and microRNA expression studies, that metformin treatment reduced the differences between the chemoresistant ALDHbright cells and the chemosensitive ALDHlow cells. This works adds on the potential therapeutic relevance of metformin and shows the potential for metabolic reprogramming to modulate cancer chemoresistance. PMID:24980829

  2. Roles of p53 and ASF1A in the Reprogramming of Sheep Kidney Cells to Pluripotent Cells.

    Science.gov (United States)

    Shi, Huijun; Fu, Qiang; Li, Guozhong; Ren, Yan; Hu, Shengwei; Ni, Wei; Guo, Fei; Shi, Mengting; Meng, Luping; Zhang, Hui; Qiao, Jun; Guo, Zhiru; Chen, Chuangfu

    2015-12-01

    Since the first report of induced pluripotent stem cells (iPSCs) by Takahashi and Yamanaka, numerous attempts have been made to derive iPSCs from other species via the ectopic expression of defined factors. Sheep iPSCs (siPSCs) have significant potential for biotechnology and agriculture. Although several groups have described siPSCs, the reprogramming efficiency was extremely low. The exogenous transgenes could be not silenced in the iPSCs, which hampered their development and application. Here, we report that p53 knockdown and antisilencing function 1A (ASF1A) overexpression promoted iPSC generation from sheep kidney cells (SKCs). Compared with transduction with eight human defined transcription factors (Oct4, Sox2, Klf4, c-Myc, Nanog, Lin28, hTERT, and SV40LT), the additional introduction of p53 RNA interference (RNAi) and/or ASF1A in the presence of small-molecule compounds [vitamin C (Vc) and valproic acid (VPA)] greatly improved the efficiency of sheep iPSC generation. The siPSCs exhibited morphological features similar to mouse embryonic stem cells (ESCs) and were positive for alkaline phosphatase and, pluripotent marker genes (Oct4, Nanog, Sox2, Rex1, TRA-1-60, TRA-1-81, and E-cadherin). Furthermore, these cells exhibited a normal karyotype of 54 chromosomes and were able to differentiate into all three germ layers both in vitro and in vivo. Moreover, the exogenous genes were silenced in siPSCs when p53 small hairpin RNA (shRNA) and ASF1A were added. Our results may help to reveal the role of p53 and ASF1A in sheep somatic cell reprogramming and provide an efficient approach to reprogramming sheep somatic cells. PMID:26580119

  3. Generation of human induced pluripotent stem cells by simple transient transfection of plasmid DNA encoding reprogramming factors

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    Lough John W

    2010-08-01

    Full Text Available Abstract Background The use of lentiviruses to reprogram human somatic cells into induced pluripotent stem (iPS cells could limit their therapeutic usefulness due to the integration of viral DNA sequences into the genome of the recipient cell. Recent work has demonstrated that human iPS cells can be generated using episomal plasmids, excisable transposons, adeno or sendai viruses, mRNA, or recombinant proteins. While these approaches offer an advance, the protocols have some drawbacks. Commonly the procedures require either subcloning to identify human iPS cells that are free of exogenous DNA, a knowledge of virology and safe handling procedures, or a detailed understanding of protein biochemistry. Results Here we report a simple approach that facilitates the reprogramming of human somatic cells using standard techniques to transfect expression plasmids that encode OCT4, NANOG, SOX2, and LIN28 without the need for episomal stability or selection. The resulting human iPS cells are free of DNA integration, express pluripotent markers, and form teratomas in immunodeficient animals. These iPS cells were also able to undergo directed differentiation into hepatocyte-like and cardiac myocyte-like cells in culture. Conclusions Simple transient transfection of plasmid DNA encoding reprogramming factors is sufficient to generate human iPS cells from primary fibroblasts that are free of exogenous DNA integrations. This approach is highly accessible and could expand the use of iPS cells in the study of human disease and development.

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

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    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.

  5. Reprogramming anti-tumor immunity

    OpenAIRE

    Crompton, Joseph G.; Clever, David; Vizcardo, Raul; Rao, Mahendra; Restifo, Nicholas P

    2014-01-01

    Regenerative medicine holds great promise in replacing tissues and organs lost to degenerative disease and injury. Applying principles of cellular reprogramming for the treatment of cancer, however, are not well established. Here we present an overview of cell-based reprogramming techniques (i.e. lineage reprogramming and stimulus-triggered acquisition of pluripotency) used in regenerative medicine, and within this context, envision how the scope of regenerative medicine may be expanded to tr...

  6. Conditionally Stabilized dCas9 Activator for Controlling Gene Expression in Human Cell Reprogramming and Differentiation

    OpenAIRE

    Diego Balboa; Jere Weltner; Solja Eurola; Ras Trokovic; Kirmo Wartiovaara; Timo Otonkoski

    2015-01-01

    Summary CRISPR/Cas9 protein fused to transactivation domains can be used to control gene expression in human cells. In this study, we demonstrate that a dCas9 fusion with repeats of VP16 activator domains can efficiently activate human genes involved in pluripotency in various cell types. This activator in combination with guide RNAs targeted to the OCT4 promoter can be used to completely replace transgenic OCT4 in human cell reprogramming. Furthermore, we generated a chemically controllable ...

  7. Defined nuclear changes accompany the reprogramming of the microspore to embryogenesis.

    Science.gov (United States)

    Testillano, P S; Coronado, M J; Seguí, J M; Domenech, J; González-Melendi, P; Raska, I; Risueño, M C

    2000-04-01

    The switch of the gametophytic developmental program toward pollen embryogenesis to form a haploid plant represents an important alternative for plant breeding. In the present study, the switch of the gametophytic developmental program toward a sporophytic pathway, "embryogenesis," has been studied in three different plant species, Brassica, tobacco, and pepper. The switch has been induced by stress (heat shock) at the very responsive stage of the microspore, which is the vacuolate period. As a result, the cell nucleus undergoes striking structural changes with regard to late gametophytic development, including alterations of biosynthetic activities and proliferative activity. An enrichment in HSP70 heat-shock protein and in the presence of Ntf6-MAP kinase was observed after inductive treatment in the nuclei during early embryogenesis. This apparently reflected the possible roles of these proteins, specifically the protective role of HSP70 for the nuclear machinery, and signal transduction of Ntf6-MAPK for the entry of cells into proliferation. Importantly, the observed nuclear changes were similar in the three species investigated and represented convenient markers for early monitoring of embryogenesis and selection purposes for obtaining double-haploid plants in plant breeding. PMID:10806072

  8. Expression of Two Classes of Pax6 Transcripts in Reprogramming Retinal Pigment Epithelium Cells of the Adult Newt.

    Science.gov (United States)

    Inami, Wataru; Islam, Md Rafiqul; Nakamura, Kenta; Yoshikawa, Taro; Yasumuro, Hirofumi; Casco-Robles, Martin Miguel; Toyama, Fubito; Maruo, Fumiaki; Chiba, Chikafumi

    2016-02-01

    The adult newt has the remarkable ability to regenerate a functional retina from retinal pigment epithelium (RPE) cells, even when the neural retina (NR) is completely lost from the eye. In this system, RPE cells are reprogrammed into a unique state of multipotent cells, named RPESCs, in an early phase of retinal regeneration. However, the signals that trigger reprogramming remain unknown. Here, to approach this issue we focused on Pax6, a transcription factor known to be expressed in RPESCs. We first identified four classes (v1, v2, v3 and v4) of Pax6 variants in the eye of adult newt, Cynops pyrrhogaster. These variants were expressed in most tissues of the intact eye in different combinations but not in the RPE, choroid or sclera. On the basis of this information, we investigated the expression of Pax6 in RPE cells after the NR was removed from the eye by surgery (retinectomy), and found that two classes (v1 and v2) of Pax6 variants were newly expressed in RPE cells 10 days after retinectomy, both in vivo and in vitro (RLEC system). In the RLEC system, we found that Pax6 expression is mediated through a pathway separate from the MEK-ERK pathway, which is required for cell cycle re-entry of RPE cells. These results predict the existence of a pathway that may be of fundamental importance to a better understanding of the reprogramming of RPE cells in vivo. PMID:26853865

  9. Suppression of PGC-1α is critical for reprogramming oxidative metabolism in renal cell carcinoma

    Science.gov (United States)

    LaGory, Edward L.; Wu, Colleen; Taniguchi, Cullen M.; Ding, Chien-Kuang Cornelia; Chi, Jen-Tsan; von Eyben, Rie; Scott, David A.; Richardson, Adam D.; Giaccia, Amato J.

    2015-01-01

    Summary Long believed to be a byproduct of malignant transformation, reprogramming of cellular metabolism is now recognized as a driving force in tumorigenesis. In clear cell renal cell carcinoma (ccRCC) frequent activation of HIF-signaling induces a metabolic switch that promotes tumorigenesis. Here we demonstrate that PGC-1α, a central regulator of energy metabolism, is suppressed in VHL-deficient ccRCC by a HIF/Dec1-dependent mechanism. In VHL wild type cells, PGC-1α suppression leads to decreased expression of the mitochondrial transcription factor Tfam and impaired mitochondrial respiration. Conversely, PGC-1α expression in VHL-deficient cells restores mitochondrial function and induces oxidative stress. ccRCC cells expressing PGC-1α exhibit impaired tumor growth and enhanced sensitivity to cytotoxic therapies. In patients, low levels of PGC-1α expression are associated with poor outcome. These studies demonstrate that suppression of PGC-1α recapitulates key metabolic phenotypes of ccRCC and highlight the potential of targeting PGC-1α expression as a therapeutic modality for the treatment of ccRCC. PMID:26119730

  10. Suppression of PGC-1α Is Critical for Reprogramming Oxidative Metabolism in Renal Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Edward L. LaGory

    2015-07-01

    Full Text Available Long believed to be a byproduct of malignant transformation, reprogramming of cellular metabolism is now recognized as a driving force in tumorigenesis. In clear cell renal cell carcinoma (ccRCC, frequent activation of HIF signaling induces a metabolic switch that promotes tumorigenesis. Here, we demonstrate that PGC-1α, a central regulator of energy metabolism, is suppressed in VHL-deficient ccRCC by a HIF/Dec1-dependent mechanism. In VHL wild-type cells, PGC-1α suppression leads to decreased expression of the mitochondrial transcription factor Tfam and impaired mitochondrial respiration. Conversely, PGC-1α expression in VHL-deficient cells restores mitochondrial function and induces oxidative stress. ccRCC cells expressing PGC-1α exhibit impaired tumor growth and enhanced sensitivity to cytotoxic therapies. In patients, low levels of PGC-1α expression are associated with poor outcome. These studies demonstrate that suppression of PGC-1α recapitulates key metabolic phenotypes of ccRCC and highlight the potential of targeting PGC-1α expression as a therapeutic modality for the treatment of ccRCC.

  11. Hybrid modeling of cell signaling and transcriptional reprogramming and its application in C. elegans development

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    Elana J Fertig

    2011-11-01

    Full Text Available Modeling of signal driven transcriptional reprogramming is critical for understanding of organism development, human disease, and cell biology. Many current modeling techniques discount key features of the biological sub-systems when modeling multi-scale, organism level processes. We present a mechanistic hybrid model, GESSA, which integrates a novel pooled probabilistic Boolean network model of cell signaling and a stochastic simulation of transcription and translation responding to a diffusion model of extra-cellular signals. We apply the model to simulate the well studied cell fate decision process of the vulval precursor cells (VPCs in C. elegans, using experimentally derived rate constants wherever possible and shared parameters to avoid overfitting. We demonstrate that GESSA recovers (1 the effects of varying scaffold protein concentration on signal strength, (2 amplification of signals in expression, (3 the relative external ligand concentration in a known geometry, and (4 feedback in biochemical networks. We demonstrate that setting model parameters based on wild-type and LIN-12 loss-of-function mutants in C. elegans leads to correct prediction of a wide variety of mutants including partial penetrance of phenotypes. Moreover, the model is relatively insensitive to parameters, retaining the wild-type phenotype for a wide range of cell signaling rate parameters.

  12. Optimized Hepatocyte-Like Cells with Functional Drug Transporters Directly-Reprogrammed from Mouse Fibroblasts and their Potential in Drug Disposition and Toxicology

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    Zhi-Tao Wu

    2016-05-01

    Full Text Available Background/Aims: To develop a suitable hepatocyte-like cell model that could be a substitute for primary hepatocytes with essential transporter expression and functions. Induced hepatocyte-like (iHep cells directly reprogrammed from mice fibroblast cells were fully characterized. Methods: Naïve iHep cells were transfected with nuclear hepatocyte factor 4 alpha (Hnf4α and treated with selected small molecules. Sandwich cultured configuration was applied. The mRNA and protein expression of transporters were determined by Real Time PCR and confocal. The functional transporters were estimated by drug biliary excretion measurement. The inhibition of bile acid efflux transporters by cholestatic drugs were assessed. Results: The expression and function of p-glycoprotein (P-gp, bile salt efflux pump (Bsep, multidrug resistance-associated protein 2 (Mrp2, Na+-dependent taurocholate cotransporting polypeptide (Ntcp, and organic anion transporter polypedtides (Oatps in iHep cells were significantly improved after transfection of hepatocyte nuclear factor 4 alpha (Hnf4α and treatment with selected inducers. In vitro intrinsic biliary clearances (CLb,int of optimized iHep cells for rosuvastatin, methotrexate, d8-TCA (deuterium-labeled sodium taurocholate acid and DPDPE ([D-Pen2,5] enkephalin hydrate correlated well with that of sandwich-cultured primary mouse hepatocytes (SCMHs (r2 = 0.984. Cholestatic drugs were evaluated and the results were compared well with primary mice hepatocytes. Conclusion: The optimized iHep cells expressed functional drug transporters and were comparable to primary mice hepatocytes. This study suggested direct reprogramming could provide a potential alternative to primary hepatocytes for drug candidate hepatobiliary disposition and hepatotoxicity screening.

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

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    Matteo Vecellio

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

  14. Ionizing Particle Radiation as a Modulator of Endogenous Bone Marrow Cell Reprogramming: Implications for Hematological Cancers.

    Science.gov (United States)

    Muralidharan, Sujatha; Sasi, Sharath P; Zuriaga, Maria A; Hirschi, Karen K; Porada, Christopher D; Coleman, Matthew A; Walsh, Kenneth X; Yan, Xinhua; Goukassian, David A

    2015-01-01

    Exposure of individuals to ionizing radiation (IR), as in the case of astronauts exploring space or radiotherapy cancer patients, increases their risk of developing secondary cancers and other health-related problems. Bone marrow (BM), the site in the body where hematopoietic stem cell (HSC) self-renewal and differentiation to mature blood cells occurs, is extremely sensitive to low-dose IR, including irradiation by high-charge and high-energy particles. Low-dose IR induces DNA damage and persistent oxidative stress in the BM hematopoietic cells. Inefficient DNA repair processes in HSC and early hematopoietic progenitors can lead to an accumulation of mutations whereas long-lasting oxidative stress can impair hematopoiesis itself, thereby causing long-term damage to hematopoietic cells in the BM niche. We report here that low-dose (1)H- and (56)Fe-IR significantly decreased the hematopoietic early and late multipotent progenitor (E- and L-MPP, respectively) cell numbers in mouse BM over a period of up to 10 months after exposure. Both (1)H- and (56)Fe-IR increased the expression of pluripotent stem cell markers Sox2, Nanog, and Oct4 in L-MPPs and 10 months post-IR exposure. We postulate that low doses of (1)H- and (56)Fe-IR may induce endogenous cellular reprogramming of BM hematopoietic progenitor cells to assume a more primitive pluripotent phenotype and that IR-induced oxidative DNA damage may lead to mutations in these BM progenitors. This could then be propagated to successive cell lineages. Persistent impairment of BM progenitor cell populations can disrupt hematopoietic homeostasis and lead to hematologic disorders, and these findings warrant further mechanistic studies into the effects of low-dose IR on the functional capacity of BM-derived hematopoietic cells including their self-renewal and pluripotency. PMID:26528440

  15. Ionizing Particle Radiation as a Modulator of Endogenous Bone Marrow Cell Reprogramming: Implications for Hematological Cancers

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    Sujatha eMuralidharan

    2015-10-01

    Full Text Available Exposure of individuals to ionizing radiation (IR, as in the case of astronauts exploring space or radiotherapy cancer patients, increases their risk of developing secondary cancers and other health-related problems. Bone marrow (BM, the site in the body where hematopoietic stem cell (HSC self-renewal and differentiation to mature blood cells occurs, is extremely sensitive to low dose IR, including irradiation by high-charge and high-energy particles (HZE. Low dose IR induces DNA damage and persistent oxidative stress in the BM hematopoietic cells. Inefficient DNA repair processes in HSC and early hematopoietic progenitors can lead to an accumulation of mutations whereas long-lasting oxidative stress can impair hematopoiesis itself, thereby causing long term damage to hematopoietic cells in the BM niche. We report here that low dose 1H- and 56Fe-IR significantly decreased the hematopoietic early and late multipotent progenitor (E- and L-MPP, respectively cell numbers in mouse BM over a period of up to 10 months after exposure. Both 1H- and 56Fe-IR increased the expression of pluripotent stem cell markers Sox2, Nanog and Oct-4 in Late-MPPs 2 and 10 months post-IR exposure. We postulate that low doses of 1H- and 56Fe-IR may induce endogenous cellular reprogramming of BM hematopoietic progenitor cells to assume a more primitive pluripotent phenotype; IR-induced oxidative DNA damage may lead to mutations in these BM progenitors. This could then be propagated to successive cell lineages. Persistent impairment of BM progenitor cell populations can disrupt hematopoietic homeostasis and lead to hematologic disorders and these findings warrant further mechanistic studies into the effects of low dose IR on the functional capacity of BM-derived hematopoietic cells including their self-renewal and pluripotency.

  16. Farnesoid X receptor activation promotes cell proliferation via PDK4-controlled metabolic reprogramming

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    Xie, Yang; Wang, Hong; Cheng, Xuefang; Wu, Yuzheng; Cao, Lijuan; Wu, Mengqiu; Xie, Wen; Wang, Guangji; Hao, Haiping

    2016-01-01

    Farnesoid X receptor (FXR) plays a pivotal role in the regulation of various metabolic pathways as well as liver regeneration. However, the casual link between cell proliferative effects during liver regeneration and metabolic regulation of FXR was elusive. In this study, we found that FXR activation significantly promotes HepG2 cell proliferation accompanied with metabolic switch towards the excessive accumulation of aerobic glycolytic intermediates including lactic acid, pyruvate and the subsequently increased biosynthesis of glycine. This FXR-induced metabolic switch was found dependent on an up-regulation of pyruvate dehydrogenate kinase 4 (PDK4), a FXR target gene. FXR agonists were found to promote liver regeneration in the murine model of APAP induced liver injury, which was associated with a metabolic switch favoring the accumulation of glycolytic intermediates as precursors for generation of biomass. However, FXR activation has little effect on the glycolytic metabolism in healthy primary hepatocytes in vitro and the liver of healthy mice in vivo. Therefore, we conclude that FXR may promote the proliferation of tumor cells and the hepatocytes in the process of liver regeneration by activating the PDK4-mediated metabolic reprogramming to generate glycolytic intermediates essential for rapid biomass generation, establishing a mechanistic link between cell proliferation and metabolic switch. PMID:26728993

  17. GRIM-19 opposes reprogramming of glioblastoma cell metabolism via HIF1α destabilization.

    Science.gov (United States)

    Liu, Qian; Wang, Lulu; Wang, Zhaojuan; Yang, Yang; Tian, Jingxia; Liu, Guoliang; Guan, Dongshi; Cao, Xinmin; Zhang, Yanmin; Hao, Aijun

    2013-08-01

    The metabolism that sustains cancer cells is adapted preferentially to glycolysis, even under aerobic conditions (Warburg effect). This effect was one of the first alterations in cancer cells recognized as conferring a survival advantage. In this study, we show that gene associated with retinoid-interferon-induced mortality-19 (GRIM-19), which was previously identified as a tumor suppressor protein associated with growth inhibition and cell apoptosis, contributes to the switch between oxidative and glycolytic pathways. In parallel to this, vascular endothelial growth factor, which promotes neovascularization, is also regulated. We have identified hypoxia-inducible factor 1α (HIF1α) as the downstream factor of GRIM-19 in human glioblastoma cell lines. Downregulation of GRIM-19 promotes HIF1α synthesis in a STAT3-dependent manner, which acts as a potential competitive inhibitor for von Hippel-Lindau (pVHL)-HIF1α interaction, and thereby prevents HIF1α from pVHL-mediated ubiquitination and proteasomal degradation. Taken together, it is concluded that GRIM-19, a potential tumor suppressor gene, performs its function in part via regulating glioblastoma metabolic reprogramming through STAT3-HIF1α signaling axis, and this has added new perspective to its role in tumorigenesis, thus providing potential strategies for tumor metabolic therapy. PMID:23580587

  18. Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells.

    Science.gov (United States)

    Klawitter, Sabine; Fuchs, Nina V; Upton, Kyle R; Muñoz-Lopez, Martin; Shukla, Ruchi; Wang, Jichang; Garcia-Cañadas, Marta; Lopez-Ruiz, Cesar; Gerhardt, Daniel J; Sebe, Attila; Grabundzija, Ivana; Merkert, Sylvia; Gerdes, Patricia; Pulgarin, J Andres; Bock, Anja; Held, Ulrike; Witthuhn, Anett; Haase, Alexandra; Sarkadi, Balázs; Löwer, Johannes; Wolvetang, Ernst J; Martin, Ulrich; Ivics, Zoltán; Izsvák, Zsuzsanna; Garcia-Perez, Jose L; Faulkner, Geoffrey J; Schumann, Gerald G

    2016-01-08

    Human induced pluripotent stem cells (hiPSCs) are capable of unlimited proliferation and can differentiate in vitro to generate derivatives of the three primary germ layers. Genetic and epigenetic abnormalities have been reported by Wissing and colleagues to occur during hiPSC derivation, including mobilization of engineered LINE-1 (L1) retrotransposons. However, incidence and functional impact of endogenous retrotransposition in hiPSCs are yet to be established. Here we apply retrotransposon capture sequencing to eight hiPSC lines and three human embryonic stem cell (hESC) lines, revealing endogenous L1, Alu and SINE-VNTR-Alu (SVA) mobilization during reprogramming and pluripotent stem cell cultivation. Surprisingly, 4/7 de novo L1 insertions are full length and 6/11 retrotransposition events occurred in protein-coding genes expressed in pluripotent stem cells. We further demonstrate that an intronic L1 insertion in the CADPS2 gene is acquired during hiPSC cultivation and disrupts CADPS2 expression. These experiments elucidate endogenous retrotransposition, and its potential consequences, in hiPSCs and hESCs.

  19. ¹H NMR spectroscopy profiling of metabolic reprogramming of Chinese hamster ovary cells upon a temperature shift during culture.

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    Jane L Wagstaff

    Full Text Available We report an NMR based approach to determine the metabolic reprogramming of Chinese hamster ovary cells upon a temperature shift during culture by investigating the extracellular cell culture media and intracellular metabolome of CHOK1 and CHO-S cells during culture and in response to cold-shock and subsequent recovery from hypothermic culturing. A total of 24 components were identified for CHOK1 and 29 components identified for CHO-S cell systems including the observation that CHO-S media contains 5.6 times the level of glucose of CHOK1 media at time zero. We confirm that an NMR metabolic approach provides quantitative analysis of components such as glucose and alanine with both cell lines responding in a similar manner and comparable to previously reported data. However, analysis of lactate confirms a differentiation between CHOK1 and CHO-S and that reprogramming of metabolism in response to temperature was cell line specific. The significance of our results is presented using principal component analysis (PCA that confirms changes in metabolite profile in response to temperature and recovery. Ultimately, our approach demonstrates the capability of NMR providing real-time analysis to detect reprogramming of metabolism upon cellular perception of cold-shock/sub-physiological temperatures. This has the potential to allow manipulation of metabolites in culture supernatant to improve growth or productivity.

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

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    Akira Shimamoto

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

  1. Substrate-mediated reprogramming of human fibroblasts into neural crest stem-like cells and their applications in neural repair.

    Science.gov (United States)

    Tseng, Ting-Chen; Hsieh, Fu-Yu; Dai, Niann-Tzyy; Hsu, Shan-Hui

    2016-09-01

    Cell- and gene-based therapies have emerged as promising strategies for treating neurological diseases. The sources of neural stem cells are limited while the induced pluripotent stem (iPS) cells have risk of tumor formation. Here, we proposed the generation of self-renewable, multipotent, and neural lineage-related neural crest stem-like cells by chitosan substrate-mediated gene transfer of a single factor forkhead box D3 (FOXD3) for the use in neural repair. A simple, non-toxic, substrate-mediated method was applied to deliver the naked FOXD3 plasmid into human fibroblasts. The transfection of FOXD3 increased cell proliferation and up-regulated the neural crest marker genes (FOXD3, SOX2, and CD271), stemness marker genes (OCT4, NANOG, and SOX2), and neural lineage-related genes (Nestin, β-tubulin and GFAP). The expression levels of stemness marker genes and neural crest maker genes in the FOXD3-transfected fibroblasts were maintained until the fifth passage. The FOXD3 reprogrammed fibroblasts based on the new method significantly rescued the neural function of the impaired zebrafish. The chitosan substrate-mediated delivery of naked plasmid showed feasibility in reprogramming somatic cells. Particularly, the FOXD3 reprogrammed fibroblasts hold promise as an easily accessible cellular source with neural crest stem-like behavior for treating neural diseases in the future. PMID:27341268

  2. Somatic Cell Nuclear Transfer in the Mouse

    Science.gov (United States)

    Kishigami, Satoshi; Wakayama, Teruhiko

    Somatic cell nuclear transfer (SCNT) has become a unique and powerful tool for epigenetic reprogramming research and gene manipulation in animals since “Dolly,” the first animal cloned from an adult cell was reported in 1997. Although the success rates of somatic cloning have been inefficient and the mechanism of reprogramming is still largely unknown, this technique has been proven to work in more than 10 mammalian species. Among them, the mouse provides the best model for both basic and applied research of somatic cloning because of its abounding genetic resources, rapid sexual maturity and propagation, minimal requirements for housing, etc. This chapter describes a basic protocol for mouse cloning using cumulus cells, the most popular cell type for NT, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. In particular, we focus on a new, more efficient mouse cloning protocol using trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, which increases both in vitro and in vivo developmental rates from twofold to fivefold. This new method including TSA will be helpful to establish mouse cloning in many laboratories.

  3. Isonitrosoacetophenone drives transcriptional reprogramming in Nicotiana tabacum cells in support of innate immunity and defense.

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    Arnaud T Djami-Tchatchou

    Full Text Available Plants respond to various stress stimuli by activating broad-spectrum defense responses both locally as well as systemically. As such, identification of expressed genes represents an important step towards understanding inducible defense responses and assists in designing appropriate intervention strategies for disease management. Genes differentially expressed in tobacco cell suspensions following elicitation with isonitrosoacetophenone (INAP were identified using mRNA differential display and pyro-sequencing. Sequencing data produced 14579 reads, which resulted in 198 contigs and 1758 singletons. Following BLAST analyses, several inducible plant defense genes of interest were identified and classified into functional categories including signal transduction, transcription activation, transcription and protein synthesis, protein degradation and ubiquitination, stress-responsive, defense-related, metabolism and energy, regulation, transportation, cytoskeleton and cell wall-related. Quantitative PCR was used to investigate the expression of 17 selected target genes within these categories. Results indicate that INAP has a sensitising or priming effect through activation of salicylic acid-, jasmonic acid- and ethylene pathways that result in an altered transcriptome, with the expression of genes involved in perception of pathogens and associated cellular re-programming in support of defense. Furthermore, infection assays with the pathogen Pseudomonas syringae pv. tabaci confirmed the establishment of a functional anti-microbial environment in planta.

  4. Novel HDAd/EBV Reprogramming Vector and Highly Efficient Ad/CRISPR-Cas Sickle Cell Disease Gene Correction.

    Science.gov (United States)

    Li, Chao; Ding, Lei; Sun, Chiao-Wang; Wu, Li-Chen; Zhou, Dewang; Pawlik, Kevin M; Khodadadi-Jamayran, Alireza; Westin, Erik; Goldman, Frederick D; Townes, Tim M

    2016-01-01

    CRISPR/Cas enhanced correction of the sickle cell disease (SCD) genetic defect in patient-specific induced Pluripotent Stem Cells (iPSCs) provides a potential gene therapy for this debilitating disease. An advantage of this approach is that corrected iPSCs that are free of off-target modifications can be identified before differentiating the cells into hematopoietic progenitors for transplantation. In order for this approach to be practical, iPSC generation must be rapid and efficient. Therefore, we developed a novel helper-dependent adenovirus/Epstein-Barr virus (HDAd/EBV) hybrid reprogramming vector, rCLAE-R6, that delivers six reprogramming factors episomally. HDAd/EBV transduction of keratinocytes from SCD patients resulted in footprint-free iPSCs with high efficiency. Subsequently, the sickle mutation was corrected by delivering CRISPR/Cas9 with adenovirus followed by nucleoporation with a 70 nt single-stranded oligodeoxynucleotide (ssODN) correction template. Correction efficiencies of up to 67.9% (β(A)/[β(S)+β(A)]) were obtained. Whole-genome sequencing (WGS) of corrected iPSC lines demonstrated no CRISPR/Cas modifications in 1467 potential off-target sites and no modifications in tumor suppressor genes or other genes associated with pathologies. These results demonstrate that adenoviral delivery of reprogramming factors and CRISPR/Cas provides a rapid and efficient method of deriving gene-corrected, patient-specific iPSCs for therapeutic applications. PMID:27460639

  5. Novel HDAd/EBV Reprogramming Vector and Highly Efficient Ad/CRISPR-Cas Sickle Cell Disease Gene Correction

    Science.gov (United States)

    Li, Chao; Ding, Lei; Sun, Chiao-Wang; Wu, Li-Chen; Zhou, Dewang; Pawlik, Kevin M.; Khodadadi-Jamayran, Alireza; Westin, Erik; Goldman, Frederick D.; Townes, Tim M.

    2016-01-01

    CRISPR/Cas enhanced correction of the sickle cell disease (SCD) genetic defect in patient-specific induced Pluripotent Stem Cells (iPSCs) provides a potential gene therapy for this debilitating disease. An advantage of this approach is that corrected iPSCs that are free of off-target modifications can be identified before differentiating the cells into hematopoietic progenitors for transplantation. In order for this approach to be practical, iPSC generation must be rapid and efficient. Therefore, we developed a novel helper-dependent adenovirus/Epstein-Barr virus (HDAd/EBV) hybrid reprogramming vector, rCLAE-R6, that delivers six reprogramming factors episomally. HDAd/EBV transduction of keratinocytes from SCD patients resulted in footprint-free iPSCs with high efficiency. Subsequently, the sickle mutation was corrected by delivering CRISPR/Cas9 with adenovirus followed by nucleoporation with a 70 nt single-stranded oligodeoxynucleotide (ssODN) correction template. Correction efficiencies of up to 67.9% (βA/[βS+βA]) were obtained. Whole-genome sequencing (WGS) of corrected iPSC lines demonstrated no CRISPR/Cas modifications in 1467 potential off-target sites and no modifications in tumor suppressor genes or other genes associated with pathologies. These results demonstrate that adenoviral delivery of reprogramming factors and CRISPR/Cas provides a rapid and efficient method of deriving gene-corrected, patient-specific iPSCs for therapeutic applications. PMID:27460639

  6. Different developmental potential of pluripotent stem cells generated by different reprogramming strategies

    Institute of Scientific and Technical Information of China (English)

    Jing Jiang; Yixue Li; Jiarui Wu; Jinsong Li; Guohui Ding; Jiangwei Lin; Man Zhang; Linyu Shi; Wenjian Lv; Hui Yang; Huasheng Xiao; Gang Pei

    2011-01-01

    @@ Dear Editor, Recent studies show that induced pluripotent stem cells (iPSCs) generated through ectopic expression of transcription factors retain an epigenetic memory of their original somatic cells (Kim et al., 2010; Polo et al., 2010) or aberrant silencing of a single imprinted gene cluster (Liu et al.,2010; Stadtfeld et al., 2010), which affects their developmental and differentiation potentials.In contrast, nuclear transfer can more faithfully reprogramme somatic cells into embryonic stem (ES)cells (nuclear transfer ES cells, ntESCs)(Brambrink et al., 2006; Wakayama et al.,2006).

  7. Genetic reprogramming of human amniotic cells with episomal vectors: neural rosettes as sentinels in candidate selection for validation assays

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    Patricia G. Wilson

    2014-11-01

    Full Text Available The promise of genetic reprogramming has prompted initiatives to develop banks of induced pluripotent stem cells (iPSCs from diverse sources. Sentinel assays for pluripotency could maximize available resources for generating iPSCs. Neural rosettes represent a primitive neural tissue that is unique to differentiating PSCs and commonly used to identify derivative neural/stem progenitors. Here, neural rosettes were used as a sentinel assay for pluripotency in selection of candidates to advance to validation assays. Candidate iPSCs were generated from independent populations of amniotic cells with episomal vectors. Phase imaging of living back up cultures showed neural rosettes in 2 of the 5 candidate populations. Rosettes were immunopositive for the Sox1, Sox2, Pax6 and Pax7 transcription factors that govern neural development in the earliest stage of development and for the Isl1/2 and Otx2 transcription factors that are expressed in the dorsal and ventral domains, respectively, of the neural tube in vivo. Dissociation of rosettes produced cultures of differentiation competent neural/stem progenitors that generated immature neurons that were immunopositive for βIII-tubulin and glia that were immunopositive for GFAP. Subsequent validation assays of selected candidates showed induced expression of endogenous pluripotency genes, epigenetic modification of chromatin and formation of teratomas in immunodeficient mice that contained derivatives of the 3 embryonic germ layers. Validated lines were vector-free and maintained a normal karyotype for more than 60 passages. The credibility of rosette assembly as a sentinel assay for PSCs is supported by coordinate loss of nuclear-localized pluripotency factors Oct4 and Nanog in neural rosettes that emerge spontaneously in cultures of self-renewing validated lines. Taken together, these findings demonstrate value in neural rosettes as sentinels for pluripotency and selection of promising candidates for advance

  8. Cell direct reprogramming:a new technique for treating diabetes%细胞直接重编程--治疗糖尿病的新技术

    Institute of Scientific and Technical Information of China (English)

    任丽伟; 杨晓菲; 李富荣

    2014-01-01

    [ ABSTRACT] Diabetes is characterized by an absolute or relative deficiency inβ-cell mass, which cannot be re-versed with existing therapeutic strategies.The restoration of the endogenous islet β-cells can stabilize the level of blood glucose.The isletβ-cells can be obtained from the directional differentiation of stem cells, but the process is complex and has the risk of teratomas generation.Cell direct reprogramming, one terminal differentiated cell can transdifferentiate into another kind of terminal differentiated cell, which is other than directional differentiation from stem cells.Direct reprogram-ming gives rise to the generation of isletβ-cells from one terminal differentiated cell, may be preferable for diabetes therapy because of its unique advantage.

  9. 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. PMID:27160443

  10. Differential nuclear remodeling of mammalian somatic cells by Xenopus laevis oocyte and egg cytoplasm

    International Nuclear Information System (INIS)

    The mechanisms governing nuclear reprogramming have not been fully elucidated yet; however, recent studies show a universally conserved ability of both oocyte and egg components to reprogram gene expression in somatic cells. The activation of genes associated with pluripotency by oocyte/egg components may require the remodeling of nuclear structures, such that they can acquire the features of early embryos and pluripotent cells. Here, we report on the remodeling of the nuclear lamina of mammalian cells by Xenopus oocyte and egg extracts. Lamin A/C is removed from somatic cells incubated in oocyte and egg extracts in an active process that requires permeable nuclear pores. Removal of lamin A/C is specific, since B-type lamins are not changed, and it is not dependent on the incorporation Xenopus egg specific lamin III. Moreover, transcriptional activity is differentially regulated in somatic cells incubated in the extracts. Pol I and II transcriptions are maintained in cells in oocyte extracts; however, both activities are abolished in egg extracts. Our study shows that components of oocyte and egg extracts can modify the nuclear lamina of somatic cells and that this nuclear remodeling induces a structural change in the nucleus which may have implications for transcriptional activity. These experiments suggest that modifications in the nuclear lamina structure by the removal of somatic proteins and the incorporation of oocyte/egg components may contribute to the reprogramming of somatic cell nuclei and may define a characteristic configuration of pluripotent cells

  11. Ectopic expression of Cvh (Chicken Vasa homologue) mediates the reprogramming of chicken embryonic stem cells to a germ cell fate.

    Science.gov (United States)

    Lavial, Fabrice; Acloque, Hervé; Bachelard, Elodie; Nieto, M Angela; Samarut, Jacques; Pain, Bertrand

    2009-06-01

    When they are derived from blastodermal cells of the pre-primitive streak in vitro, the pluripotency of Chicken Embryonic Stem Cells (cESC) can be controlled by the cPouV and Nanog genes. These cESC can differentiate into derivatives of the three germ layers both in vitro and in vivo, but they only weakly colonize the gonads of host embryos. By contrast, non-cultured blastodermal cells and long-term cultured chicken primordial germ cells maintain full germline competence. This restriction in the germline potential of the cESC may result from either early germline determination in the donor embryos or it may occur as a result of in vitro culture. We are interested in understanding the genetic determinants of germline programming. The RNA binding protein Cvh (Chicken Vasa Homologue) is considered as one such determinant, although its role in germ cell physiology is still unclear. Here we show that the exogenous expression of Cvh, combined with appropriate culture conditions, induces cESC reprogramming towards a germ cell fate. Indeed, these cells express the Dazl, Tudor and Sycp3 germline markers, and they display improved germline colonization and adopt a germ cell fate when injected into recipient embryos. Thus, our results demonstrate that Vasa can drive ES cell differentiation towards the germ cell lineage, both in vitro and in vivo.

  12. Residual expression of reprogramming factors affects the transcriptional program and epigenetic signatures of induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Cesar A Sommer

    Full Text Available Delivery of the transcription factors Oct4, Klf4, Sox2 and c-Myc via integrating viral vectors has been widely employed to generate induced pluripotent stem cell (iPSC lines from both normal and disease-specific somatic tissues, providing an invaluable resource for medical research and drug development. Residual reprogramming transgene expression from integrated viruses nevertheless alters the biological properties of iPSCs and has been associated with a reduced developmental competence both in vivo and in vitro. We performed transcriptional profiling of mouse iPSC lines before and after excision of a polycistronic lentiviral reprogramming vector to systematically define the overall impact of persistent transgene expression on the molecular features of iPSCs. We demonstrate that residual expression of the Yamanaka factors prevents iPSCs from acquiring the transcriptional program exhibited by embryonic stem cells (ESCs and that the expression profiles of iPSCs generated with and without c-Myc are indistinguishable. After vector excision, we find 36% of iPSC clones show normal methylation of the Gtl2 region, an imprinted locus that marks ESC-equivalent iPSC lines. Furthermore, we show that the reprogramming factor Klf4 binds to the promoter region of Gtl2. Regardless of Gtl2 methylation status, we find similar endodermal and hepatocyte differentiation potential comparing syngeneic Gtl2(ON vs Gtl2(OFF iPSC clones. Our findings provide new insights into the reprogramming process and emphasize the importance of generating iPSCs free of any residual transgene expression.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-01-17

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

  14. Growth factor-activated stem cell circuits and stromal signals cooperatively accelerate non-integrated iPSC reprogramming of human myeloid progenitors.

    Directory of Open Access Journals (Sweden)

    Tea Soon Park

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

  15. Quantitative 1H NMR metabolomics reveals extensive metabolic reprogramming of primary and secondary metabolism in elicitor-treated opium poppy cell cultures

    Directory of Open Access Journals (Sweden)

    Vogel Hans J

    2008-01-01

    Full Text Available Abstract Background Opium poppy (Papaver somniferum produces a diverse array of bioactive benzylisoquinoline alkaloids and has emerged as a model system to study plant alkaloid metabolism. The plant is cultivated as the only commercial source of the narcotic analgesics morphine and codeine, but also produces many other alkaloids including the antimicrobial agent sanguinarine. Modulations in plant secondary metabolism as a result of environmental perturbations are often associated with the altered regulation of other metabolic pathways. As a key component of our functional genomics platform for opium poppy we have used proton nuclear magnetic resonance (1H NMR metabolomics to investigate the interplay between primary and secondary metabolism in cultured opium poppy cells treated with a fungal elicitor. Results Metabolite fingerprinting and compound-specific profiling showed the extensive reprogramming of primary metabolic pathways in association with the induction of alkaloid biosynthesis in response to elicitor treatment. Using Chenomx NMR Suite v. 4.6, a software package capable of identifying and quantifying individual compounds based on their respective signature spectra, the levels of 42 diverse metabolites were monitored over a 100-hour time course in control and elicitor-treated opium poppy cell cultures. Overall, detectable and dynamic changes in the metabolome of elicitor-treated cells, especially in cellular pools of carbohydrates, organic acids and non-protein amino acids were detected within 5 hours after elicitor treatment. The metabolome of control cultures also showed substantial modulations 80 hours after the start of the time course, particularly in the levels of amino acids and phospholipid pathway intermediates. Specific flux modulations were detected throughout primary metabolism, including glycolysis, the tricarboxylic acid cycle, nitrogen assimilation, phospholipid/fatty acid synthesis and the shikimate pathway, all of which

  16. 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.

  17. 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

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    Exposure to ionizing radiation increases the risk of chronic metabolic disorders such as insulin resistance and type 2 diabetes later in life. We hypothesized that irradiation reprograms the epigenome of metabolic progenitor cells, which could account for impaired metabolism after cancer treatment....... C57Bl/6 mice were treated with a single dose of irradiation and subjected to high fat diet (HFD). RNA Sequencing and Reduced Representation Bisulfite Sequencing were used to create transcriptomic and epigenomic profiles of preadipocytes and skeletal muscle satellite cells collected from irradiated...

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

    Directory of Open Access Journals (Sweden)

    Lee K

    2015-03-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  1. Oxidative modifications of glyceraldehyde 3-phosphate dehydrogenase regulate metabolic reprogramming of stored red blood cells.

    Science.gov (United States)

    Reisz, Julie A; Wither, Matthew J; Dzieciatkowska, Monika; Nemkov, Travis; Issaian, Aaron; Yoshida, Tatsuro; Dunham, Andrew J; Hill, Ryan C; Hansen, Kirk C; D'Alessandro, Angelo

    2016-09-22

    Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) plays a key regulatory function in glucose oxidation by mediating fluxes through glycolysis or the pentose phosphate pathway (PPP) in an oxidative stress-dependent fashion. Previous studies documented metabolic reprogramming in stored red blood cells (RBCs) and oxidation of GAPDH at functional residues upon exposure to pro-oxidants diamide and H2O2 Here we hypothesize that routine storage of erythrocyte concentrates promotes metabolic modulation of stored RBCs by targeting functional thiol residues of GAPDH. Progressive increases in PPP/glycolysis ratios were determined via metabolic flux analysis after spiking (13)C1,2,3-glucose in erythrocyte concentrates stored in Additive Solution-3 under blood bank conditions for up to 42 days. Proteomics analyses revealed a storage-dependent oxidation of GAPDH at functional Cys152, 156, 247, and His179. Activity loss by oxidation occurred with increasing storage duration and was progressively irreversible. Irreversibly oxidized GAPDH accumulated in stored erythrocyte membranes and supernatants through storage day 42. By combining state-of-the-art ultra-high-pressure liquid chromatography-mass spectrometry metabolic flux analysis with redox and switch-tag proteomics, we identify for the first time ex vivo functionally relevant reversible and irreversible (sulfinic acid; Cys to dehydroalanine) oxidations of GAPDH without exogenous supplementation of excess pro-oxidant compounds in clinically relevant blood products. Oxidative and metabolic lesions, exacerbated by storage under hyperoxic conditions, were ameliorated by hypoxic storage. Storage-dependent reversible oxidation of GAPDH represents a mechanistic adaptation in stored erythrocytes to promote PPP activation and generate reducing equivalents. Removal of irreversibly oxidized, functionally compromised GAPDH identifies enhanced vesiculation as a self-protective mechanism in ex vivo aging erythrocytes.

  2. Reprogramming anti-tumor immunity

    Science.gov (United States)

    Crompton, Joseph G.; Clever, David; Vizcardo, Raul; Rao, Mahendra; Restifo, Nicholas P.

    2014-01-01

    Regenerative medicine holds great promise in replacing tissues and organs lost to degenerative disease and injury. Applying principles of cellular reprogramming for the treatment of cancer, however, are not well established. Here we present an overview of cell-based reprogramming techniques (i.e. lineage reprogramming and stimulus-triggered acquisition of pluripotency) used in regenerative medicine, and within this context, envision how the scope of regenerative medicine may be expanded to treat metastatic cancer by revitalizing an exhausted and senescent immune system. PMID:24661777

  3. Human mesenchymal stem cells reprogram adult cardiomyocytes toward a progenitor-like state through partial cell fusion and mitochondria transfer : Cell fusion-mediated cardiomyocyte reprogramming.

    OpenAIRE

    Acquistapace, Adrien; Bru, Thierry; Lesault, Pierre-François; Figeac, Florence; Coudert, Amélie,; Le Coz, Olivier; Christov, Christo; Baudin, Xavier; Auber, Fréderic; Yiou, René; Dubois-Randé, Jean-Luc; Rodriguez, Anne-Marie

    2011-01-01

    International audience Because stem cells are often found to improve repair tissue including heart without evidence of engraftment or differentiation, mechanisms underlying wound healing are still elusive. Several studies have reported that stem cells can fuse with cardiomyocytes either by permanent or partial cell fusion processes. However, the respective physiological impact of these two processes remains unknown in part because of the lack of knowledge of the resulting hybrid cells. To ...

  4. Somatic cell nuclear transfer: pros and cons.

    Science.gov (United States)

    Sumer, Huseyin; Liu, Jun; Tat, Pollyanna; Heffernan, Corey; Jones, Karen L; Verma, Paul J

    2009-01-01

    Even though the technique of mammalian SCNT is just over a decade old it has already resulted in numerous significant advances. Despite the recent advances in the reprogramming field, SCNT remains the bench-mark for the generation of both genetically unmodified autologous pluripotent stem cells for transplantation and for the production of cloned animals. In this review we will discuss the pros and cons of SCNT, drawing comparisons with other reprogramming methods. PMID:20232594

  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. Sustained ERK Activation Underlies Reprogramming in Regeneration-Competent Salamander Cells and Distinguishes Them from Their Mammalian Counterparts

    Science.gov (United States)

    Yun, Maximina H.; Gates, Phillip B.; Brockes, Jeremy P.

    2014-01-01

    Summary In regeneration-competent vertebrates, such as salamanders, regeneration depends on the ability of various differentiated adult cell types to undergo natural reprogramming. This ability is rarely observed in regeneration-incompetent species such as mammals, providing an explanation for their poor regenerative potential. To date, little is known about the molecular mechanisms mediating natural reprogramming during regeneration. Here, we have identified the extent of extracellular signal-regulated kinase (ERK) activation as a key component of such mechanisms. We show that sustained ERK activation following serum induction is required for re-entry into the cell cycle of postmitotic salamander muscle cells, partially by promoting the downregulation of p53 activity. Moreover, ERK activation induces epigenetic modifications and downregulation of muscle-specific genes such as Sox6. Remarkably, while long-term ERK activation is found in salamander myotubes, only transient activation is seen in their mammalian counterparts, suggesting that the extent of ERK activation could underlie differences in regenerative competence between species. PMID:25068118

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

    OpenAIRE

    Maria ePoulou; Nikolaos eMandalos; Theodoros eKarnavas; Marannia eSaridaki; Ronald G. McKay; Eumorphia eRemboutsika

    2016-01-01

    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 nest...

  8. Conditionally Stabilized dCas9 Activator for Controlling Gene Expression in Human Cell Reprogramming and Differentiation

    Directory of Open Access Journals (Sweden)

    Diego Balboa

    2015-09-01

    Full Text Available CRISPR/Cas9 protein fused to transactivation domains can be used to control gene expression in human cells. In this study, we demonstrate that a dCas9 fusion with repeats of VP16 activator domains can efficiently activate human genes involved in pluripotency in various cell types. This activator in combination with guide RNAs targeted to the OCT4 promoter can be used to completely replace transgenic OCT4 in human cell reprogramming. Furthermore, we generated a chemically controllable dCas9 activator version by fusion with the dihydrofolate reductase (DHFR destabilization domain. Finally, we show that the destabilized dCas9 activator can be used to control human pluripotent stem cell differentiation into endodermal lineages.

  9. Bright/Arid3A Acts as a Barrier to Somatic Cell Reprogramming through Direct Regulation of Oct4, Sox2, and Nanog

    Directory of Open Access Journals (Sweden)

    Melissa Popowski

    2014-01-01

    Full Text Available We show here that singular loss of the Bright/Arid3A transcription factor leads to reprograming of mouse embryonic fibroblasts (MEFs and enhancement of standard four-factor (4F reprogramming. Bright-deficient MEFs bypass senescence and, under standard embryonic stem cell (ESC culture conditions, spontaneously form clones that in vitro express pluripotency markers, differentiate to all germ lineages, and in vivo form teratomas and chimeric mice. We demonstrate that BRIGHT binds directly to the promoter/enhancer regions of Oct4, Sox2, and Nanog to contribute to their repression in both MEFs and ESCs. Thus, elimination of the BRIGHT barrier may provide an approach for somatic cell reprogramming.

  10. 重编程细胞的命运%Fate of reprogramming cells

    Institute of Scientific and Technical Information of China (English)

    阮光萍; 姚翔; 刘菊芬; 胡媛媛; 王金祥; 何洁; 赵晶; 潘兴华

    2015-01-01

    背景:体细胞重新编程技术也称细胞重组技术,使已经完成分化的体细胞回到原始的全能性或多能性状态,并可以重新分化成与原来不一样的细胞。通过重编程技术可以获得患者特异性诱导多能干细胞和疾病特异性诱导多能干细胞,显著减少了免疫排斥反应。目的:探讨关于直接重编程到特定系的方法,总结参与重编程的分子机制。方法:以“重编程”为中文检索词,“reprogramming”为英文检索词,应用计算机检索维普(VIP)期刊全文数据库、万方全文数据库、中国知网全文数据库、PubMed 数据库、Springer 数据库1958年1月至2015年4月有关细胞重编程技术的文献,排除与研究目的无关及重复性研究,保留40篇文献进一步分析。结果与结论:当前重编程的步骤效率很低,在特定群只有相对少量的细胞能进行重编程,重编程的完整性和程度也有待证实。直接重编程成体、定系的细胞从一种细胞到另一种细胞一直是发育生物学很难达到的目标。最近的研究证明分化的细胞强制表达特异转录因子能促进细胞分化。这些发现使再生医学领域有了重大进展,可以提供替代细胞治疗各种再生紊乱。目前,基本的分子机制需要进一步阐明,在直接重编程被应用于临床之前还有许多问题需要解决。%BACKGROUND:Somatic cel reprogramming technology, also known as recombinant technology, has completed differentiated somatic cels back to the original totipotent or pluripotent state, and can be re-differentiated into cels different from original ones. Re-programming techniques are able to harvest specificaly induced pluripotent stem cels and disease-specific induced pluripotent stem cels from patients, which can significantly reduce the immune rejection. OBJECTIVE: To explore the method from direct reprogramming to specific cel lines and to conclude the

  11. Efficient non-viral reprogramming of myoblasts to stemness with a single small molecule to generate cardiac progenitor cells.

    Directory of Open Access Journals (Sweden)

    Zeeshan Pasha

    Full Text Available UNLABELLED: The current protocols for generation of induced pluripotent stem (iPS cells involve genome integrating viral vectors which may induce tumorgenesis. The aim of this study was to develop and optimize a non-viral method without genetic manipulation for reprogramming of skeletal myoblasts (SMs using small molecules. METHODS AND RESULTS: SMs from young male Oct3/4-GFP(+ transgenic mouse were treated with DNA methyltransferase (DNMT inhibitor, RG108. Two weeks later, GFP(+ colonies of SM derived iPS cells (SiPS expressing GFP and with morphological similarity of mouse embryonic stem (ESCs were formed and propagated in vitro. SiPS were positive for alkaline phosphatase activity, expressed SSEA1, displayed ES cell specific pluripotency markers and formed teratoma in nude mice. Optimization of culture conditions for embryoid body (EBs formation yielded spontaneously contracting EBs having morphological, molecular, and ultra-structural similarities with cardiomyocytes and expressed early and late cardiac markers. miR profiling showed abrogation of let-7 family and upregulation of ESCs specific miR-290-295 cluster thus indicating that SiPS were similar to ESCs in miR profile. Four weeks after transplantation into the immunocompetent mice model of acute myocardial infarction (n = 12 per group, extensive myogenesis was observed in SiPS transplanted hearts as compared to DMEM controls (n = 6 per group. A significant reduction in fibrosis and improvement in global heart function in the hearts transplanted with SiPS derived cardiac progenitor cells were observed. CONCLUSIONS: Reprogramming of SMs by DNMT inhibitor is a simple, reproducible and efficient technique more likely to generate transgene integration-free iPS cells. Cardiac progenitors derived from iPS cells propagated extensively in the infarcted myocardium without tumorgenesis and improved cardiac function.

  12. LET-418/Mi2 and SPR-5/LSD1 Cooperatively Prevent Somatic Reprogramming of C. elegans Germline Stem Cells

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    Stéphanie Käser-Pébernard

    2014-04-01

    Full Text Available Throughout their journey to forming new individuals, germline stem cells must remain totipotent, particularly by maintaining a specific chromatin structure. However, the place epigenetic factors occupy in this process remains elusive. So far, “sensitization” of chromatin by modulation of histone arrangement and/or content was believed to facilitate transcription-factor-induced germ cell reprogramming. Here, we demonstrate that the combined reduction of two epigenetic factors suffices to reprogram C. elegans germ cells. The histone H3K4 demethylase SPR-5/LSD1 and the chromatin remodeler LET-418/Mi2 function together in an early process to maintain germ cell status and act as a barrier to block precocious differentiation. This epigenetic barrier is capable of limiting COMPASS-mediated H3K4 methylation, because elevated H3K4me3 levels correlate with germ cell reprogramming in spr-5; let-418 mutants. Interestingly, germ cells deficient for spr-5 and let-418 mainly reprogram as neurons, suggesting that neuronal fate might be the first to be derepressed in early embryogenesis.

  13. C/EBPα creates elite cells for iPSC reprogramming by upregulating Klf4 and increasing the levels of Lsd1 and Brd4

    DEFF Research Database (Denmark)

    Di Stefano, Bruno; Collombet, Samuel; Jakobsen, Janus Schou;

    2016-01-01

    reprogrammed into iPSCs by the Yamanaka factors OSKM. Here we show that C/EBPα post-transcriptionally increases the abundance of several hundred proteins, including Lsd1, Hdac1, Brd4, Med1 and Cdk9, components of chromatin-modifying complexes present at super-enhancers. Lsd1 was found to be required for B cell...

  14. Bmil puSHHes reprogramming

    Institute of Scientific and Technical Information of China (English)

    Han Li; Manuel Serrano

    2011-01-01

    In 2006,the group of Shinya Yamanaka demonstrated that somatic cells could be reprogrammed into induced pluripotent stem cells (iPSCs) by ectopic expression of four transcription factors associated to stemness:Oct4,Sox2,Klf4 and c-Myc [1].This groundbreaking discovery opened the possibility of generating patient-specific cells for research,drug development and regenerative medicine.Due to the tremendous potential of its clinical applications,understanding the process of reprogramming has become a priority and one of the most fascinating biomedical research topics.

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

    Directory of Open Access Journals (Sweden)

    Hyojung Jeon

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

  16. Metabolic reprogramming for producing energy and reducing power in fumarate hydratase null cells from hereditary leiomyomatosis renal cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Youfeng Yang

    Full Text Available Fumarate hydratase (FH-deficient kidney cancer undergoes metabolic remodeling, with changes in mitochondrial respiration, glucose, and glutamine metabolism. These changes represent multiple biochemical adaptations in glucose and fatty acid metabolism that supports malignant proliferation. However, the metabolic linkages between altered mitochondrial function, nucleotide biosynthesis and NADPH production required for proliferation and survival have not been elucidated. To characterize the alterations in glycolysis, the Krebs cycle and the pentose phosphate pathways (PPP that either generate NADPH (oxidative or do not (non-oxidative, we utilized [U-(13C]-glucose, [U-(13C,(15N]-glutamine, and [1,2- (13C2]-glucose tracers with mass spectrometry and NMR detection to track these pathways, and measured the oxygen consumption rate (OCR and extracellular acidification rate (ECAR of growing cell lines. This metabolic reprogramming in the FH null cells was compared to cells in which FH has been restored. The FH null cells showed a substantial metabolic reorganization of their intracellular metabolic fluxes to fulfill their high ATP demand, as observed by a high rate of glucose uptake, increased glucose turnover via glycolysis, high production of glucose-derived lactate, and low entry of glucose carbon into the Krebs cycle. Despite the truncation of the Krebs cycle associated with inactivation of fumarate hydratase, there was a small but persistent level of mitochondrial respiration, which was coupled to ATP production from oxidation of glutamine-derived α-ketoglutarate through to fumarate. [1,2- (13C2]-glucose tracer experiments demonstrated that the oxidative branch of PPP initiated by glucose-6-phosphate dehydrogenase activity is preferentially utilized for ribose production (56-66% that produces increased amounts of ribose necessary for growth and NADPH. Increased NADPH is required to drive reductive carboxylation of α-ketoglutarate and fatty acid

  17. Rapid and efficient reprogramming of human fetal and adult blood CD34+ cells into mesenchymal stem cells with a single factor

    Institute of Scientific and Technical Information of China (English)

    Xianmei Meng; Rui-Jun Su; David J Baylink; Amanda Neises; Jason B Kiroyan; Wayne Yuk-Wai Lee; Kimberly J Payne

    2013-01-01

    The direct conversion of skin cells into somatic stem cells has opened new therapeutic possibilities in regenerative medicine.Here,we show that human induced mesenchymal stem cells (iMSCs) can be efficiently generated from cord blood (CB)-or adult peripheral blood (PB)-CD34+ cells by direct reprogramming with a single factor,OCT4.In the presence of a GSK3 inhibitor,16% of the OCT4-transduced CD34+ cells are converted into iMSCs within 2 weeks.Efficient direct reprogramming is achieved with both episomal vector-mediated transient OCT4 expression and lentiviral vector-mediated OCT4 transduction.The iMSCs express MSC markers,resemble bone marrow (BM)-MSCs in morphology,and possess in vitro multilineage differentiation capacity,yet have a greater proliferative capacity compared with BM-MSCs.Similar to BM-MSCs,the implanted iMSCs form bone and connective tissues,and are non-tumorigenic in mice.However,BM-MSCs do not,whereas iMSCs do form muscle fibers,indicating a potential functional advantage of iMSCs.In addition,we observed that a high level of OCT4 expression is required for the initial reprogramming and the optimal iMSC self-renewal,while a reduction of OCT4 expression is required for multilineage differentiation.Our method will contribute to the generation of patient-specific iMSCs,which could have applications in regenerative medicine.This discovery may also facilitate the development of strategies for direct conversion of blood cells into other types of cells of clinical importance.

  18. Cumulus-specific genes are transcriptionally silent following somatic cell nuclear transfer in a mouse model*

    OpenAIRE

    Tong, Guo-qing; Heng, Boon-chin; Ng, Soon-chye

    2007-01-01

    This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 µmol/L strontium chloride for 5 h and sub...

  19. Asymmetric Reprogramming Capacity of Parental Pronuclei in Mouse Zygotes

    Directory of Open Access Journals (Sweden)

    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.

  20. PD-1 alters T-cell metabolic reprogramming by inhibiting glycolysis and promoting lipolysis and fatty acid oxidation

    Science.gov (United States)

    Patsoukis, Nikolaos; Bardhan, Kankana; Chatterjee, Pranam; Sari, Duygu; Liu, Bianling; Bell, Lauren N.; Karoly, Edward D.; Freeman, Gordon J.; Petkova, Victoria; Seth, Pankaj; Li, Lequn; Boussiotis, Vassiliki A.

    2015-01-01

    During activation, T cells undergo metabolic reprogramming, which imprints distinct functional fates. We determined that on PD-1 ligation, activated T cells are unable to engage in glycolysis or amino acid metabolism but have an increased rate of fatty acid β-oxidation (FAO). PD-1 promotes FAO of endogenous lipids by increasing expression of CPT1A, and inducing lipolysis as indicated by elevation of the lipase ATGL, the lipolysis marker glycerol and release of fatty acids. Conversely, CTLA-4 inhibits glycolysis without augmenting FAO, suggesting that CTLA-4 sustains the metabolic profile of non-activated cells. Because T cells utilize glycolysis during differentiation to effectors, our findings reveal a metabolic mechanism responsible for PD-1-mediated blockade of T-effector cell differentiation. The enhancement of FAO provides a mechanistic explanation for the longevity of T cells receiving PD-1 signals in patients with chronic infections and cancer, and for their capacity to be reinvigorated by PD-1 blockade. PMID:25809635

  1. Cumulus-specific genes are transcriptionally silent following somatic cell nuclear transfer in a mouse model

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 μmol/L strontium chloride for 5 h and subsequent in vitro culture up to the blastocyst stage. Expression of cumulus-specific genes in SCNT-derived embryos at 2-cell, 4-cell and day 4.5 blastocyst stages was compared with corresponding in vivo fertilized embryos by real-time PCR. It was demonstrated that immediately after the first cell cycle, SCNT-derived 2-cell stage embryos did not express all four cumulus-specific genes, which continually remained silent at the 4-cell and blastocyst stages. It is therefore concluded that all four cumulus-specific genes were correctly reprogrammed to be silent following nuclear transfer with cumulus donor cells in the mouse model. This would imply that the poor preimplantation developmental competence of SCNT embryos derived from cumulus cells is due to incomplete reprogramming of other embryonic genes, rather than cumulus-specific genes.

  2. Epigenetic Reprogramming of Human Embryonic Stem Cells into Skeletal Muscle Cells and Generation of Contractile Myospheres

    Directory of Open Access Journals (Sweden)

    Sonia Albini

    2013-03-01

    Full Text Available Direct generation of a homogeneous population of skeletal myoblasts from human embryonic stem cells (hESCs and formation of three-dimensional contractile structures for disease modeling in vitro are current challenges in regenerative medicine. Previous studies reported on the generation of myoblasts from ESC-derived embryoid bodies (EB, but not from undifferentiated ESCs, indicating the requirement for mesodermal transition to promote skeletal myogenesis. Here, we show that selective absence of the SWI/SNF component BAF60C (encoded by SMARCD3 confers on hESCs resistance to MyoD-mediated activation of skeletal myogenesis. Forced expression of BAF60C enables MyoD to directly activate skeletal myogenesis in hESCs by instructing MyoD positioning and allowing chromatin remodeling at target genes. BAF60C/MyoD-expressing hESCs are epigenetically committed myogenic progenitors, which bypass the mesodermal requirement and, when cultured as floating clusters, give rise to contractile three-dimensional myospheres composed of skeletal myotubes. These results identify BAF60C as a key epigenetic determinant of hESC commitment to the myogenic lineage and establish the molecular basis for the generation of hESC-derived myospheres exploitable for “disease in a dish” models of muscular physiology and dysfunction.

  3. Epigenetic reprogramming of human embryonic stem cells into skeletal muscle cells and generation of contractile myospheres.

    Science.gov (United States)

    Albini, Sonia; Coutinho, Paula; Malecova, Barbora; Giordani, Lorenzo; Savchenko, Alex; Forcales, Sonia Vanina; Puri, Pier Lorenzo

    2013-03-28

    Direct generation of a homogeneous population of skeletal myoblasts from human embryonic stem cells (hESCs) and formation of three-dimensional contractile structures for disease modeling in vitro are current challenges in regenerative medicine. Previous studies reported on the generation of myoblasts from ESC-derived embryoid bodies (EB), but not from undifferentiated ESCs, indicating the requirement for mesodermal transition to promote skeletal myogenesis. Here, we show that selective absence of the SWI/SNF component BAF60C (encoded by SMARCD3) confers on hESCs resistance to MyoD-mediated activation of skeletal myogenesis. Forced expression of BAF60C enables MyoD to directly activate skeletal myogenesis in hESCs by instructing MyoD positioning and allowing chromatin remodeling at target genes. BAF60C/MyoD-expressing hESCs are epigenetically committed myogenic progenitors, which bypass the mesodermal requirement and, when cultured as floating clusters, give rise to contractile three-dimensional myospheres composed of skeletal myotubes. These results identify BAF60C as a key epigenetic determinant of hESC commitment to the myogenic lineage and establish the molecular basis for the generation of hESC-derived myospheres exploitable for "disease in a dish" models of muscular physiology and dysfunction.

  4. Violacein induces death of resistant leukaemia cells via kinome reprogramming, endoplasmic reticulum stress and Golgi apparatus collapse.

    Directory of Open Access Journals (Sweden)

    Karla C S Queiroz

    Full Text Available It is now generally recognised that different modes of programmed cell death (PCD are intimately linked to the cancerous process. However, the mechanism of PCD involved in cancer chemoprevention is much less clear and may be different between types of chemopreventive agents and tumour cell types involved. Therefore, from a pharmacological view, it is crucial during the earlier steps of drug development to define the cellular specificity of the candidate as well as its capacity to bypass dysfunctional tumoral signalling pathways providing insensitivity to death stimuli. Studying the cytotoxic effects of violacein, an antibiotic dihydro-indolone synthesised by an Amazon river Chromobacterium, we observed that death induced in CD34(+/c-Kit(+/P-glycoprotein(+/MRP1(+ TF1 leukaemia progenitor cells is not mediated by apoptosis and/or autophagy, since biomarkers of both types of cell death were not significantly affected by this compound. To clarify the working mechanism of violacein, we performed kinome profiling using peptide arrays to yield comprehensive descriptions of cellular kinase activities. Pro-death activity of violacein is actually carried out by inhibition of calpain and DAPK1 and activation of PKA, AKT and PDK, followed by structural changes caused by endoplasmic reticulum stress and Golgi apparatus collapse, leading to cellular demise. Our results demonstrate that violacein induces kinome reprogramming, overcoming death signaling dysfunctions of intrinsically resistant human leukaemia cells.

  5. The mitochondrial H(+)-ATP synthase and the lipogenic switch: new core components of metabolic reprogramming in induced pluripotent stem (iPS) cells.

    Science.gov (United States)

    Vazquez-Martin, Alejandro; Corominas-Faja, Bruna; Cufi, Sílvia; Vellon, Luciano; Oliveras-Ferraros, Cristina; Menendez, Octavio J; Joven, Jorge; Lupu, Ruth; Menendez, Javier A

    2013-01-15

    Induced pluripotent stem (iPS) cells share some basic properties, such as self-renewal and pluripotency, with cancer cells, and they also appear to share several metabolic alterations that are commonly observed in human tumors. The cancer cells' glycolytic phenotype, first reported by Otto Warburg, is necessary for the optimal routing of somatic cells to pluripotency. However, how iPS cells establish a Warburg-like metabolic phenotype and whether the metabolic pathways that support the bioenergetics of iPS cells are produced by the same mechanisms that are selected during the tumorigenic process remain largely unexplored. We recently investigated whether the reprogramming-competent metabotype of iPS cells involves changes in the activation/expression status of the H(+)-ATPase, which is a core component of mitochondrial oxidative phosphorylation that is repressed at both the activity and protein levels in human carcinomas, and of the lipogenic switch, which refers to a marked overexpression and hyperactivity of the acetyl-CoA carboxylase (ACACA) and fatty acid synthase (FASN) lipogenic enzymes that has been observed in nearly all examined cancer types. A comparison of a starting population of mouse embryonic fibroblasts and their iPS cell progeny revealed that somatic cell reprogramming involves a significant increase in the expression of ATPase inhibitor factor 1 (IF1), accompanied by extremely low expression levels of the catalytic β-F1-ATPase subunit. The pharmacological inhibition of ACACA and FASN activities markedly decreases reprogramming efficiency, and ACACA and FASN expression are notably upregulated in iPS cells. Importantly, iPS cells exhibited a significant intracellular accumulation of neutral lipid bodies; however, these bodies may be a reflection of intense lysosomal/autophagocytic activity rather than bona fide lipid droplet formation in iPS cells, as they were largely unresponsive to pharmacological modulation of PPARgamma and FASN activities. The

  6. Cancer stem cell molecular reprogramming of the Warburg effect in glioblastomas: a new target gleaned from an old concept.

    Science.gov (United States)

    Yuen, Carlen A; Asuthkar, Swapna; Guda, Maheedhara R; Tsung, Andrew J; Velpula, Kiran K

    2016-01-01

    Prior targeted treatment for glioblastoma multiforme (GBM) with anti-angiogenic agents, such as bevacizumab, has been met with limited success potentially owing to GBM tumor's ability to develop a hypoxia-induced escape mechanism--a glycolytic switch from oxidative phosphorylation to glycolysis, an old concept known as the Warburg effect. New studies points to a subpopulation of cells as a source for treatment-resistance, cancer stem cells (CSCs). Taken together, the induction of the Warburg effect leads to the promotion of CSC self-renewal and undifferentiation. In response to hypoxia, hypoxia-inducible transcription factor is upregulated and is the central driver in setting off the cascade of events in CSC metabolic reprogramming. Hypoxia-inducible transcription factor upregulates GLUT1 to increase glucose uptake into the cell, upregulates HK2 and PK during glycolysis, upregulates LDHA in the termination of glycolysis, and downregulates PDH to redirect energy production toward glycolysis. This review aims to unite these old and new concepts simultaneously and examine potential enzyme targets driven by hypoxia in the glycolytic phenotype of CSCs to reverse the metabolic shift induced by the Warburg effect. PMID:26997129

  7. High in vitro development after somatic cell nuclear transfer and trichostatin A treatment of reconstructed porcine embryos

    DEFF Research Database (Denmark)

    Li, J.; Østrup, Olga; Villemoes, Klaus;

    2008-01-01

    Abnormal epigenetic modification is supposed to be one of factors accounting for inefficient reprogramming of the donor cell nuclei in ooplasm after somatic cell nuclear transfer (SCNT). Trichostatin A (TSA) is an inhibitor of histone deacetylase, potentially enhancing cloning efficiency. The aim...

  8. Metabolic Reprogramming and Dependencies Associated with Epithelial Cancer Stem Cells Independent of the Epithelial-Mesenchymal Transition Program.

    Science.gov (United States)

    Aguilar, Esther; Marin de Mas, Igor; Zodda, Erika; Marin, Silvia; Morrish, Fionnuala; Selivanov, Vitaly; Meca-Cortés, Óscar; Delowar, Hossain; Pons, Mònica; Izquierdo, Inés; Celià-Terrassa, Toni; de Atauri, Pedro; Centelles, Josep J; Hockenbery, David; Thomson, Timothy M; Cascante, Marta

    2016-05-01

    In solid tumors, cancer stem cells (CSCs) can arise independently of epithelial-mesenchymal transition (EMT). In spite of recent efforts, the metabolic reprogramming associated with CSC phenotypes uncoupled from EMT is poorly understood. Here, by using metabolomic and fluxomic approaches, we identify major metabolic profiles that differentiate metastatic prostate epithelial CSCs (e-CSCs) from non-CSCs expressing a stable EMT. We have found that the e-CSC program in our cellular model is characterized by a high plasticity in energy substrate metabolism, including an enhanced Warburg effect, a greater carbon and energy source flexibility driven by fatty acids and amino acid metabolism and an essential reliance on the proton buffering capacity conferred by glutamine metabolism. An analysis of transcriptomic data yielded a metabolic gene signature for our e-CSCs consistent with the metabolomics and fluxomics analyses that correlated with tumor progression and metastasis in prostate cancer and in 11 additional cancer types. Interestingly, an integrated metabolomics, fluxomics, and transcriptomics analysis allowed us to identify key metabolic players regulated at the post-transcriptional level, suggesting potential biomarkers and therapeutic targets to effectively forestall metastasis. Stem Cells 2016;34:1163-1176. PMID:27146024

  9. Metabolic reprogramming towards aerobic glycolysis correlates with greater proliferative ability and resistance to metabolic inhibition in CD8 versus CD4 T cells.

    Directory of Open Access Journals (Sweden)

    Yilin Cao

    Full Text Available T lymphocytes (T cells undergo metabolic reprogramming after activation to provide energy and biosynthetic materials for growth, proliferation and differentiation. Distinct T cell subsets, however, adopt metabolic programs specific to support their needs. As CD4 T cells coordinate adaptive immune responses while CD8 T cells become cytotoxic effectors, we compared activation-induced proliferation and metabolic reprogramming of these subsets. Resting CD4 and CD8 T cells were metabolically similar and used a predominantly oxidative metabolism. Following activation CD8 T cells proliferated more rapidly. Stimulation led both CD4 and CD8 T cells to sharply increase glucose metabolism and adopt aerobic glycolysis as a primary metabolic program. Activated CD4 T cells, however, remained more oxidative and had greater maximal respiratory capacity than activated CD8 T cells. CD4 T cells were also associated with greater levels of ROS and increased mitochondrial content, irrespective of the activation context. CD8 cells were better able, however, to oxidize glutamine as an alternative fuel source. The more glycolytic metabolism of activated CD8 T cells correlated with increased capacity for growth and proliferation, along with reduced sensitivity of cell growth to metabolic inhibition. These specific metabolic programs may promote greater growth and proliferation of CD8 T cells and enhance survival in diverse nutrient conditions.

  10. Live embryo imaging to follow cell cycle and chromosomes stability after nuclear transfer.

    Science.gov (United States)

    Balbach, Sebastian T; Boiani, Michele

    2015-01-01

    Nuclear transfer (NT) into mouse oocytes yields a transcriptionally and functionally heterogeneous population of cloned embryos. Most studies of NT embryos consider only embryos at predefined key stages (e.g., morula or blastocyst), that is, after the bulk of reprogramming has taken place. These retrospective approaches are of limited use to elucidate mechanisms of reprogramming and to predict developmental success. Observing cloned embryo development using live embryo cinematography has the potential to reveal otherwise undetectable embryo features. However, light exposure necessary for live cell cinematography is highly toxic to cloned embryos. Here we describe a protocol for combined bright-field and fluorescence live-cell imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This protocol, which can be adapted to observe other reporters such as Oct4-GFP or Nanog-GFP, allowed us to quantitatively analyze cleavage kinetics of cloned embryos. PMID:25287344

  11. Reprogramming of mouse amniotic fluid cells using a PiggyBac transposon system

    Directory of Open Access Journals (Sweden)

    E. Bertin

    2015-11-01

    Full Text Available Induced pluripotent stem (iPS cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Amniotic fluid (AF cells are easy to obtain from routinely scheduled procedures for prenatal diagnosis and iPS cells have been generated from human AF. Here, we generated iPS cells from mouse AF cells, using a non-viral-based approach constituted by the PiggyBac (PB transposon system. All iPS cell lines obtained exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo.

  12. WOX13-like genes are required for reprogramming of leaf and protoplast cells into stem cells in the moss Physcomitrella patens.

    Science.gov (United States)

    Sakakibara, Keiko; Reisewitz, Pascal; Aoyama, Tsuyoshi; Friedrich, Thomas; Ando, Sayuri; Sato, Yoshikatsu; Tamada, Yosuke; Nishiyama, Tomoaki; Hiwatashi, Yuji; Kurata, Tetsuya; Ishikawa, Masaki; Deguchi, Hironori; Rensing, Stefan A; Werr, Wolfgang; Murata, Takashi; Hasebe, Mitsuyasu; Laux, Thomas

    2014-04-01

    Many differentiated plant cells can dedifferentiate into stem cells, reflecting the remarkable developmental plasticity of plants. In the moss Physcomitrella patens, cells at the wound margin of detached leaves become reprogrammed into stem cells. Here, we report that two paralogous P. patens WUSCHEL-related homeobox 13-like (PpWOX13L) genes, homologs of stem cell regulators in flowering plants, are transiently upregulated and required for the initiation of cell growth during stem cell formation. Concordantly, Δppwox13l deletion mutants fail to upregulate genes encoding homologs of cell wall loosening factors during this process. During the moss life cycle, most of the Δppwox13l mutant zygotes fail to expand and initiate an apical stem cell to form the embryo. Our data show that PpWOX13L genes are required for the initiation of cell growth specifically during stem cell formation, in analogy to WOX stem cell functions in seed plants, but using a different cellular mechanism.

  13. C/EBPα creates elite cells for iPSC reprogramming by upregulating Klf4 and increasing the levels of Lsd1 and Brd4.

    Science.gov (United States)

    Di Stefano, Bruno; Collombet, Samuel; Jakobsen, Janus Schou; Wierer, Michael; Sardina, Jose Luis; Lackner, Andreas; Stadhouders, Ralph; Segura-Morales, Carolina; Francesconi, Mirko; Limone, Francesco; Mann, Matthias; Porse, Bo; Thieffry, Denis; Graf, Thomas

    2016-04-01

    Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) is typically inefficient and has been explained by elite-cell and stochastic models. We recently reported that B cells exposed to a pulse of C/EBPα (Bα' cells) behave as elite cells, in that they can be rapidly and efficiently reprogrammed into iPSCs by the Yamanaka factors OSKM. Here we show that C/EBPα post-transcriptionally increases the abundance of several hundred proteins, including Lsd1, Hdac1, Brd4, Med1 and Cdk9, components of chromatin-modifying complexes present at super-enhancers. Lsd1 was found to be required for B cell gene silencing and Brd4 for the activation of the pluripotency program. C/EBPα also promotes chromatin accessibility in pluripotent cells and upregulates Klf4 by binding to two haematopoietic enhancers. Bα' cells share many properties with granulocyte/macrophage progenitors, naturally occurring elite cells that are obligate targets for leukaemic transformation, whose formation strictly requires C/EBPα. PMID:26974661

  14. Superenhancer reprogramming drives a B-cell-epithelial transition and high-risk leukemia.

    Science.gov (United States)

    Hu, Yeguang; Zhang, Zhihong; Kashiwagi, Mariko; Yoshida, Toshimi; Joshi, Ila; Jena, Nilamani; Somasundaram, Rajesh; Emmanuel, Akinola Olumide; Sigvardsson, Mikael; Fitamant, Julien; El-Bardeesy, Nabeel; Gounari, Fotini; Van Etten, Richard A; Georgopoulos, Katia

    2016-09-01

    IKAROS is required for the differentiation of highly proliferative pre-B-cell precursors, and loss of IKAROS function indicates poor prognosis in precursor B-cell acute lymphoblastic leukemia (B-ALL). Here we show that IKAROS regulates this developmental stage by positive and negative regulation of superenhancers with distinct lineage affiliations. IKAROS defines superenhancers at pre-B-cell differentiation genes together with B-cell master regulators such as PAX5, EBF1, and IRF4 but is required for a highly permissive chromatin environment, a function that cannot be compensated for by the other transcription factors. IKAROS is also highly enriched at inactive enhancers of genes normally expressed in stem-epithelial cells. Upon IKAROS loss, expression of pre-B-cell differentiation genes is attenuated, while a group of extralineage transcription factors that are directly repressed by IKAROS and depend on EBF1 relocalization at their enhancers for expression is induced. LHX2, LMO2, and TEAD-YAP1, normally kept separate from native B-cell transcription regulators by IKAROS, now cooperate directly with them in a de novo superenhancer network with its own feed-forward transcriptional reinforcement. Induction of de novo superenhancers antagonizes Polycomb repression and superimposes aberrant stem-epithelial cell properties in a B-cell precursor. This dual mechanism of IKAROS regulation promotes differentiation while safeguarding against a hybrid stem-epithelial-B-cell phenotype that underlies high-risk B-ALL.

  15. The environmental carcinogen benzo[a]pyrene induces a Warburg-like metabolic reprogramming dependent on NHE1 and associated with cell survival

    Science.gov (United States)

    Hardonnière, Kévin; Saunier, Elise; Lemarié, Anthony; Fernier, Morgane; Gallais, Isabelle; Héliès-Toussaint, Cécile; Mograbi, Baharia; Antonio, Samantha; Bénit, Paule; Rustin, Pierre; Janin, Maxime; Habarou, Florence; Ottolenghi, Chris; Lavault, Marie-Thérèse; Benelli, Chantal; Sergent, Odile; Huc, Laurence; Bortoli, Sylvie; Lagadic-Gossmann, Dominique

    2016-01-01

    Cancer cells display alterations in many cellular processes. One core hallmark of cancer is the Warburg effect which is a glycolytic reprogramming that allows cells to survive and proliferate. Although the contributions of environmental contaminants to cancer development are widely accepted, the underlying mechanisms have to be clarified. Benzo[a]pyrene (B[a]P), the prototype of polycyclic aromatic hydrocarbons, exhibits genotoxic and carcinogenic effects, and it is a human carcinogen according to the International Agency for Research on Cancer. In addition to triggering apoptotic signals, B[a]P may induce survival signals, both of which are likely to be involved in cancer promotion. We previously suggested that B[a]P-induced mitochondrial dysfunctions, especially membrane hyperpolarization, might trigger cell survival signaling in rat hepatic epithelial F258 cells. Here, we further characterized these dysfunctions by focusing on energy metabolism. We found that B[a]P promoted a metabolic reprogramming. Cell respiration decreased and lactate production increased. These changes were associated with alterations in the tricarboxylic acid cycle which likely involve a dysfunction of the mitochondrial complex II. The glycolytic shift relied on activation of the Na+/H+ exchanger 1 (NHE1) and appeared to be a key feature in B[a]P-induced cell survival related to changes in cell phenotype (epithelial-to-mesenchymal transition and cell migration). PMID:27488617

  16. Short Chain Fatty Acids (SCFA) Reprogram Gene Expression in Human Malignant Epithelial and Lymphoid Cells

    Science.gov (United States)

    Astakhova, Lidiia; Ngara, Mtakai; Babich, Olga; Prosekov, Aleksandr; Asyakina, Lyudmila; Dyshlyuk, Lyubov; Midtvedt, Tore; Zhou, Xiaoying; Ernberg, Ingemar; Matskova, Liudmila

    2016-01-01

    The effect of short chain fatty acids (SCFAs) on gene expression in human, malignant cell lines was investigated, with a focus on signaling pathways. The commensal microbial flora produce high levels of SCFAs with established physiologic effects in humans. The most abundant SCFA metabolite in the human microflora is n-butyric acid. It is well known to activate endogenous latent Epstein-Barr virus (EBV), that was used as a reference read out system and extended to EBV+ epithelial cancer cell lines. N-butyric acid and its salt induced inflammatory and apoptotic responses in tumor cells of epithelial and lymphoid origin. Epithelial cell migration was inhibited. The n-butyric gene activation was reduced by knock-down of the cell membrane transporters MCT-1 and -4 by siRNA. N-butyric acid show biologically significant effects on several important cellular functions, also with relevance for tumor cell phenotype. PMID:27441625

  17. Short Chain Fatty Acids (SCFA) Reprogram Gene Expression in Human Malignant Epithelial and Lymphoid Cells.

    Science.gov (United States)

    Astakhova, Lidiia; Ngara, Mtakai; Babich, Olga; Prosekov, Aleksandr; Asyakina, Lyudmila; Dyshlyuk, Lyubov; Midtvedt, Tore; Zhou, Xiaoying; Ernberg, Ingemar; Matskova, Liudmila

    2016-01-01

    The effect of short chain fatty acids (SCFAs) on gene expression in human, malignant cell lines was investigated, with a focus on signaling pathways. The commensal microbial flora produce high levels of SCFAs with established physiologic effects in humans. The most abundant SCFA metabolite in the human microflora is n-butyric acid. It is well known to activate endogenous latent Epstein-Barr virus (EBV), that was used as a reference read out system and extended to EBV+ epithelial cancer cell lines. N-butyric acid and its salt induced inflammatory and apoptotic responses in tumor cells of epithelial and lymphoid origin. Epithelial cell migration was inhibited. The n-butyric gene activation was reduced by knock-down of the cell membrane transporters MCT-1 and -4 by siRNA. N-butyric acid show biologically significant effects on several important cellular functions, also with relevance for tumor cell phenotype. PMID:27441625

  18. The biology of cancer: metabolic reprogramming fuels cell growth and proliferation.

    Science.gov (United States)

    DeBerardinis, Ralph J; Lum, Julian J; Hatzivassiliou, Georgia; Thompson, Craig B

    2008-01-01

    Cell proliferation requires nutrients, energy, and biosynthetic activity to duplicate all macromolecular components during each passage through the cell cycle. It is therefore not surprising that metabolic activities in proliferating cells are fundamentally different from those in nonproliferating cells. This review examines the idea that several core fluxes, including aerobic glycolysis, de novo lipid biosynthesis, and glutamine-dependent anaplerosis, form a stereotyped platform supporting proliferation of diverse cell types. We also consider regulation of these fluxes by cellular mediators of signal transduction and gene expression, including the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR system, hypoxia-inducible factor 1 (HIF-1), and Myc, during physiologic cell proliferation and tumorigenesis.

  19. Metformin-induced metabolic reprogramming of chemoresistant ALDHbright breast cancer cells

    OpenAIRE

    Cioce, Mario; Valerio, MariaCristina; Casadei, Luca; Pulito, Claudio; Sacconi, Andrea; Mori, Federica; Biagioni, Francesca; Manetti, Cesare; Muti, Paola; Strano, Sabrina; Blandino, Giovanni

    2014-01-01

    Metabolic remodeling is a hallmark of cancer progression and may affect tumor chemoresistance. Here we investigated by 1H-NMR/PCA analysis the metabolic profile of chemoresistant breast cancer cell subpopulations (ALDHbright cells) and their response to metformin, a promising anticancer metabolic modulator. The purified ALDHbright cells exhibited a different metabolic profile as compared to their chemosensitive ALDHlow counterparts. Metformin treatment strongly affected the metabolism of the ...

  20. The epigenetic reprogramming of poorly aggressive melanoma cells by a metastatic microenvironment

    OpenAIRE

    Seftor, Elisabeth A.; Meltzer, PS; Kirschmann, DA; Margaryan, NV; Seftor, REB; Hendrix, Mary JC

    2007-01-01

    A dynamic, complex relationship exists between tumor cells and their microenvironment, which plays a pivotal role in cancer progression, yet remains poorly understood. Particularly perplexing is the finding that aggressive melanoma cells express genes associated with multiple cellular phenotypes, in addition to their ability to form vasculogenic-like networks in three-dimensional matrix - called vasculogenic mimicry, which is illustrative of tumor cells plasticity. This study addressed the un...

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

    OpenAIRE

    Regalo, G.; Leutz, A

    2013-01-01

    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...

  2. DNA methylation in porcine preimplantation embryos developed in vivo and produced by in vitro fertilization, parthenogenetic activation and somatic cell nuclear transfer

    DEFF Research Database (Denmark)

    Deshmukh, Rahul Shahaji; Østrup, Olga; Østrup, Esben;

    2011-01-01

    DNA demethylation and remethylation are crucial for reprogramming of the differentiated parental/somatic genome in the recipient ooplasm upon somatic cell nuclear transfer. Here, we analyzed the DNA methylation dynamics during porcine preimplantation development. Porcine in vivo developed (IV), i...

  3. DHP-derivative and low oxygen tension effectively induces human adipose stromal cell reprogramming.

    Directory of Open Access Journals (Sweden)

    Min Ki Jee

    Full Text Available BACKGROUND AND METHODS: In this study, we utilized a combination of low oxygen tension and a novel anti-oxidant, 4-(3,4-dihydroxy-phenyl-derivative (DHP-d to directly induce adipose tissue stromal cells (ATSC to de-differentiate into more primitive stem cells. De-differentiated ATSCs was overexpress stemness genes, Rex-1, Oct-4, Sox-2, and Nanog. Additionally, demethylation of the regulatory regions of Rex-1, stemnesses, and HIF1alpha and scavenging of reactive oxygen species were finally resulted in an improved stem cell behavior of de-differentiate ATSC (de-ATSC. Proliferation activity of ATSCs after dedifferentiation was induced by REX1, Oct4, and JAK/STAT3 directly or indirectly. De-ATSCs showed increased migration activity that mediated by P38/JUNK and ERK phosphorylation. Moreover, regenerative efficacy of de-ATSC engrafted spinal cord-injured rats and chemical-induced diabetes animals were significantly restored their functions. CONCLUSIONS/SIGNIFICANCE: Our stem cell remodeling system may provide a good model which would provide insight into the molecular mechanisms underlying ATSC proliferation and transdifferentiation. Also, these multipotent stem cells can be harvested may provide us with a valuable reservoir of primitive and autologous stem cells for use in a broad spectrum of regenerative cell-based disease therapy.

  4. Intraperitoneal delivery of a novel liposome-encapsulated paclitaxel redirects metabolic reprogramming and effectively inhibits cancer stem cells in Taxol(®)-resistant ovarian cancer.

    Science.gov (United States)

    Shen, Yao-An; Li, Wai-Hou; Chen, Po-Hung; He, Chun-Lin; Chang, Yen-Hou; Chuang, Chi-Mu

    2015-01-01

    Taxol(®) remained as the mainstay therapeutic agent in the treatment of ovarian cancer, however recurrence rate is still high. Cancer stem cells (CSCs) represent a subset of cells in the bulk of tumors and play a central role in inducing drug resistance and recurrence. Furthermore, cancer metabolism has been an area under intensive investigation, since accumulating evidence has shown that CSCs and cancer metabolism are closely linked, an effect named as metabolic reprogramming. In this work, we aimed to investigate the impacts of a novel liposome-encapsulated paclitaxel (Nano-Taxol) on the stemness phenotype and metabolic reprogramming. A paclitaxel-resistant cell line (TR) was established at first. Tumor growth was induced in the mice peritoneal cavity by inoculation of TR cells. A 2x2 factorial experiment was designed to test the therapeutic efficacy in which factor 1 represented the comparison of drugs (Taxol(®) versus Nano-Taxol), while factor 2 represented the delivery route (intravenous versus intraperitoneal delivery). In this work, we found that intraperitoneal delivery of Nano-Taxol redirects metabolic reprogramming, from glycolysis to oxidative phosphorylation, and effectively suppresses cancer stem cells. Also, intraperitoneal delivery of Nano-Taxol led to a significantly better control of tumor growth compared with intravenous delivery of Taxol(®) (current standard treatment). This translational research may serve as a novel pathway for the drug development of nanomedicine. In the future, this treatment modality may be extended to treat several relevant cancers that have been proved to be suitable for the loco-regional delivery of therapeutic agents, including colon cancer, gastric cancer, and pancreatic cancer.

  5. Ionizing Particle Radiation as a Modulator of Endogenous Bone Marrow Cell Reprogramming: Implications for Hematological Cancers

    OpenAIRE

    Muralidharan, Sujatha; Sharath P. Sasi; Zuriaga, Maria A.; Hirschi, Karen K.; Porada, Christopher D.; Matthew A. Coleman; Kenneth X Walsh; Yan, Xinhua; Goukassian, David A.

    2015-01-01

    Exposure of individuals to ionizing radiation (IR), as in the case of astronauts exploring space or radiotherapy cancer patients, increases their risk of developing secondary cancers and other health-related problems. Bone marrow (BM), the site in the body where hematopoietic stem cell (HSC) self-renewal and differentiation to mature blood cells occurs, is extremely sensitive to low-dose IR, including irradiation by high-charge and high-energy particles. Low-dose IR induces DNA damage and per...

  6. Ionizing Particle Radiation as a Modulator of Endogenous Bone Marrow Cell Reprogramming: Implications for Hematological Cancers

    OpenAIRE

    Sujatha eMuralidharan; Sharath ePankajavihar Sasi; Zuriaga, Maria A.; Hirschi, Karen K.; Porada, Christopher D.; Matthew A. Coleman; Kenneth X Walsh; Xinhua eYan; Goukassian, David A.

    2015-01-01

    Exposure of individuals to ionizing radiation (IR), as in the case of astronauts exploring space or radiotherapy cancer patients, increases their risk of developing secondary cancers and other health-related problems. Bone marrow (BM), the site in the body where hematopoietic stem cell (HSC) self-renewal and differentiation to mature blood cells occurs, is extremely sensitive to low dose IR, including irradiation by high-charge and high-energy particles (HZE). Low dose IR induces DNA damage a...

  7. Reprogramming human amniotic fluid stem cells to functional pluripotency by manipulation of culture conditions.

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Dafni Moschidou & Pascale V Guillot ### Abstract Pluripotent stem cells have potential applications in regenerative medicine, disease modelling and drug screening. Induced pluripotent stem (iPS) cells have first been generated from fibroblasts using retroviral insertion of OCT4A, SOX2, c-MYC and KLF4. Since then, a number of methods have been developed to avoid the random integration of ectopic factors in the genome and the low efficiency of the process. Those include alt...

  8. Mitochondrial uncoupling and the reprogramming of intermediary metabolism in leukemia cells

    Directory of Open Access Journals (Sweden)

    Juliana eVélez

    2013-04-01

    Full Text Available Nearly 60 years ago Otto Warburg proposed, in a seminal publication, that an irreparable defect in the oxidative capacity of normal cells supported the switch to glycolysis for energy generation and the appearance of the malignant phenotype (Warburg, 1956. Curiously, this phenotype was also observed by Warburg in embryonic tissues, and recent research demonstrated that normal stem cells may indeed rely on aerobic glycolysis – fermenting pyruvate to lactate in the presence of ample oxygen - rather than on the complete oxidation of pyruvate in the Krebs cycle - to generate cellular energy (Folmes et al., 2012. However, it remains to be determined whether this phenotype is causative for neoplastic development, or rather the result of malignant transformation. In addition, in light of mounting evidence demonstrating that cancer cells can carry out electron transport and oxidative phosphorylation, although in some cases predominantly using electrons from non-glucose carbon sources (Bloch-Frankenthal et al., 1965, Warburg´s hypothesis needs to be revisited. Lastly, recent evidence suggests that the leukemia bone marrow microenvironment promotes the Warburg phenotype adding another layer of complexity to the study of metabolism in hematological malignancies. In this review we will discuss some of the evidence for alterations in the intermediary metabolism of leukemia cells and present evidence for a concept put forth decades ago by lipid biochemist Feodor Lynen, and acknowledged by Warburg himself, that cancer cell mitochondria uncouple ATP synthesis from electron transport and therefore depend on glycolysis to meet their energy demands (Lynen, 1951;Warburg, 1956.

  9. Kinetic Measurement and Real Time Visualization of Somatic Reprogramming.

    Science.gov (United States)

    Quintanilla, Rene H; Asprer, Joanna; Sylakowski, Kyle; Lakshmipathy, Uma

    2016-01-01

    Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease phenotypes. Recent advances have identified more efficient and safe methods for introduction of reprogramming factors. However, there are few tools to monitor and track the progression of reprogramming. Current methods for monitoring reprogramming rely on the qualitative inspection of morphology or staining with stem cell-specific dyes and antibodies. Tools to dissect the progression of iPSC generation can help better understand the process under different conditions from diverse cell sources. This study presents key approaches for kinetic measurement of reprogramming progression using flow cytometry as well as real-time monitoring via imaging. To measure the kinetics of reprogramming, flow analysis was performed at discrete time points using antibodies against positive and negative pluripotent stem cell markers. The combination of real-time visualization and flow analysis enables the quantitative study of reprogramming at different stages and provides a more accurate comparison of different systems and methods. Real-time, image-based analysis was used for the continuous monitoring of fibroblasts as they are reprogrammed in a feeder-free medium system. The kinetics of colony formation was measured based on confluence in the phase contrast or fluorescence channels after staining with live alkaline phosphatase dye or antibodies against SSEA4 or TRA-1-60. The results indicated that measurement of confluence provides semi-quantitative metrics to monitor the progression of reprogramming. PMID:27500543

  10. Kinetic Measurement and Real Time Visualization of Somatic Reprogramming.

    Science.gov (United States)

    Quintanilla, Rene H; Asprer, Joanna; Sylakowski, Kyle; Lakshmipathy, Uma

    2016-07-30

    Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease phenotypes. Recent advances have identified more efficient and safe methods for introduction of reprogramming factors. However, there are few tools to monitor and track the progression of reprogramming. Current methods for monitoring reprogramming rely on the qualitative inspection of morphology or staining with stem cell-specific dyes and antibodies. Tools to dissect the progression of iPSC generation can help better understand the process under different conditions from diverse cell sources. This study presents key approaches for kinetic measurement of reprogramming progression using flow cytometry as well as real-time monitoring via imaging. To measure the kinetics of reprogramming, flow analysis was performed at discrete time points using antibodies against positive and negative pluripotent stem cell markers. The combination of real-time visualization and flow analysis enables the quantitative study of reprogramming at different stages and provides a more accurate comparison of different systems and methods. Real-time, image-based analysis was used for the continuous monitoring of fibroblasts as they are reprogrammed in a feeder-free medium system. The kinetics of colony formation was measured based on confluence in the phase contrast or fluorescence channels after staining with live alkaline phosphatase dye or antibodies against SSEA4 or TRA-1-60. The results indicated that measurement of confluence provides semi-quantitative metrics to monitor the progression of reprogramming.

  11. The use of Xenopus oocytes and embryos as a route towards cell replacement

    Indian Academy of Sciences (India)

    J B Gurdon

    2005-02-01

    When nuclei of somatic cells are transplanted to enucleated eggs of Xenopus, a complete reprogramming of nuclear function can take place. To identify mechanisms of nuclear reprogramming, somatic nuclei can be transplanted to growing meiotic oocytes of Xenopus, and stem cell genes activated without DNA replication. The combination of somatic cell nuclear transfer with morphogen signalling and the community effect may lead towards the possibility of cell replacement therapy. When mechanisms of nuclear reprogramming are understood, it may eventually be possible to directly reprogramme human somatic cell nuclei without the use of eggs.

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

    DEFF Research Database (Denmark)

    Ilieva, Mirolyuba

    2013-01-01

    cell level is molecular beacons (MBs). They are stem-loop structured antisense oligonucleotide probes labelled with a reporter fluorophore at one end and with quencher at the other end. Upon hybridization with complementary target, hydrogen bonds between stem nucleotide bases brake, resulting...

  13. 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.

  14. Arginine deiminase resistance in melanoma cells is associated with metabolic reprogramming, glucose dependence, and glutamine addiction.

    Science.gov (United States)

    Long, Yan; Tsai, Wen-Bin; Wangpaichitr, Medhi; Tsukamoto, Takashi; Savaraj, Niramol; Feun, Lynn G; Kuo, Macus Tien

    2013-11-01

    Many malignant human tumors, including melanomas, are auxotrophic for arginine due to reduced expression of argininosuccinate synthetase-1 (ASS1), the rate-limiting enzyme for arginine biosynthesis. Pegylated arginine deiminase (ADI-PEG20), which degrades extracellular arginine, resulting in arginine deprivation, has shown favorable results in clinical trials for treating arginine-auxotrophic tumors. Drug resistance is the major obstacle for effective ADI-PEG20 usage. To elucidate mechanisms of resistance, we established several ADI-PEG20-resistant (ADI(R)) variants from A2058 and SK-Mel-2 melanoma cells. Compared with the parental lines, these ADI(R) variants showed the following characteristics: (i) all ADI(R) cell lines showed elevated ASS1 expression, resulting from the constitutive binding of the transcription factor c-Myc on the ASS1 promoter, suggesting that elevated ASS1 is the major mechanism of resistance; (ii) the ADI(R) cell lines exhibited enhanced AKT signaling and were preferentially sensitive to PI3K/AKT inhibitors, but reduced mTOR signaling, and were preferentially resistant to mTOR inhibitor; (iii) these variants showed enhanced expression of glucose transporter-1 and lactate dehydrogenase-A, reduced expression of pyruvate dehydrogenase, and elevated sensitivity to the glycolytic inhibitors 2-deoxy-glucose and 3-bromopyruvate, consistent with the enhanced glycolytic pathway (the Warburg effect); (iv) the resistant cells showed higher glutamine dehydrogenase and glutaminase expression and were preferentially vulnerable to glutamine inhibitors. We showed that c-Myc, not elevated ASS1 expression, is involved in upregulation of many of these enzymes because knockdown of c-Myc reduced their expression, whereas overexpressed ASS1 by transfection reduced their expression. This study identified multiple targets for overcoming ADI-PEG resistance in cancer chemotherapy using recombinant arginine-degrading enzymes.

  15. Arginine deiminase resistance in melanoma cells is associated with metabolic reprogramming, glucose dependence, and glutamine addiction.

    Science.gov (United States)

    Long, Yan; Tsai, Wen-Bin; Wangpaichitr, Medhi; Tsukamoto, Takashi; Savaraj, Niramol; Feun, Lynn G; Kuo, Macus Tien

    2013-11-01

    Many malignant human tumors, including melanomas, are auxotrophic for arginine due to reduced expression of argininosuccinate synthetase-1 (ASS1), the rate-limiting enzyme for arginine biosynthesis. Pegylated arginine deiminase (ADI-PEG20), which degrades extracellular arginine, resulting in arginine deprivation, has shown favorable results in clinical trials for treating arginine-auxotrophic tumors. Drug resistance is the major obstacle for effective ADI-PEG20 usage. To elucidate mechanisms of resistance, we established several ADI-PEG20-resistant (ADI(R)) variants from A2058 and SK-Mel-2 melanoma cells. Compared with the parental lines, these ADI(R) variants showed the following characteristics: (i) all ADI(R) cell lines showed elevated ASS1 expression, resulting from the constitutive binding of the transcription factor c-Myc on the ASS1 promoter, suggesting that elevated ASS1 is the major mechanism of resistance; (ii) the ADI(R) cell lines exhibited enhanced AKT signaling and were preferentially sensitive to PI3K/AKT inhibitors, but reduced mTOR signaling, and were preferentially resistant to mTOR inhibitor; (iii) these variants showed enhanced expression of glucose transporter-1 and lactate dehydrogenase-A, reduced expression of pyruvate dehydrogenase, and elevated sensitivity to the glycolytic inhibitors 2-deoxy-glucose and 3-bromopyruvate, consistent with the enhanced glycolytic pathway (the Warburg effect); (iv) the resistant cells showed higher glutamine dehydrogenase and glutaminase expression and were preferentially vulnerable to glutamine inhibitors. We showed that c-Myc, not elevated ASS1 expression, is involved in upregulation of many of these enzymes because knockdown of c-Myc reduced their expression, whereas overexpressed ASS1 by transfection reduced their expression. This study identified multiple targets for overcoming ADI-PEG resistance in cancer chemotherapy using recombinant arginine-degrading enzymes. PMID:23979920

  16. Reprogramming of cassava (Manihot esculenta) microspores towards sporophytic development.

    Science.gov (United States)

    Perera, P I P; Ordoñez, C A; Dedicova, B; Ortega, P E M

    2014-05-21

    Gametes have the unique potential to enter the sporophytic pathway, called androgenesis. The plants produced are usually haploid and recombinant due to the preceding meiosis and they can double their chromosome number to form doubled haploids, which are completely homozygous. Availability of the doubled haploids facilitates mapping the genes of agronomically important traits, shortening the time of the breeding process required to produce new hybrids and homozygous varieties, and saving the time and cost for inbreeding. This study aimed to test the feasibility of using isolated and in vitro cultured immature cassava (Manihot esculenta) microspores to reprogramme and initiate sporophytic development. Different culture media and different concentrations of two ion components (Cu(2+) and Fe(2+)) were tested in two genotypes of cassava. External structural changes, nuclear divisions and cellular changes during reprogramming were analysed by scanning electron microscopy, by staining with 4',6-diamidino-2-phenylindole, and through classical histology and transmission electron microscopy. In two cassava genotypes, different developmental stages of microspores were found to initiate sporophytic cell divisions, that is, with tetrads of TMS 60444 and with mid or late uni-nucleate microspores of SM 1219-9. In the modified NLN medium (NLNS), microspore enlargements were observed. The medium supplemented with either sodium ferrous ethylene-diamine-tetraacetic acid (NaFeEDTA) or CuSO4·5H2O induced sporophytic cell division in both genotypes. A low frequency of the reprogramming and the presence of non-responsive microspores among the responsive ones in tetrads were found to be related to the viability and exine formation of the microspores. The present study clearly demonstrated that reprogramming occurs much faster in isolated microspore culture than in anther culture. This paves the way for the development of an efficient technique for the production of homozygous lines in

  17. Patient-Specific Therapy via Cell-Reprogramming Technology: a Curative Potential for Patients with Diabetes

    Science.gov (United States)

    Luo, Haizhao; Wang, Xianbao; Zhang, Ruyi; Chen, Youping; Shu, Yi; Li, Huixian; Chen, Hong

    2015-12-01

    Gene therapeutics provides great opportunities for curing diabetes. Numerous attempts have been made to establish a safe and high-efficiency gene delivery strategy, but all of them are unsuccessful. To achieve an ideal transfection, a novel gene delivery strategy was presented in this research. The novel system proposed was transfection mediated by the combination of ultrasound with microbubbles and cross-linked polyethylenimines (PEIs). Ultrasound with microbubbles enhances the permeability of target cells; moreover, cross-linked PEIs enabled DNA to escape from endosomes into the cytoplasm. If the proposed method is feasible and effective, the endogenous secretion system of insulin would be re-established in patients with diabetes.

  18. Cardiac Niche Influences the Direct Reprogramming of Canine Fibroblasts into Cardiomyocyte-Like Cells

    Directory of Open Access Journals (Sweden)

    Giacomo Palazzolo

    2016-01-01

    Full Text Available The Duchenne and Becker muscular dystrophies are caused by mutation of dystrophin gene and primarily affect skeletal and cardiac muscles. Cardiac involvement in dystrophic GRMD dogs has been demonstrated by electrocardiographic studies with the onset of a progressive cardiomyopathy similar to the cardiac disease in DMD patients. In this respect, GRMD is a useful model to explore cardiac and skeletal muscle pathogenesis and for developing new therapeutic protocols. Here we describe a protocol to convert GRMD canine fibroblasts isolated from heart and skin into induced cardiac-like myocytes (ciCLMs. We used a mix of transcription factors (GATA4, HAND2, TBX5, and MEF2C, known to be able to differentiate mouse and human somatic cells into ciCLMs. Exogenous gene expression was obtained using four lentiviral vectors carrying transcription factor genes and different resistance genes. Our data demonstrate a direct switch from fibroblast into ciCLMs with no activation of early cardiac genes. ciCLMs were unable to contract spontaneously, suggesting, differently from mouse and human cells, an incomplete differentiation process. However, when transplanted in neonatal hearts of SCID/Beige mice, ciCLMs participate in cardiac myogenesis.

  19. Effects of Integrating and Non-Integrating Reprogramming Methods on Copy Number Variation and Genomic Stability of Human Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Kang, Xiangjin; Yu, Qian; Huang, Yuling; Song, Bing; Chen, Yaoyong; Gao, Xingcheng; He, Wenyin; Sun, Xiaofang; Fan, Yong

    2015-01-01

    Human-induced pluripotent stem cells (iPSCs) are derived from differentiated somatic cells using defined factors and provide a renewable source of autologous cells for cell therapy. Many reprogramming methods have been employed to generate human iPSCs, including the use of integrating vectors and non-integrating vectors. Maintenance of the genomic integrity of iPSCs is highly desirable if the cells are to be used in clinical applications. Here, using the Affymetrix Cytoscan HD array, we investigated the genomic aberration profiles of 19 human cell lines: 5 embryonic stem cell (ESC) lines, 6 iPSC lines derived using integrating vectors ("integrating iPSC lines"), 6 iPSC lines derived using non-integrating vectors ("non-integrating iPSC lines"), and the 2 parental cell lines from which the iPSCs were derived. The genome-wide copy number variation (CNV), loss of heterozygosity (LOH) and mosaicism patterns of integrating and non-integrating iPSC lines were investigated. The maximum sizes of CNVs in the genomes of the integrating iPSC lines were 20 times higher than those of the non-integrating iPSC lines. Moreover, the total number of CNVs was much higher in integrating iPSC lines than in other cell lines. The average numbers of novel CNVs with a low degree of overlap with the DGV and of likely pathogenic CNVs with a high degree of overlap with the ISCA (International Symposium on Computer Architecture) database were highest in integrating iPSC lines. Different single nucleotide polymorphisms (SNP) calls revealed that, using the parental cell genotype as a reference, integrating iPSC lines displayed more single nucleotide variations and mosaicism than did non-integrating iPSC lines. This study describes the genome stability of human iPSCs generated using either a DNA-integrating or non-integrating reprogramming method, of the corresponding somatic cells, and of hESCs. Our results highlight the importance of using a high-resolution method to monitor genomic aberrations

  20. Effect of biophysical cues on reprogramming to cardiomyocytes.

    Science.gov (United States)

    Sia, Junren; Yu, Pengzhi; Srivastava, Deepak; Li, Song

    2016-10-01

    Reprogramming of fibroblasts to cardiomyocytes offers exciting potential in cell therapy and regenerative medicine, but has low efficiency. We hypothesize that physical cues may positively affect the reprogramming process, and studied the effects of periodic mechanical stretch, substrate stiffness and microgrooved substrate on reprogramming yield. Subjecting reprogramming fibroblasts to periodic mechanical stretch and different substrate stiffness did not improve reprogramming yield. On the other hand, culturing the cells on microgrooved substrate enhanced the expression of cardiomyocyte genes by day 2 and improved the yield of partially reprogrammed cells at day 10. By combining microgrooved substrate with an existing optimized culture protocol, yield of reprogrammed cardiomyocytes with striated cardiac troponin T staining and spontaneous contractile activity was increased. We identified the regulation of Mkl1 activity as a new mechanism by which microgroove can affect reprogramming. Biochemical approach could only partially recapitulate the effect of microgroove. Microgroove demonstrated an additional effect of enhancing organization of sarcomeric structure, which could not be recapitulated by biochemical approach. This study provides insights into new mechanisms by which topographical cues can affect cellular reprogramming.

  1. Twist promotes reprogramming of glucose metabolism in breast cancer cells through PI3K/AKT and p53 signaling pathways.

    Science.gov (United States)

    Yang, Li; Hou, Yixuan; Yuan, Jie; Tang, Shifu; Zhang, Hailong; Zhu, Qing; Du, Yan-e; Zhou, Mingli; Wen, Siyang; Xu, Liyun; Tang, Xi; Cui, Xiaojiang; Liu, Manran

    2015-09-22

    Twist, a key regulator of epithelial-mesenchymal transition (EMT), plays an important role in the development of a tumorigenic phenotype. Energy metabolism reprogramming (EMR), a newly discovered hallmark of cancer cells, potentiates cancer cell proliferation, survival, and invasion. Currently little is known about the effects of Twist on tumor EMR. In this study, we found that glucose consumption and lactate production were increased and mitochondrial mass was decreased in Twist-overexpressing MCF10A mammary epithelial cells compared with vector-expressing MCF10A cells. Moreover, these Twist-induced phenotypic changes were augmented by hypoxia. The expression of some glucose metabolism-related genes such as PKM2, LDHA, and G6PD was also found to be upregulated. Mechanistically, activated β1-integrin/FAK/PI3K/AKT/mTOR and suppressed P53 signaling were responsible for the observed EMR. Knockdown of Twist reversed the effects of Twist on EMR in Twist-overexpressing MCF10A cells and Twist-positive breast cancer cells. Furthermore, blockage of the β1-integrin/FAK/PI3K/AKT/mTOR pathway by siRNA or specific chemical inhibitors, or rescue of p53 activation can partially reverse the switch of glucose metabolism and inhibit the migration of Twist-overexpressing MCF10A cells and Twist-positive breast cancer cells. Thus, our data suggest that Twist promotes reprogramming of glucose metabolism in MCF10A-Twist cells and Twist-positive breast cancer cells via activation of the β1-integrin/FAK/PI3K/AKT/mTOR pathway and inhibition of the p53 pathway. Our study provides new insight into EMR. PMID:26342198

  2. Cellular Reprogramming Employing Recombinant Sox2 Protein

    Directory of Open Access Journals (Sweden)

    Marc Thier

    2012-01-01

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

  3. In Vivo Reprogramming for Brain and Spinal Cord Repair.

    Science.gov (United States)

    Chen, Gong; Wernig, Marius; Berninger, Benedikt; Nakafuku, Masato; Parmar, Malin; Zhang, Chun-Li

    2015-01-01

    Cell reprogramming technologies have enabled the generation of various specific cell types including neurons from readily accessible patient cells, such as skin fibroblasts, providing an intriguing novel cell source for autologous cell transplantation. However, cell transplantation faces several difficult hurdles such as cell production and purification, long-term survival, and functional integration after transplantation. Recently, in vivo reprogramming, which makes use of endogenous cells for regeneration purpose, emerged as a new approach to circumvent cell transplantation. There has been evidence for in vivo reprogramming in the mouse pancreas, heart, and brain and spinal cord with various degrees of success. This mini review summarizes the latest developments presented in the first symposium on in vivo reprogramming glial cells into functional neurons in the brain and spinal cord, held at the 2014 annual meeting of the Society for Neuroscience in Washington, DC. PMID:26730402

  4. miR-155 Drives Metabolic Reprogramming of ER+ Breast Cancer Cells Following Long-Term Estrogen Deprivation and Predicts Clinical Response to Aromatase Inhibitors.

    Science.gov (United States)

    Bacci, Marina; Giannoni, Elisa; Fearns, Antony; Ribas, Ricardo; Gao, Qiong; Taddei, Maria Letizia; Pintus, Gianfranco; Dowsett, Mitch; Isacke, Clare M; Martin, Lesley-Ann; Chiarugi, Paola; Morandi, Andrea

    2016-03-15

    Aromatase inhibitors (AI) have become the first-line endocrine treatment of choice for postmenopausal estrogen receptor-positive (ER(+)) breast cancer patients, but resistance remains a major challenge. Metabolic reprogramming is a hallmark of cancer and may contribute to drug resistance. Here, we investigated the link between altered breast cancer metabolism and AI resistance using AI-resistant and sensitive breast cancer cells, patient tumor samples, and AI-sensitive human xenografts. We found that long-term estrogen deprivation (LTED), a model of AI resistance, was associated with increased glycolysis dependency. Targeting the glycolysis-priming enzyme hexokinase-2 (HK2) in combination with the AI, letrozole, synergistically reduced cell viability in AI-sensitive models. Conversely, MCF7-LTED cells, which displayed a high degree of metabolic plasticity, switched to oxidative phosphorylation when glycolysis was impaired. This effect was ER dependent as breast cancer cells with undetectable levels of ER failed to exhibit metabolic plasticity. MCF7-LTED cells were also more motile than their parental counterparts and assumed amoeboid-like invasive abilities upon glycolysis inhibition with 2-deoxyglucose (2-DG). Mechanistic investigations further revealed an important role for miR-155 in metabolic reprogramming. Suppression of miR-155 resulted in sensitization of MCF7-LTED cells to metformin treatment and impairment of 2-DG-induced motility. Notably, high baseline miR-155 expression correlated with poor response to AI therapy in a cohort of ER(+) breast cancers treated with neoadjuvant anastrozole. These findings suggest that miR-155 represents a biomarker potentially capable of identifying the subset of breast cancers most likely to adapt to and relapse on AI therapy. PMID:26795347

  5. Reprogramming of bone marrow-derived mesenchymal stem cells into functional insulin-producing cells by chemical regimen

    OpenAIRE

    Wang, Qiwei; Ye, Lingling; Liu, Hong; Liu, Xingmao; Li, Shichong; Chen, Zhaolie

    2012-01-01

    Beta-cell transplantation is considered to be the most effective approach to cure type 1 diabetes (T1D). Unfortunately, the scarce availability of donor tissue limits the applicability of this therapy. Recent stem cell research progress shows stem cell therapy may be a potential means to solve this problem. Bone marrow-derived mesenchymal stem cells (MSCs) are self-renewable and multipotent adult stem cells which can differentiate into the three germ layers. Here we aimed to investigate wheth...

  6. Cells from the adult corneal stroma can be reprogrammed to a neuron-like cell using exogenous growth factors

    Energy Technology Data Exchange (ETDEWEB)

    Greene, Carol Ann, E-mail: carol.greene@auckland.ac.nz; Chang, Chuan-Yuan; Fraser, Cameron J.; Nelidova, Dasha E.; Chen, Jing A.; Lim, Angela; Brebner, Alex; McGhee, Jennifer; Sherwin, Trevor; Green, Colin R.

    2014-03-10

    Cells thought to be stem cells isolated from the cornea of the eye have been shown to exhibit neurogenic potential. We set out to uncover the identity and location of these cells within the cornea and to elucidate their neuronal protein and gene expression profile during the process of switching to a neuron-like cell. Here we report that every cell of the adult human and rat corneal stroma is capable of differentiating into a neuron-like cell when treated with neurogenic differentiation specifying growth factors. Furthermore, the expression of genes regulating neurogenesis and mature neuronal structure and function was increased. The switch from a corneal stromal cell to a neuron-like cell was also shown to occur in vivo in intact corneas of living rats. Our results clearly indicate that lineage specifying growth factors can affect changes in the protein and gene expression profiles of adult cells, suggesting that possibly many adult cell populations can be made to switch into another type of mature cell by simply modifying the growth factor environment. - Highlights: • Adult corneal stromal cells can differentiated into neuron-like cells. • Neuronal specification of the adult stromal cell population is stochastic. • Neuronal specification in an adult cell population can be brought about by growth factors.

  7. A “Hit and Run” Approach to Inducible Direct Reprogramming of Astrocytes to Neural Stem Cells

    OpenAIRE

    Poulou, Maria; Mandalos, Nikolaos P.; Karnavas, Theodoros; Saridaki, Marannia; Ronald D G McKay; Remboutsika, Eumorphia

    2016-01-01

    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, 2 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 nesti...

  8. Generation of embryonic stem cells from mouse adipose-tissue derived cells via somatic cell nuclear transfer.

    Science.gov (United States)

    Qin, Yiren; Qin, Jilong; Zhou, Chikai; Li, Jinsong; Gao, Wei-Qiang

    2015-01-01

    Somatic cells can be reprogrammed into embryonic stem cells (ESCs) by nuclear transfer (NT-ESCs), or into induced pluripotent stem cells (iPSCs) by the "Yamanaka method." However, recent studies have indicated that mouse and human iPSCs are prone to epigenetic and transcriptional aberrations, and that NT-ESCs correspond more closely to ESCs derived from in vitro fertilized embryos than iPSCs. In addition, the procedure of NT-ESCs does not involve gene modification. Demonstration of generation of NT-ESCs using an easily-accessible source of adult cell types would be very important. Adipose tissue is a source of readily accessible donor cells and can be isolated from both males and females at different ages. Here we report that NT-ESCs can be generated from adipose tissue-derived cells (ADCs). At morphological, mRNA and protein levels, these NT-ESCs show classic ESC colonies, exhibit alkaline phosphatase (AP) activity, and display normal diploid karyotypes. Importantly, these cells express pluripotent markers including Oct4, Sox2, Nanog and SSEA-1. Furthermore, they can differentiate in vivo into various types of cells from 3 germinal layers by teratoma formation assays. This study demonstrates for the first time that ESCs can be generated from the adipose tissue by somatic cell nuclear transfer (SCNT) and suggests that ADCs can be a new donor-cell type for potential therapeutic cloning.

  9. Direct neuronal reprogramming: learning from and for development.

    Science.gov (United States)

    Masserdotti, Giacomo; Gascón, Sergio; Götz, Magdalena

    2016-07-15

    The key signalling pathways and transcriptional programmes that instruct neuronal diversity during development have largely been identified. In this Review, we discuss how this knowledge has been used to successfully reprogramme various cell types into an amazing array of distinct types of functional neurons. We further discuss the extent to which direct neuronal reprogramming recapitulates embryonic development, and examine the particular barriers to reprogramming that may exist given a cell's unique developmental history. We conclude with a recently proposed model for cell specification called the 'Cook Islands' model, and consider whether it is a fitting model for cell specification based on recent results from the direct reprogramming field. PMID:27436039

  10. Regulatory T cell reprogramming towards a Th2 cell-like lineage impairs oral tolerance and promotes food allergy

    OpenAIRE

    Rivas, Magali Noval; Burton, Oliver T.; Wise, Petra; Charbonnier, Louis-Marie; Georgiev, Peter; Oettgen, Hans C.; Rachid, Rima; Chatila, Talal

    2015-01-01

    Oral immunotherapy has had limited success in establishing tolerance in food allergy, reflecting failure to elicit an effective regulatory T (Treg) cell response. We show that disease-susceptible mice (Il4raF709) with enhanced IL-4 receptor (IL-4R) signaling exhibited STAT6-dependent impaired generation and function of mucosal allergen-specific Treg cells. This failure was associated with the acquisition by Treg cells of T helper 2 (Th2) cell-like phenotype, also found in peripheral blood all...

  11. Nuclear lamina in plant cells

    Institute of Scientific and Technical Information of China (English)

    汪健; 杨澄; 翟中和

    1996-01-01

    By using selective extraction and diethylene glycol distearate (DGD) embedment and embedment-free electron microscopy, the nuclear lamina was demonstrated in carrot and Ginkgo male generative cells. Western blotting revealed that the nuclear lamina was composed of A-type and B-type lamins which contained at least 66-ku and 84-ku or 66-ku and 86-ku polypeptides, respectively. These lamin proteins were localized at the nudear periphery as shown by immunogold-labelling. In situ hybridization for light microscope and electron microscope showed that plant cells have the homologous sequences of animal lamin cDNA. The sorting site of lamin mRNA is mainly distributed in the cytoplasm near the nudear envelope. The data have verified that there indeed exists nudear lamina in plant cells.

  12. The Aberrant DNA Methylation Profile of Human Induced Pluripotent Stem Cells Is Connected to the Reprogramming Process and Is Normalized During In Vitro Culture.

    Directory of Open Access Journals (Sweden)

    Lenka Tesarova

    Full Text Available The potential clinical applications of human induced pluripotent stem cells (hiPSCs are limited by genetic and epigenetic variations among hiPSC lines and the question of their equivalency with human embryonic stem cells (hESCs. We used MethylScreen technology to determine the DNA methylation profile of pluripotency and differentiation markers in hiPSC lines from different source cell types compared to hESCs and hiPSC source cells. After derivation, hiPSC lines compromised a heterogeneous population characterized by variable levels of aberrant DNA methylation. These aberrations were induced during somatic cell reprogramming and their levels were associated with the type of hiPSC source cells. hiPSC population heterogeneity was reduced during prolonged culture and hiPSCs acquired an hESC-like methylation profile. In contrast, the expression of differentiation marker genes in hiPSC lines remained distinguishable from that in hESCs. Taken together, in vitro culture facilitates hiPSC acquisition of hESC epigenetic characteristics. However, differences remain between both pluripotent stem cell types, which must be considered before their use in downstream applications.

  13. Tet-mediated imprinting erasure in H19 locus following reprogramming of spermatogonial stem cells to induced pluripotent stem cells

    Science.gov (United States)

    Selective methylation of CpG islands at imprinting control regions (ICR) determines the monoparental expression of a subset of genes. The imprinting marks are protected from global demethylation taking place during pre-implantation development before being reset in primordial germ cells. However, it...

  14. Pancreatic Cancer Cell Exosome-Mediated Macrophage Reprogramming and the Role of MicroRNAs 155 and 125b2 Transfection using Nanoparticle Delivery Systems.

    Science.gov (United States)

    Su, Mei-Ju; Aldawsari, Hibah; Amiji, Mansoor

    2016-01-01

    Exosomes are nano-sized endosome-derived small intraluminal vesicles, which are important facilitators of intercellular communication by transporting contents, such as protein, mRNA, and microRNAs, between neighboring cells, such as in the tumor microenvironment. The purpose of this study was to understand the mechanisms of exosomes-mediated cellular communication between human pancreatic cancer (Panc-1) cells and macrophages (J771.A1) using a Transwell co-culture system. Following characterization of exosome-mediated cellular communication and pro-tumoral baseline M2 macrophage polarization, the Panc-1 cells were transfected with microRNA-155 (miR-155) and microRNA-125b-2 (miR-125b2) expressing plasmid DNA using hyaluronic acid-poly(ethylene imine)/hyaluronic acid-poly(ethylene glycol) (HA-PEI/HA-PEG) self-assembling nanoparticle-based non-viral vectors. Our results show that upon successful transfection of Panc-1 cells, the exosome content was altered leading to differential communication and reprogramming of the J774.A1 cells to an M1 phenotype. Based on these results, genetic therapies targeted towards selective manipulation of tumor cell-derived exosome content may be very promising for cancer therapy. PMID:27443190

  15. Pancreatic Cancer Cell Exosome-Mediated Macrophage Reprogramming and the Role of MicroRNAs 155 and 125b2 Transfection using Nanoparticle Delivery Systems

    Science.gov (United States)

    Su, Mei-Ju; Aldawsari, Hibah; Amiji, Mansoor

    2016-01-01

    Exosomes are nano-sized endosome-derived small intraluminal vesicles, which are important facilitators of intercellular communication by transporting contents, such as protein, mRNA, and microRNAs, between neighboring cells, such as in the tumor microenvironment. The purpose of this study was to understand the mechanisms of exosomes-mediated cellular communication between human pancreatic cancer (Panc-1) cells and macrophages (J771.A1) using a Transwell co-culture system. Following characterization of exosome-mediated cellular communication and pro-tumoral baseline M2 macrophage polarization, the Panc-1 cells were transfected with microRNA-155 (miR-155) and microRNA-125b-2 (miR-125b2) expressing plasmid DNA using hyaluronic acid-poly(ethylene imine)/hyaluronic acid-poly(ethylene glycol) (HA-PEI/HA-PEG) self-assembling nanoparticle-based non-viral vectors. Our results show that upon successful transfection of Panc-1 cells, the exosome content was altered leading to differential communication and reprogramming of the J774.A1 cells to an M1 phenotype. Based on these results, genetic therapies targeted towards selective manipulation of tumor cell-derived exosome content may be very promising for cancer therapy. PMID:27443190

  16. Research progress in reprogramming somatic cells into stem cells%体细胞重编程为干细胞的方法研究与进展***

    Institute of Scientific and Technical Information of China (English)

    阮光萍; 王金祥; 杨晓燕; 宋巧巧; 姚翔; 庞荣清; 潘兴华

    2013-01-01

    BACKGROUND:The use of cloned human embryos gives rise to ethical issues and al ows scientists to find alternative ways to reverse differentiation of cel s into multi/pluripotent stem cel s, which is known as reprogramming. The new method of reprogramming is the focus of attention. OBJECTIVE:To investigate the current status of reprogramming and review the methods of somatic cel reprogramming into stem cel s. METHODS:Using the database of CNKI and Pubmed (1983-01/2006-12) to search the related articles about reprogramming. The retrieval words include reprogramming, method, somatic cel , stem cel , differentiation. Papers related reprogramming published recently or in high-impact journals were initial y surveyed and 17 papers were included in the final analysis. RESULTS AND CONCLUSION:The reprogramming of a pluripotent stem cel is the consequence of gradual reconstruction of cel structure, genetic change of chromatin, transcription expression and posttranscriptional control. Remodeling of targeted cel s requires a stable status of reprogramming and a final reorientation into specific differentiation. Sufficient evidence demonstrates that cel identification is influenced by in vitro operation. The fate of reprogrammed cel s needs further in vivo determination.%  背景:克隆人类胚胎会引起伦理问题,这使科学家寻找替代的方法来逆向分化细胞为多能/全能干细胞,这个过程称为重编程。重编程的新方法是研究者关注的焦点。目的:探讨重编程技术的研究现状,并对体细胞重编程为干细胞的各种方法做一综述。方法:应用计算机检索CNKI和Pubmed数据库中1983年1月至2006年12月关于重编程的文章,在标题和中以“重编程,方法,体细胞,干细胞,分化”或“reprogramming, method, somatic cel , stem cel , differentiation”为检索词进行检索。选择文章内容与重编程有关者,同一领域文献则选择近期发表或发表在权

  17. Gnotobiotic Miniature Pig Interbreed Somatic Cell Nuclear Transfer for Xenotransplantation.

    Science.gov (United States)

    Hwang, Jeong Ho; Kim, Sang Eun; Gupta, Mukesh Kumar; Lee, HoonTaek

    2016-08-01

    Transgenic animal producing technology has improved consistently over the last couple of decades. Among the available methods, somatic cell nuclear transfer (SCNT) technology was officially the most popular. However, SCNT has low efficiency and requires a highly skilled individual. Additionally, the allo-SCNT nuclear reprogramming mechanism is poorly understood in the gnotobiotic miniature pig, which is a candidate for xenotransplantation, making sampling in oocytes very difficult compared to commercial hybrid pigs. Therefore, interbreed SCNT (ibSCNT), which is a combination of miniature pig and commercial pig (Landrace based), was analyzed and was found to be similar to SCNT in terms of the rate of blastocyst formation (12.6% ± 2.9% vs. 15.5% ± 2.2%; p > 0.05). However, a significantly lower fusion rate was observed in the ibSCNT compared to normal SCNT with Landrace pig somatic cells (29.6% ± 0.8% vs. 65.0% ± 4.9%). Thus, the optimization of fusion parameters was necessary for efficient SCNT. Our results further revealed that ibSCNT by the whole-cell intracytoplasmic injection (WCICI) method had a significantly higher blastocyst forming efficiency than the electrofusion method (31.1 ± 8.5 vs. 15.5% ± 2.2%). The nuclear remodeling and the pattern of changes in acetylation at H3K9 residue were similar in both SCNT and ibSCNT embryos. PMID:27459580

  18. DNA methylation status of H19 and Xist genos in lungs of somatic cell nuclear transfer bovines

    Institute of Scientific and Technical Information of China (English)

    CHEN Jie; LI DongJie; LIU YanQin; ZHANG Cui; DAI YunPing; LI ShiJie; LINing

    2008-01-01

    In somatic cell nuclear transfer (SCNT) technologies, the donor cell's nuclei need to be epigenetically reprogrammed for embryonic development. The incomplete reprogramming of donor cell nuclei has been implicated as a primary reason for the low efficiency of SCNT. DNA methylation is a major epige- netic modification of the genome that regulates crucial aspects of genome function, including estab-lishment of genomic imprinting. In order to make sure whether the DNA methylation reprogramming is efficient in SCNT animals, we analyzed the DNA methylation status of two imprinting genes, H19 and Xist, in lungs of deceased SCNT bovines that died within 48 h of birth using bisulfite sequencing analysis. Our findings demonstrated that cloned bovines showed significantly lower DNA methylation of H19 than controls (P<0.05), and three tested CpGs sites (1, 2, 3) exhibited unmethylation in one cloned bovine (9C3); however, Xist showed similar DNA methylation levels between clones and con- trols, and both showed hypermethylation (96.11% and 86.67%).

  19. Telomere Elongation and Naive Pluripotent Stem Cells Achieved from Telomerase Haplo-Insufficient Cells by Somatic Cell Nuclear Transfer

    Directory of Open Access Journals (Sweden)

    Li-Ying Sung

    2014-12-01

    Full Text Available Haplo-insufficiency of telomerase genes in humans leads to telomere syndromes such as dyskeratosis congenital and idiopathic pulmonary fibrosis. Generation of pluripotent stem cells from telomerase haplo-insufficient donor cells would provide unique opportunities toward the realization of patient-specific stem cell therapies. Recently, pluripotent human embryonic stem cells (ntESCs have been efficiently achieved by somatic cell nuclear transfer (SCNT. We tested the hypothesis that SCNT could effectively elongate shortening telomeres of telomerase haplo-insufficient cells in the ntESCs with relevant mouse models. Indeed, telomeres of telomerase haplo-insufficient (Terc+/− mouse cells are elongated in ntESCs. Moreover, ntESCs derived from Terc+/− cells exhibit naive pluripotency as evidenced by generation of Terc+/− ntESC clone pups by tetraploid embryo complementation, the most stringent test of naive pluripotency. These data suggest that SCNT could offer a powerful tool to reprogram telomeres and to discover the factors for robust restoration of telomeres and pluripotency of telomerase haplo-insufficient somatic cells.

  20. Cell Cycle Regulates Nuclear Stability of AID and Determines the Cellular Response to AID.

    Directory of Open Access Journals (Sweden)

    Quy Le

    2015-09-01

    Full Text Available AID (Activation Induced Deaminase deaminates cytosines in DNA to initiate immunoglobulin gene diversification and to reprogram CpG methylation in early development. AID is potentially highly mutagenic, and it causes genomic instability evident as translocations in B cell malignancies. Here we show that AID is cell cycle regulated. By high content screening microscopy, we demonstrate that AID undergoes nuclear degradation more slowly in G1 phase than in S or G2-M phase, and that mutations that affect regulatory phosphorylation or catalytic activity can alter AID stability and abundance. We directly test the role of cell cycle regulation by fusing AID to tags that destabilize nuclear protein outside of G1 or S-G2/M phases. We show that enforced nuclear localization of AID in G1 phase accelerates somatic hypermutation and class switch recombination, and is well-tolerated; while nuclear AID compromises viability in S-G2/M phase cells. We identify AID derivatives that accelerate somatic hypermutation with minimal impact on viability, which will be useful tools for engineering genes and proteins by iterative mutagenesis and selection. Our results further suggest that use of cell cycle tags to regulate nuclear stability may be generally applicable to studying DNA repair and to engineering the genome.

  1. Nuclear Mechanics and Stem Cell Differentiation.

    Science.gov (United States)

    Mao, Xinjian; Gavara, Nuria; Song, Guanbin

    2015-12-01

    Stem cells are characterized by their self-renewal and multi-lineage differentiation potential. Stem cell differentiation is a prerequisite for the application of stem cells in regenerative medicine and clinical therapy. In addition to chemical stimulation, mechanical cues play a significant role in regulating stem cell differentiation. The integrity of mechanical sensors is necessary for the ability of cells to respond to mechanical signals. The nucleus, the largest and stiffest cellular organelle, interacts with the cytoskeleton as a key mediator of cell mechanics. Nuclear mechanics are involved in the complicated interactions of lamins, chromatin and nucleoskeleton-related proteins. Thus, stem cell differentiation is intimately associated with nuclear mechanics due to its indispensable role in mechanotransduction and mechanical response. This paper reviews several main contributions of nuclear mechanics, highlights the hallmarks of the nuclear mechanics of stem cells, and provides insight into the relationship between nuclear mechanics and stem cell differentiation, which may guide clinical applications in the future.

  2. Resveratrol Impedes the Stemness, Epithelial-Mesenchymal Transition, and Metabolic Reprogramming of Cancer Stem Cells in Nasopharyngeal Carcinoma through p53 Activation

    Directory of Open Access Journals (Sweden)

    Yao-An Shen

    2013-01-01

    Full Text Available Cancer stem cells (CSCs are able to self-renew and are refractory to cancer treatment. To investigate the effects of resveratrol on CSCs of nasopharyngeal carcinoma (NPC, we employed a behavior selection strategy to isolate CSCs based on radioresistance, chemoresistance, and tumor sphere formation ability. These NPC CSCs displayed stem cell properties and underwent metabolic shift to predominately rely on glycolysis for energy supply. Intriguingly, we found that resveratrol turned off the metabolic switch, increased the reactive oxygen species (ROS level, and depolarized mitochondrial membranes. These alterations in metabolism occurred concomitantly with the suppression of CSC properties including resistance to therapy, self-renewal capacity, tumor initiation capacity, and metastatic potential in NPC CSCs. We found that resveratrol impeded CSC properties through the activation of p53 and this effect could be reversed by knockdown of p53. Furthermore, resveratrol suppressed the stemness and EMT through reactivating p53 and inducing miR-145 and miR-200c, which were downregulated in NPC CSCs. In conclusion, we demonstrated that resveratrol employed the p53 pathway in regulating stemness, EMT, and metabolic reprogramming. Further investigation of the molecular mechanism of p53 activation by resveratrol may provide useful information for the development of novel therapies for cancer treatment through targeting to CSCs.

  3. Effects of Histone Deacetylase Inhibitor Oxamflatin on In Vitro Porcine Somatic Cell Nuclear Transfer Embryos

    Science.gov (United States)

    Hou, Liming; Ma, Fanhua; Yang, Jinzeng; Riaz, Hasan; Wang, Yongliang; Wu, Wangjun; Xia, Xiaoliang; Ma, Zhiyuan; Zhou, Ying; Zhang, Lin; Ying, Wenqin; Xu, Dequan; Zuo, Bo; Ren, Zhuqing

    2014-01-01

    Abstract Low cloning efficiency is considered to be caused by the incomplete or aberrant epigenetic reprogramming of differentiated donor cells in somatic cell nuclear transfer (SCNT) embryos. Oxamflatin, a novel class of histone deacetylase inhibitor (HDACi), has been found to improve the in vitro and full-term developmental potential of SCNT embryos. In the present study, we studied the effects of oxamflatin treatment on in vitro porcine SCNT embryos. Our results indicated that the rate of in vitro blastocyst formation of SCNT embryos treated with 1 μM oxamflatin for 15 h postactivation was significantly higher than all other treatments. Treatment of oxamflatin decreased the relative histone deacetylase (HDAC) activity in cloned embryos and resulted in hyperacetylation levels of histone H3 at lysine 9 (AcH3K9) and histone H4 at lysine 5 (AcH4K5) at pronuclear, two-cell, and four-cell stages partly through downregulating HDAC1. The suppression of HDAC6 through oxamflatin increased the nonhistone acetylation level of α-tubulin during the mitotic cell cycle of early SCNT embryos. In addition, we demonstrated that oxamflatin downregulated DNA methyltransferase 1 (DNMT1) expression and global DNA methylation level (5-methylcytosine) in two-cell-stage porcine SCNT embryos. The pluripotency-related gene POU5F1 was found to be upregulated in the oxamflatin-treated group with a decreased DNA methylation tendency in its promoter regions. Treatment of oxamflatin did not change the locus-specific DNA methylation levels of Sus scrofa heterochromatic satellite DNA sequences at the blastocyst stage. Meanwhile, our findings suggest that treatment with HDACi may contribute to maintaining the stable status of cytoskeleton-associated elements, such as acetylated α-tubulin, which may be the crucial determinants of donor nuclear reprogramming in early SCNT embryos. In summary, oxamflatin treatment improves the developmental potential of porcine SCNT embryos in vitro. PMID

  4. Fishing Fish Stem Cells and Nuclear Transplants

    OpenAIRE

    Yunhan Hong

    2011-01-01

    Fish has been the subject of various research fields, ranging from ecology, evolution, physiology and toxicology to aquaculture. In the past decades fish has attracted considerable attention for functional genomics, cancer biology and developmental genetics, in particular nuclear transfer for understanding of cytoplasmic-nuclear relationship. This special issue reports on recent progress made in fish stem cells and nuclear transfer.

  5. Oncogenic H-ras reprograms Madin-Darby canine kidney (MDCK) cell-derived exosomal proteins following epithelial-mesenchymal transition.

    Science.gov (United States)

    Tauro, Bow J; Mathias, Rommel A; Greening, David W; Gopal, Shashi K; Ji, Hong; Kapp, Eugene A; Coleman, Bradley M; Hill, Andrew F; Kusebauch, Ulrike; Hallows, Janice L; Shteynberg, David; Moritz, Robert L; Zhu, Hong-Jian; Simpson, Richard J

    2013-08-01

    together, our findings reveal that exosomes from Ras-transformed MDCK cells are reprogrammed with factors which may be capable of inducing EMT in recipient cells. PMID:23645497

  6. Acquired resistance to metformin in breast cancer cells triggers transcriptome reprogramming toward a degradome-related metastatic stem-like profile.

    Science.gov (United States)

    Oliveras-Ferraros, Cristina; Vazquez-Martin, Alejandro; Cuyàs, Elisabet; Corominas-Faja, Bruna; Rodríguez-Gallego, Esther; Fernández-Arroyo, Salvador; Martin-Castillo, Begoña; Joven, Jorge; Menendez, Javier A

    2014-01-01

    Therapeutic interventions based on metabolic inhibitor-based therapies are expected to be less prone to acquired resistance. However, there has not been any study assessing the possibility that the targeting of the tumor cell metabolism may result in unforeseeable resistance. We recently established a pre-clinical model of estrogen-dependent MCF-7 breast cancer cells that were chronically adapted to grow (> 10 months) in the presence of graded, millimolar concentrations of the anti-diabetic biguanide metformin, an AMPK agonist/mTOR inhibitor that has been evaluated in multiple in vitro and in vivo cancer studies and is now being tested in clinical trials. To assess what impact the phenomenon of resistance might have on the metformin-like "dirty" drugs that are able to simultaneously hit several metabolic pathways, we employed the ingenuity pathway analysis (IPA) software to functionally interpret the data from Agilent whole-human genome arrays in the context of biological processes, networks, and pathways. Our findings establish, for the first time, that a "global" targeting of metabolic reprogramming using metformin certainly imposes a great selective pressure for the emergence of new breast cancer cellular states. Intriguingly, acquired resistance to metformin appears to trigger a transcriptome reprogramming toward a metastatic stem-like profile, as many genes encoding the components of the degradome (KLK11, CTSF, FREM1, BACE-2, CASP, TMPRSS4, MMP16, HTRA1), cancer cell migration and invasion factors (TP63, WISP2, GAS3, DKK1, BCAR3, PABPC1, MUC1, SPARCL1, SEMA3B, SEMA6A), stem cell markers (DCLK1, FAK), and key pro-metastatic lipases (MAGL and Cpla2) were included in the signature. Because this convergent activation of pathways underlying tumor microenvironment interactions occurred in low-proliferative cancer cells exhibiting a notable downregulation of the G 2/M DNA damage checkpoint regulators that maintain genome stability (CCNB1, CCNB2, CDC20, CDC25C, AURKA

  7. Acquired resistance to metformin in breast cancer cells triggers transcriptome reprogramming toward a degradome-related metastatic stem-like profile

    Science.gov (United States)

    Oliveras-Ferraros, Cristina; Vazquez-Martin, Alejandro; Cuyàs, Elisabet; Corominas-Faja, Bruna; Rodríguez-Gallego, Esther; Fernández-Arroyo, Salvador; Martin-Castillo, Begoña; Joven, Jorge; Menendez, Javier A

    2014-01-01

    Therapeutic interventions based on metabolic inhibitor-based therapies are expected to be less prone to acquired resistance. However, there has not been any study assessing the possibility that the targeting of the tumor cell metabolism may result in unforeseeable resistance. We recently established a pre-clinical model of estrogen-dependent MCF-7 breast cancer cells that were chronically adapted to grow (> 10 months) in the presence of graded, millimolar concentrations of the anti-diabetic biguanide metformin, an AMPK agonist/mTOR inhibitor that has been evaluated in multiple in vitro and in vivo cancer studies and is now being tested in clinical trials. To assess what impact the phenomenon of resistance might have on the metformin-like “dirty” drugs that are able to simultaneously hit several metabolic pathways, we employed the ingenuity pathway analysis (IPA) software to functionally interpret the data from Agilent whole-human genome arrays in the context of biological processes, networks, and pathways. Our findings establish, for the first time, that a “global” targeting of metabolic reprogramming using metformin certainly imposes a great selective pressure for the emergence of new breast cancer cellular states. Intriguingly, acquired resistance to metformin appears to trigger a transcriptome reprogramming toward a metastatic stem-like profile, as many genes encoding the components of the degradome (KLK11, CTSF, FREM1, BACE-2, CASP, TMPRSS4, MMP16, HTRA1), cancer cell migration and invasion factors (TP63, WISP2, GAS3, DKK1, BCAR3, PABPC1, MUC1, SPARCL1, SEMA3B, SEMA6A), stem cell markers (DCLK1, FAK), and key pro-metastatic lipases (MAGL and Cpla2) were included in the signature. Because this convergent activation of pathways underlying tumor microenvironment interactions occurred in low-proliferative cancer cells exhibiting a notable downregulation of the G2/M DNA damage checkpoint regulators that maintain genome stability (CCNB1, CCNB2, CDC20, CDC25C

  8. Manipulation of KLF4 Expression Generates iPSCs Paused at Successive Stages of Reprogramming

    Directory of Open Access Journals (Sweden)

    Ken Nishimura

    2014-11-01

    Full Text Available The detailed mechanism of reprogramming somatic cells into induced pluripotent stem cells (iPSCs remains largely unknown. Partially reprogrammed iPSCs are informative and useful for understanding the mechanism of reprogramming but remain technically difficult to generate in a predictable and reproducible manner. Using replication-defective and persistent Sendai virus (SeVdp vectors, we analyzed the effect of decreasing the expression levels of OCT4, SOX2, KLF4, and c-MYC and found that low KLF4 expression reproducibly gives rise to a homogeneous population of partially reprogrammed iPSCs. Upregulation of KLF4 allows these cells to resume reprogramming, indicating that they are paused iPSCs that remain on the path toward pluripotency. Paused iPSCs with different KLF4 expression levels remain at distinct intermediate stages of reprogramming. This SeVdp-based stage-specific reprogramming system (3S reprogramming system is applicable for both mouse and human somatic cells and will facilitate the mechanistic analysis of reprogramming.

  9. The human application of gene therapy to re-program T-cell specificity using chimeric antigen receptors

    Institute of Scientific and Technical Information of China (English)

    Alan DGuerrero; Judy SMoyes; Laurence JN Cooper

    2014-01-01

    The adoptive transfer of T cells is a promising approach to treat cancers. Primary human T cells can be modified using viral and non-viral vectors to promote the specific targeting of cancer cells via the introduction of exogenous T-cell receptors (TCRs) or chimeric antigen receptors (CARs). This gene transfer displays the potential to increase the specificity and potency of the anticancer response while decreasing the systemic adverse effects that arise from conventional treatments that target both cancerous and healthy cells. This review highlights the generation of clinical-grade T cells expressing CARs for immunotherapy, the use of these cels to target B-cellmalignancies and, particularly, the first clinical trials deploying the Sleeping Beauty gene transfer system, which engineers T cells to target CD19+ leukemia and non-Hodgkin’s lymphoma.

  10. DNA methylation at a bovine alpha satellite I repeat CpG site during development following fertilization and somatic cell nuclear transfer.

    Directory of Open Access Journals (Sweden)

    Christine Couldrey

    Full Text Available Incomplete epigenetic reprogramming is postulated to contribute to the low developmental success following somatic cell nuclear transfer (SCNT. Here, we describe the epigenetic reprogramming of DNA methylation at an alpha satellite I CpG site (αsatI-5 during development of cattle generated either by artificial insemination (AI or in vitro fertilization (IVF and SCNT. Quantitative methylation analysis identified that SCNT donor cells were highly methylated at αsatI-5 and resulting SCNT blastocysts showed significantly more methylation than IVF blastocysts. At implantation, no difference in methylation was observed between SCNT and AI in trophoblast tissue at αsatI-5, however, SCNT embryos were significantly hyper-methylated compared to AI controls at this time point. Following implantation, DNA methylation at αsatI-5 decreased in AI but not SCNT placental tissues. In contrast to placenta, the proportion of methylation at αsatI-5 remained high in adrenal, kidney and muscle tissues during development. Differences in the average proportion of methylation were smaller in somatic tissues than placental tissues but, on average, SCNT somatic tissues were hyper-methylated at αsatI-5. Although sperm from all bulls was less methylated than somatic tissues at αsatI-5, on average this site remained hyper-methylated in sperm from cloned bulls compared with control bulls. This developmental time course confirms that epigenetic reprogramming does occur, at least to some extent, following SCNT. However, the elevated methylation levels observed in SCNT blastocysts and cellular derivatives implies that there is either insufficient time or abundance of appropriate reprogramming factors in oocytes to ensure complete reprogramming. Incomplete reprogramming at this CpG site may be a contributing factor to low SCNT success rates, but more likely represents the tip of the iceberg in terms of incompletely reprogramming. Until protocols ensure the epigenetic

  11. Epigenetic Regulation of B Lymphocyte Differentiation, Transdifferentiation, and Reprogramming

    Directory of Open Access Journals (Sweden)

    Bruna Barneda-Zahonero

    2012-01-01

    Full Text Available B cell development is a multistep process that is tightly regulated at the transcriptional level. In recent years, investigators have shed light on the transcription factor networks involved in all the differentiation steps comprising B lymphopoiesis. The interplay between transcription factors and the epigenetic machinery involved in establishing the correct genomic landscape characteristic of each cellular state is beginning to be dissected. The participation of “epigenetic regulator-transcription factor” complexes is also crucial for directing cells during reprogramming into pluripotency or lineage conversion. In this context, greater knowledge of epigenetic regulation during B cell development, transdifferentiation, and reprogramming will enable us to understand better how epigenetics can control cell lineage commitment and identity. Herein, we review the current knowledge about the epigenetic events that contribute to B cell development and reprogramming.

  12. Podoplanin is a component of extracellular vesicles that reprograms cell-derived exosomal proteins and modulates lymphatic vessel formation

    Science.gov (United States)

    Andrés, Germán; Gopal, Shashi K.; Martín-Villar, Ester; Renart, Jaime; Simpson, Richard J.; Quintanilla, Miguel

    2016-01-01

    Podoplanin (PDPN) is a transmembrane glycoprotein that plays crucial roles in embryonic development, the immune response, and malignant progression. Here, we report that cells ectopically or endogenously expressing PDPN release extracellular vesicles (EVs) that contain PDPN mRNA and protein. PDPN incorporates into membrane shed microvesicles (MVs) and endosomal-derived exosomes (EXOs), where it was found to colocalize with the canonical EV marker CD63 by immunoelectron microscopy. We have previously found that expression of PDPN in MDCK cells induces an epithelial-mesenchymal transition (EMT). Proteomic profiling of MDCK-PDPN cells compared to control cells shows that PDPN-induced EMT is associated with upregulation of oncogenic proteins and diminished expression of tumor suppressors. Proteomic analysis of exosomes reveals that MDCK-PDPN EXOs were enriched in protein cargos involved in cell adhesion, cytoskeletal remodeling, signal transduction and, importantly, intracellular trafficking and EV biogenesis. Indeed, expression of PDPN in MDCK cells stimulated both EXO and MV production, while knockdown of endogenous PDPN in human HN5 squamous carcinoma cells reduced EXO production and inhibited tumorigenesis. EXOs released from MDCK-PDPN and control cells both stimulated in vitro angiogenesis, but only EXOs containing PDPN were shown to promote lymphatic vessel formation. This effect was mediated by PDPN on the surface of EXOs, as demonstrated by a neutralizing specific monoclonal antibody. These results contribute to our understanding of PDPN-induced EMT in association to tumor progression, and suggest an important role for PDPN in EV biogenesis and/or release and for PDPN-EXOs in modulating lymphangiogenesis. PMID:26893367

  13. 再生医学研究新进展:DNA甲基化与细胞重编程%New development of regenerative medicine: DNA methylation and cell reprogramming

    Institute of Scientific and Technical Information of China (English)

    陈丽丽; 崔淯夏; 杨水祥

    2011-01-01

    背景:使用一些生长因子能使终分化的体细胞重编程而产生多能性干细胞.目的:讨论表观遗传机制在细胞重编程和调节中的作用,揭示表观遗传基因调控的变化、表观遗传修饰标记的稳定性及其对基因组表达的影响.方法:在PubMed数据库及CNKI数据库,以"DNA甲基化,细胞重编程,干细胞"为关键词检索1990/2008相关的文章.结果与结论:尽管在体内细胞分化通常是单向和不可逆的,但这个过程可被重编程而改变.使用一些生长因子能使终分化的体细胞重编程而产生多能性干细胞.目前为止,导入转录因子、DNA去甲基化、表观基因改变已被用于诱导重编程.通过了解其分子机制,揭示表观遗传基因调控的变化、表观遗传修饰标记的稳定性及其对基因组表达的影响,对基因治疗的发展有重要意义.然而依然许多问题有待深入研究,如:是AID还是 DNA去甲基酶对DNA去甲基化起重要作用?DNA去甲基化需要什么条件?肿瘤细胞中CpG片段能否去甲基化,癌细胞是否更难重编程?%BACKGROUND: A small number of growth factors are sufficient to reprogram somatic cells into pluripotentstem cells. OBJECTIVE: To explore the e pi genetic mechanism in cellular re prog ramming and regulating, and to reveal epigenetic alterations, the stability of epigenetic modifications and their effects on genome expression.METHODS: Pubmed database and CNK) database from 1990 to 2008 were retrieved by computer vurth the keywords of'DNA methylation; cell reprogramming; stem cells".RESULTS AND CONCLUSION: Though cellular differentiation is unidirectional and irreversible, this process can be changed by reprogramming. Some growth factors are sufficient to reprogram so malic cells into pluripotentstem cells. So far, the introduction of transcription factors, DNA de methylation and the apparent gen etic modification have been used to induce reprogramming. By understanding the molecular

  14. Bidirectional reprogramming of fusion cells of pluripotent stem cells/primary cardiac myocytes%诱导多能干细胞/原代心肌细胞的融合细胞表现出双向重建

    Institute of Scientific and Technical Information of China (English)

    熊挺淋; 张晓刚; 赵霞; 马红芬

    2011-01-01

    Objective To construct fusion cells with induced pluripotent stem cells (iPSc) and primary cardiac myocytes in vitro, and to investigate biological features of the fusion cells. Methods Polyethylene glycol (PEG-4000) was used to mediate the cell fusion of iPSc derived from green fluorescent protein (GFP) transgenes (octamer-binding transcription factor-4, Oct-4) mouse and cardiac myocytes from neonatal mouse. Morphological changes of the fusion cells were observed dynamically after alkaline phosphatase (AKP) staining. Specific proteins of stem cells and cardiac myocytes in fusion cells were detected by immunofluores-cence. Chromosome karyotype analysis were performed to determine whether the occurrence of nuclear fusion and degree of integration. Results Fusion cells were constructed successfully by polyethylene glycol mediation. Colony-like cell clusters appeared in 4 d after fusion. The AKP positive rate of iPSc were 0.935 ±0.039, 0.939 ± 0.022, 0.954 ± 0.017, and 0.944 ± 0.027 at the 2nd, 3rd, 4th and 5th days respectively, and that of fusion cells were 0.761 ±0.044, 0.740 ±0.023, 0.681 ±0.034, and 0.748 ±0.045 at the corresponding days respectively. At the same time points, there were significant differences between iPSc AKP-positive rates and those of fusion cells ( P < 0. 05). In the initial stage, fusion cells mainly displayed iPSc characteristics, with Oct-4 positive while cTnT negative. Then the fusion cells began to display both characteristics of iPSc and cardiac myocytes in 7 d after fusion, with positive expression of Oct-4 and cTnT. More than 80% of fusion cells had 76 to 80 chromosomes. Conclusion Fusion cells from diploid iPSc and diploid myocardial cells display the characteristics of the two parental cells and show bidirectional reprogramming.%目的 体外构建诱导多能干细胞(induced pluripotent stem cells,iPSc)与原代心肌细胞的融合细胞,初步探讨融合细胞体外生物学特性.方法

  15. Human monocytes undergo functional re-programming during differentiation to dendritic cell mediated by human extravillous trophoblasts

    Science.gov (United States)

    Zhao, Lei; Shao, Qianqian; Zhang, Yun; Zhang, Lin; He, Ying; Wang, Lijie; Kong, Beihua; Qu, Xun

    2016-01-01

    Maternal immune adaptation is required for a successful pregnancy to avoid rejection of the fetal–placental unit. Dendritic cells within the decidual microenvironment lock in a tolerogenic profile. However, how these tolerogenic DCs are induced and the underlying mechanisms are largely unknown. In this study, we show that human extravillous trophoblasts redirect the monocyte-to-DC transition and induce regulatory dendritic cells. DCs differentiated from blood monocytes in the presence of human extravillous trophoblast cell line HTR-8/SVneo displayed a DC-SIGN+CD14+CD1a− phenotype, similar with decidual DCs. HTR8-conditioned DCs were unable to develop a fully mature phenotype in response to LPS, and altered the cytokine secretory profile significantly. Functionally, conditioned DCs poorly induced the proliferation and activation of allogeneic T cells, whereas promoted CD4+CD25+Foxp3+ Treg cells generation. Furthermore, the supernatant from DC and HTR-8/SVneo coculture system contained significant high amount of M-CSF and MCP-1. Using neutralizing antibodies, we discussed the role of M-CSF and MCP-1 during monocyte-to-DCs differentiation mediated by extravillous trophoblasts. Our data indicate that human extravillous trophoblasts play an important role in modulating the monocyte-to-DC differentiation through M-CSF and MCP-1, which facilitate the establishment of a tolerogenic microenvironment at the maternal–fetal interface. PMID:26857012

  16. Human monocytes undergo functional re-programming during differentiation to dendritic cell mediated by human extravillous trophoblasts.

    Science.gov (United States)

    Zhao, Lei; Shao, Qianqian; Zhang, Yun; Zhang, Lin; He, Ying; Wang, Lijie; Kong, Beihua; Qu, Xun

    2016-01-01

    Maternal immune adaptation is required for a successful pregnancy to avoid rejection of the fetal-placental unit. Dendritic cells within the decidual microenvironment lock in a tolerogenic profile. However, how these tolerogenic DCs are induced and the underlying mechanisms are largely unknown. In this study, we show that human extravillous trophoblasts redirect the monocyte-to-DC transition and induce regulatory dendritic cells. DCs differentiated from blood monocytes in the presence of human extravillous trophoblast cell line HTR-8/SVneo displayed a DC-SIGN(+)CD14(+)CD1a(-) phenotype, similar with decidual DCs. HTR8-conditioned DCs were unable to develop a fully mature phenotype in response to LPS, and altered the cytokine secretory profile significantly. Functionally, conditioned DCs poorly induced the proliferation and activation of allogeneic T cells, whereas promoted CD4(+)CD25(+)Foxp3(+) Treg cells generation. Furthermore, the supernatant from DC and HTR-8/SVneo coculture system contained significant high amount of M-CSF and MCP-1. Using neutralizing antibodies, we discussed the role of M-CSF and MCP-1 during monocyte-to-DCs differentiation mediated by extravillous trophoblasts. Our data indicate that human extravillous trophoblasts play an important role in modulating the monocyte-to-DC differentiation through M-CSF and MCP-1, which facilitate the establishment of a tolerogenic microenvironment at the maternal-fetal interface. PMID:26857012

  17. Oreocnide integrifolia Flavonoids Augment Reprogramming for Islet Neogenesis and β-Cell Regeneration in Pancreatectomized BALB/c Mice

    Directory of Open Access Journals (Sweden)

    Ansarullah

    2012-01-01

    Full Text Available Agents which can either trigger proliferation of β-cells or induce neogenesis of β-cells from precursors would be of pivotal role in reversing diabetic manifestations. We examined the role of flavonoid rich fraction (FRF of Oreocnide integrifolia leaves using a mice model of experimental regeneration. BALB/c mice were subjected to ~70% pancreatectomy (Px and supplemented with FRF for 7, 14, and 21 days after pancreatectomy. Px animals displayed increased blood glucose levels and decreased insulin titres which were ameliorated by FRF supplementation. FRF-treated mice demonstrated prominent newly formed islets budding off from ducts and depicting increased BrdU incorporation. Additionally, transcripts levels of Ins1/2, Reg-3α/γ, Ngn-3, and Pdx-1 were upregulated during the initial 1 week. The present study provides evidence of a nutraceutical contributing to islet neogenesis from ductal cells as the mode of β-cell regeneration and a potential therapeutic for clinical trials in management of diabetic manifestations.

  18. Immortality, but not oncogenic transformation, of primary human cells leads to epigenetic reprogramming of DNA methylation and gene expression

    OpenAIRE

    Gordon, Katrina; Clouaire, Thomas; Bao, Xun X.; Kemp, Sadie E.; Xenophontos, Maria; de Las Heras, Jose Ignacio; Stancheva, Irina

    2013-01-01

    Tumourigenic transformation of normal cells into cancer typically involves several steps resulting in acquisition of unlimited growth potential, evasion of apoptosis and non-responsiveness to growth inhibitory signals. Both genetic and epigenetic changes can contribute to cancer development and progression. Given the vast genetic heterogeneity of human cancers and difficulty to monitor cancer-initiating events in vivo, the precise relationship between acquisition of genetic mutations and the ...

  19. Development of a Piggybac based direct reprogramming system for derivation of integration free induced pluripotent stem cells

    OpenAIRE

    Matias, Dino Emanuel Santos

    2013-01-01

    Induced pluripotent stem cells (iPSc) have great potential for applications in regenerative medicine, disease modeling and basic research. Several methods have been developed for their derivation. The original method of Takahashi and Yamanaka involved the use of retroviral vectors which result in insertional mutagenesis, presence in the genome of potential oncogenes and effects of residual transgene expression on differentiation bias of each particular iPSc line. Other methods have been devel...

  20. Advances in MicroRNA-Mediated Reprogramming Technology

    Directory of Open Access Journals (Sweden)

    Chih-Hao Kuo

    2012-01-01

    Full Text Available The use of somatic cells to generate induced-pluripotent stem cells (iPSCs, which have gene characteristic resembling those of human embryonic stem cells (hESCs, has opened up a new avenue to produce patient-specific stem cells for regenerative medicine. MicroRNAs (miRNAs have gained much attention over the past few years due to their pivotal role in many biological activites, including metabolism, host immunity, and cancer. Soon after the discovery of embryonic-stem-cell- (ESC- specific miRNAs, researchers began to investigate their functions in embryonic development and differentiation, as well as their potential roles in somatic cell reprogramming (SCR. Several approaches for ESC-specific miRNA-mediated reprogramming have been developed using cancer and somatic cells to generate ESC-like cells with similarity to iPSCs and/or hESCs. However, the use of virus-integration to introduce reprogramming factors limits future clinical applications. This paper discusses the possible underlying mechanism for miRNA-mediated somatic cell reprogramming and the approaches used by different groups to induce iPSCs with miRNAs.

  1. Cellular reprogramming for understanding and treating human disease.

    Directory of Open Access Journals (Sweden)

    Riya Rajan Kanherkar

    2014-11-01

    Full Text Available In the last two decades we have witnessed a paradigm shift in our understanding of cells so radical that it has rewritten the rules of biology. The study of cellular reprogramming has gone from little more than a hypothesis, to applied bioengineering, with the creation of a variety of important cell types. By way of metaphor, we can compare the discovery of reprogramming with the archaeological discovery of the Rosetta stone. This stone slab made possible the initial decipherment of Egyptian hieroglyphics because it allowed us to see this language in a way that was previously impossible. We propose that cellular reprogramming will have an equally profound impact on understanding and curing human disease, because it allows us to perceive and study molecular biological processes such as differentiation, epigenetics, and chromatin in ways that were likewise previously impossible. Stem cells could be called cellular Rosetta stones because they allow also us to perceive the connections between development, disease, cancer, aging, and regeneration in novel ways. Here we present a comprehensive historical review of stem cells and cellular reprogramming, and illustrate the developing synergy between many previously unconnected fields. We show how stem cells can be used to create in vitro models of human disease and provide examples of how reprogramming is being used to study and treat such diverse diseases as cancer, aging and accelerated aging syndromes, infectious diseases such as AIDS, and epigenetic diseases such as polycystic ovary syndrome. While the technology of reprogramming is being developed and refined there have also been significant ongoing developments in other complementary technologies such as gene editing, progenitor cell production, and tissue engineering. These technologies are the foundations of what is becoming a fully-functional field of regenerative medicine and are converging to a point that will allow us to treat almost any

  2. Cloning adult farm animals: a review of the possibilities and problems associated with somatic cell nuclear transfer.

    Science.gov (United States)

    Edwards, J L; Schrick, F N; McCracken, M D; van Amstel, S R; Hopkins, F M; Welborn, M G; Davies, C J

    2003-08-01

    In 1997, Wilmut et al. announced the birth of Dolly, the first ever clone of an adult animal. To date, adult sheep, goats, cattle, mice, pigs, cats and rabbits have been cloned using somatic cell nuclear transfer. The ultimate challenge of cloning procedures is to reprogram the somatic cell nucleus for development of the early embryo. The cell type of choice for reprogramming the somatic nucleus is an enucleated oocyte. Given that somatic cells are easily obtained from adult animals, cultured in the laboratory and then genetically modified, cloning procedures are ideal for introducing specific genetic modifications in farm animals. Genetic modification of farm animals provides a means of studying genes involved in a variety of biological systems and disease processes. Moreover, genetically modified farm animals have created a new form of 'pharming' whereby farm animals serve as bioreactors for production of pharmaceuticals or organ donors. A major limitation of cloning procedures is the extreme inefficiency for producing live offspring. Dolly was the only live offspring produced after 277 attempts. Similar inefficiencies for cloning adult animals of other species have been described by others. Many factors related to cloning procedures and culture environment contribute to the death of clones, both in the embryonic and fetal periods as well as during neonatal life. Extreme inefficiencies of this magnitude, along with the fact that death of the surrogate may occur, continue to raise great concerns with cloning humans. PMID:12846674

  3. Cloning adult farm animals: a review of the possibilities and problems associated with somatic cell nuclear transfer.

    Science.gov (United States)

    Edwards, J L; Schrick, F N; McCracken, M D; van Amstel, S R; Hopkins, F M; Welborn, M G; Davies, C J

    2003-08-01

    In 1997, Wilmut et al. announced the birth of Dolly, the first ever clone of an adult animal. To date, adult sheep, goats, cattle, mice, pigs, cats and rabbits have been cloned using somatic cell nuclear transfer. The ultimate challenge of cloning procedures is to reprogram the somatic cell nucleus for development of the early embryo. The cell type of choice for reprogramming the somatic nucleus is an enucleated oocyte. Given that somatic cells are easily obtained from adult animals, cultured in the laboratory and then genetically modified, cloning procedures are ideal for introducing specific genetic modifications in farm animals. Genetic modification of farm animals provides a means of studying genes involved in a variety of biological systems and disease processes. Moreover, genetically modified farm animals have created a new form of 'pharming' whereby farm animals serve as bioreactors for production of pharmaceuticals or organ donors. A major limitation of cloning procedures is the extreme inefficiency for producing live offspring. Dolly was the only live offspring produced after 277 attempts. Similar inefficiencies for cloning adult animals of other species have been described by others. Many factors related to cloning procedures and culture environment contribute to the death of clones, both in the embryonic and fetal periods as well as during neonatal life. Extreme inefficiencies of this magnitude, along with the fact that death of the surrogate may occur, continue to raise great concerns with cloning humans.

  4. Cellular Reprogramming Using Defined Factors and MicroRNAs.

    Science.gov (United States)

    Eguchi, Takanori; Kuboki, Takuo

    2016-01-01

    Development of human bodies, organs, and tissues contains numerous steps of cellular differentiation including an initial zygote, embryonic stem (ES) cells, three germ layers, and multiple expertized lineages of cells. Induced pluripotent stem (iPS) cells have been recently developed using defined reprogramming factors such as Nanog, Klf5, Oct3/4 (Pou5f1), Sox2, and Myc. This outstanding innovation is largely changing life science and medicine. Methods of direct reprogramming of cells into myocytes, neurons, chondrocytes, and osteoblasts have been further developed using modified combination of factors such as N-myc, L-myc, Sox9, and microRNAs in defined cell/tissue culture conditions. Mesenchymal stem cells (MSCs) and dental pulp stem cells (DPSCs) are also emerging multipotent stem cells with particular microRNA expression signatures. It was shown that miRNA-720 had a role in cellular reprogramming through targeting the pluripotency factor Nanog and induction of DNA methyltransferases (DNMTs). This review reports histories, topics, and idea of cellular reprogramming.

  5. Cellular Reprogramming Using Defined Factors and MicroRNAs

    Directory of Open Access Journals (Sweden)

    Takanori Eguchi

    2016-01-01

    Full Text Available Development of human bodies, organs, and tissues contains numerous steps of cellular differentiation including an initial zygote, embryonic stem (ES cells, three germ layers, and multiple expertized lineages of cells. Induced pluripotent stem (iPS cells have been recently developed using defined reprogramming factors such as Nanog, Klf5, Oct3/4 (Pou5f1, Sox2, and Myc. This outstanding innovation is largely changing life science and medicine. Methods of direct reprogramming of cells into myocytes, neurons, chondrocytes, and osteoblasts have been further developed using modified combination of factors such as N-myc, L-myc, Sox9, and microRNAs in defined cell/tissue culture conditions. Mesenchymal stem cells (MSCs and dental pulp stem cells (DPSCs are also emerging multipotent stem cells with particular microRNA expression signatures. It was shown that miRNA-720 had a role in cellular reprogramming through targeting the pluripotency factor Nanog and induction of DNA methyltransferases (DNMTs. This review reports histories, topics, and idea of cellular reprogramming.

  6. Cellular Reprogramming Using Defined Factors and MicroRNAs.

    Science.gov (United States)

    Eguchi, Takanori; Kuboki, Takuo

    2016-01-01

    Development of human bodies, organs, and tissues contains numerous steps of cellular differentiation including an initial zygote, embryonic stem (ES) cells, three germ layers, and multiple expertized lineages of cells. Induced pluripotent stem (iPS) cells have been recently developed using defined reprogramming factors such as Nanog, Klf5, Oct3/4 (Pou5f1), Sox2, and Myc. This outstanding innovation is largely changing life science and medicine. Methods of direct reprogramming of cells into myocytes, neurons, chondrocytes, and osteoblasts have been further developed using modified combination of factors such as N-myc, L-myc, Sox9, and microRNAs in defined cell/tissue culture conditions. Mesenchymal stem cells (MSCs) and dental pulp stem cells (DPSCs) are also emerging multipotent stem cells with particular microRNA expression signatures. It was shown that miRNA-720 had a role in cellular reprogramming through targeting the pluripotency factor Nanog and induction of DNA methyltransferases (DNMTs). This review reports histories, topics, and idea of cellular reprogramming. PMID:27382371

  7. Successful generation of cloned mice using nuclear transfer from induced pluripotent stem cells

    Institute of Scientific and Technical Information of China (English)

    Shuya Zhou; Chunjing Feng; Tang Hai; Liu Wang; Qi Zhou; Chenhui Ding; Xiaoyang Zhao; Eryao Wang; Xiangpeng Dai; Lei Liu; Wei Li; Zichuan Liu; Haifeng Wan

    2010-01-01

    Dear Editor, It is now well known that somatic cells can be efficiently reprogrammed into induced pluripotent stem cells (iPSCs) by forced expression of defined factors [1-3]. These cells, like embryonic stem cells (ESCs), have true pluripotency as shown by the live, fertile mice that can be generated through the tetraploid complementation assay using these iPSCs [4, 5].

  8. The Natural Product Resveratrol Inhibits Yeast Cell Separation by Extensively Modulating the Transcriptional Landscape and Reprogramming the Intracellular Metabolome.

    Directory of Open Access Journals (Sweden)

    Zhe Wang

    Full Text Available An increasing number of studies have shown that the promising compound resveratrol treats multiple diseases, such as cancer and aging; however, the resveratrol mode-of-action (MoA remains largely unknown. Here, by virtue of multiple omics approaches, we adopted fission yeast as a model system with the goal of dissecting the common MoA of the anti-proliferative activity of resveratrol. We found that the anti-proliferative activity of resveratrol is mainly due to its unique role of inhibiting the separation of sister cells, similar phenotype with the C2H2 zinc finger transcription factor Ace2 knock-out strain. Microarray analysis shown that resveratrol has extensive impact on the fission yeast transcription levels. Among the changed gene's list, 40% of up-regulated genes are Core Environmental Stress Responses genes, and 57% of the down-regulated genes are periodically expressed. Moreover, resveratrol leverages the metabolome, which unbalances the intracellular pool sizes of several classes of amino acids, nucleosides, sugars and lipids, thus reflecting the remodulated metabolic networks. The complexity of the resveratrol MoA displayed in previous reports and our work demonstrates that multiple omics approaches must be applied together to obtain a complete picture of resveratrol's anti-proliferative function.

  9. Direct reprogramming of fibroblasts into myocytes to reverse fibrosis.

    Science.gov (United States)

    Muraoka, Naoto; Ieda, Masaki

    2014-01-01

    Heart disease is a major cause of morbidity and mortality worldwide. The low regenerative capacity of adult human hearts has thus far limited the available therapeutic approaches for heart failure. Therefore, new therapies that can regenerate damaged myocardium and improve heart function are urgently needed. Although cell transplantation-based therapies may hold great potential, direct reprogramming of endogenous cardiac fibroblasts, which represent more than half of the cells in the heart, into functional cardiomyocytes in situ may be an alternative strategy by which to regenerate the heart. We and others demonstrated that functional cardiomyocytes can be directly generated from fibroblasts by using several combinations of cardiac-enriched factors in mouse and human. In vivo gene delivery of cardiac reprogramming factors generates new cardiac muscle and improved heart function after myocardial infarction in mouse. This article reviews recent progress in cardiac reprogramming research and discusses the perspectives and challenges of this new technology for future regenerative therapy. PMID:24079415

  10. Complete reprogramming to all-iPS mice

    Institute of Scientific and Technical Information of China (English)

    Steven Y Cheng

    2010-01-01

    @@ The landmark discoveries by Shinya Yamanaka in mice and later separately by James Thompson in humans that adult animal cells can be reprogrammed to a pluripotent state of embryonic stem (ES) cells by forced expression of just a few key transcription factors have permanently altered a long-held view about the stability of differentiated cells in developmental biology, and suggested a way of generating personalized replacement tissues from patients' own cells for the regen-erative medical treatment. However, those initial repro-grammed cells, called induced pluripotent stem (iPS) cells,were not completely identical to ES cells because of their limited ability to contribute to every type of cells in an embryo.

  11. Members of the NODE (Nanog and Oct4-associated deacetylase) complex and SOX-2 promote the initiation of a natural cellular reprogramming event in vivo.

    Science.gov (United States)

    Kagias, Konstantinos; Ahier, Arnaud; Fischer, Nadine; Jarriault, Sophie

    2012-04-24

    Differentiated cells can be forced to change identity, either to directly adopt another differentiated identity or to revert to a pluripotent state. Direct reprogramming events can also occur naturally. We recently characterized such an event in Caenorhabditis elegans, in which a rectal cell switches to a neuronal cell. Here we have used this single-cell paradigm to investigate the molecular requirements of direct cell-type conversion, with a focus on the early steps. Our genetic analyses revealed the requirement of sem-4/Sall, egl-27/Mta, and ceh-6/Oct, members of the NODE complex recently identified in embryonic stem (ES) cells, and of the OCT4 partner sox-2, for the initiation of this natural direct reprogramming event. These four factors have been shown to individually impact on ES cell pluripotency; however, whether they act together to control cellular potential during development remained an open question. We further found that, in addition to acting at the same time, these factors physically associate, suggesting that they could act together as a NODE-like complex during this in vivo process. Finally, we have elucidated the functional domains in EGL-27/MTA that mediate its reprogramming activity in this system and have found that modulation of the posterior HOX protein EGL-5 is a downstream event to allow the initiation of Y identity change. Our data reveal unique in vivo functions in a natural direct reprogramming event for these genes that impact on ES cells pluripotency and suggest that conserved nuclear events could be shared between different cell plasticity phenomena across phyla. PMID:22493276

  12. Epigenetic reprogramming in plant sexual reproduction.

    Science.gov (United States)

    Kawashima, Tomokazu; Berger, Frédéric

    2014-09-01

    Epigenetic reprogramming consists of global changes in DNA methylation and histone modifications. In mammals, epigenetic reprogramming is primarily associated with sexual reproduction and occurs during both gametogenesis and early embryonic development. Such reprogramming is crucial not only to maintain genomic integrity through silencing transposable elements but also to reset the silenced status of imprinted genes. In plants, observations of stable transgenerational inheritance of epialleles have argued against reprogramming. However, emerging evidence supports that epigenetic reprogramming indeed occurs during sexual reproduction in plants and that it has a major role in maintaining genome integrity and a potential contribution to epiallelic variation.

  13. Implications and limitations of cellular reprogramming for psychiatric drug development

    OpenAIRE

    Tobe, Brian T. D.; Brandel, Michael G.; Nye, Jeffrey S; Snyder, Evan Y.

    2013-01-01

    Human-induced pluripotent stem cells (hiPSCs) derived from somatic cells of patients have opened possibilities for in vitro modeling of the physiology of neural (and other) cells in psychiatric disease states. Issues in early stages of technology development include (1) establishing a library of cells from adequately phenotyped patients, (2) streamlining laborious, costly hiPSC derivation and characterization, (3) assessing whether mutations or other alterations introduced by reprogramming co...

  14. New balance in pluripotency: reprogramming with lineage specifiers.

    Science.gov (United States)

    Ben-David, Uri; Nissenbaum, Jonathan; Benvenisty, Nissim

    2013-05-23

    Induction of pluripotency in somatic cells has been achieved by myriad combinations of transcription factors that belong to the core pluripotency circuitry. In this issue, Shu et al. report reprogramming with lineage specifiers, lending support to the view of the pluripotent state as a fine balance between competing differentiation forces.

  15. The four reprogramming factors and embryonic development in mice.

    Science.gov (United States)

    Yan, Xingrong; Yu, Shumin; Lei, Anmin; Hua, Jinlian; Chen, Fulin; Li, Liwen; Xie, Xin; Yang, Xueyi; Geng, Wenxin; Dou, Zhongying

    2010-10-01

    The transcription factors (Oct4, Sox2, c-Myc, and Klf4) play an important role in the generation of induced pluripotent stem cells. These factors are expressed in metaphase II oocytes and embryonic stem cells (ESCs). The mechanisms responsible for the reprogramming of ooplasm during nuclear transfer are expected to be associated with the four factors. Here, we show that different paternal genetic backgrounds are able to influence the in vitro development of parthenogenetic and cloned embryos. Using real- time polymerase chain reaction (PCR) we found that the expression level of Oct4 in oocytes was less than that of ESCs, whereas oocytes from KM x C3H females showed the highest expression level of Sox2 than the other strains tested or in G1 ESCs. c-Myc mRNA levels in oocytes from KM mice were greater than those found in ESCs or oocytes of KM x C3H mice. These data demonstrate that the expression of the four transcription factors was different among the oocytes, which may be a contributing factor for the different efficiencies of parthenogenesis and the development of cloned embryos in vitro. PMID:20936906

  16. Nuclear myosin I regulates cell membrane tension

    Science.gov (United States)

    Venit, Tomáš; Kalendová, Alžběta; Petr, Martin; Dzijak, Rastislav; Pastorek, Lukáš; Rohožková, Jana; Malohlava, Jakub; Hozák, Pavel

    2016-01-01

    Plasma membrane tension is an important feature that determines the cell shape and influences processes such as cell motility, spreading, endocytosis and exocytosis. Unconventional class 1 myosins are potent regulators of plasma membrane tension because they physically link the plasma membrane with adjacent cytoskeleton. We identified nuclear myosin 1 (NM1) - a putative nuclear isoform of myosin 1c (Myo1c) - as a new player in the field. Although having specific nuclear functions, NM1 localizes predominantly to the plasma membrane. Deletion of NM1 causes more than a 50% increase in the elasticity of the plasma membrane around the actin cytoskeleton as measured by atomic force microscopy. This higher elasticity of NM1 knock-out cells leads to 25% higher resistance to short-term hypotonic environment and rapid cell swelling. In contrast, overexpression of NM1 in wild type cells leads to an additional 30% reduction of their survival. We have shown that NM1 has a direct functional role in the cytoplasm as a dynamic linker between the cell membrane and the underlying cytoskeleton, regulating the degree of effective plasma membrane tension. PMID:27480647

  17. 供体细胞来源和TSA处理对牦牛iSCNT胚胎重编程的影响%Effects of donor cell source and TSA treatment on reprogramming of yakbovine iSCNT embryos

    Institute of Scientific and Technical Information of China (English)

    熊显荣; 高川; 符梅; 李键; 字向东; 钟金城

    2012-01-01

    【Objective】The present study was to investigate the effect of different sources of donor cells and TSA treatment on in vitro development of yak interspecies somatic cell nuclear transfer(iSCNT)embryos.【Method】With cattle oocytes as recipient,yak fetal fibroblasts of 50-day-old,6 and 18-month-old adult fibroblasts as the donor nuclear,yak iSCNT embryos were reconstructed and then the effect of donor cell source on the development of cloned embryos evaluated.We compared the effect of different TSA treated concentrations(0,20,40,60 and 80 nmol/L)and times(0,6,8 and 12 h)on the development of yak cloned embryos,respectively,then determined the best program for treatment,and treated donor cells and embryos with this program for detecting the effect of TSA on the relative expression level of HDAC2 by qRT-PCR.【Result】With fetal fibroblast cells as donor cells,yak interspecies cloning embryos blastocyst rate was the highest,but there was no significant difference(P〉0.05).Treatment donor cell with 40 nmol/L TSA for 6 h significantly increased the blastocyst rate(30.6%),and significant difference existed with the control group(P〈0.05).Treatment of reconstructed embryos with 40 nmol/L TSA for 12 h was helpful for embryos development.However,the blastocyst formation rate decreased along with the treated time and concentration of TSA.The expression level of HDAC2 was reduced significantly after donor cell and embryos treated with TSA.【Conclusion】There was no significant effect of donor cells source on the development of yak iSCNT embryos.Treated donor cells for 6 h and reconstructed embryos for 12 h with 40 nmol/L TSA can significantly improve in vitro development of yak iSCNT embryos and reprogramming.%【目的】探讨不同来源的供体细胞和TSA处理对牦牛异种体细胞核移植(Interspecies nuclear transfer,iSCNT)胚胎的影响。【方法】以黄牛卵母细胞为受体,分别以牦牛50日龄胎儿及6和18月龄个体成纤维细

  18. Temporal Requirements of cMyc Protein for Reprogramming Mouse Fibroblasts

    Directory of Open Access Journals (Sweden)

    Corey Heffernan

    2012-01-01

    Full Text Available Exogenous expression of Oct4, Sox2, Klf4, and cMyc forces mammalian somatic cells to adopt molecular and phenotypic characteristics of embryonic stem cells, commencing with the required suppression of lineage-associated genes (e.g., Thy1 in mouse. Although omitting cMyc from the reprogramming cocktail minimizes risks of uncontrolled proliferation, its exclusion results in fold reductions in reprogramming efficiency. Thus, the feasibility of substituting cMyc transgene with (non-integrative recombinant “pTAT-mcMyc” protein delivery was assessed, without compromising reprogramming efficiency or the pluripotent phenotype. Purification and delivery of semisoluble/particulate pTAT-mcMyc maintained Oct4-GFP+ colony formation (i.e., reprogramming efficiency whilst supporting pluripotency by various criteria. Differential repression of Thy1 by pTAT-mcMyc ± Oct4, Sox2, and Klf4 (OSK suggested differential (and non-additive mechanisms of repression. Extending these findings, attempts to enhance reprogramming efficiency through a staggered approach (prerepression of Thy1 failed to improve reprogramming efficiency. We consider protein delivery a useful tool to decipher temporal/molecular events characterizing somatic cell reprogramming.

  19. Stress-triggered atavistic reprogramming (STAR) addiction: driving force behind head and neck cancer?

    Science.gov (United States)

    Masuda, Muneyuki; Wakasaki, Takahiro; Toh, Satoshi

    2016-01-01

    Recent results of the Cancer Genome Atlas on head and neck squamous cell carcinoma (HNSCC) revealed that HNSCC lacked predominant gain-of-function mutations in oncogenes, whereas an essential role for epigenetics in oncogenesis has become apparent. In parallel, it has gained general acceptance that cancer is considered as complex adaptive system, which evolves responding environmental selective pressures. This somatic evolution appears to proceed concurrently with the acquisition of an atavistic pluripotent state (i.e., "stemness"), which is inducible by intrinsic epigenetic reprogramming program as demonstrated by induced pluripotent stem (iPS) cells. This Nobel prize-winning discovery has markedly accelerated and expanded cancer stem cell research from the point of epigenetic reprogramming. Taken together, we hypothesize that stress-triggered atavistic reprogramming (STAR) may be the major driving force of HNSCC evolution. In this perspective, we discuss the possible mechanisms of STAR in HNSCC, focusing on recent topics of epigenetic reprogramming in developmental and cancer cell biology.

  20. Proliferating cell nuclear antigen in neutrophil fate.

    Science.gov (United States)

    Witko-Sarsat, Véronique; Ohayon, Delphine

    2016-09-01

    The life span of a neutrophil is a tightly regulated process as extended survival is beneficial for pathogen elimination and cell death necessary to prevent cytotoxic content release from activated neutrophils at the inflammatory site. Therefore, the control between survival and death must be a dynamic process. We have previously described that proliferating cell nuclear antigen (PCNA) which is known as a nuclear protein pivotal in DNA synthesis, is a key element in controlling neutrophil survival through its association with procaspases. Contrary to the dogma which asserted that PCNA has a strictly nuclear function, in mature neutrophils, PCNA is present exclusively within the cytosol due to its nuclear export at the end of the granulocytic differentiation. More recent studies are consistent with the notion that the cytosolic scaffold of PCNA is aimed at modulating neutrophil fate rather than simply preventing death. Ultimately, targeting neutrophil survival might have important applications not just in the field of immunology and inflammation, but also in hematology and transfusion. The neutrophil emerges as a unique and powerful cellular model to unravel the basic mechanisms governing the cell cycle-independent functions of PCNA and should be considered as a leader of the pack. PMID:27558345

  1. Localization of a bacterial group II intron-encoded protein in eukaryotic nuclear splicing-related cell compartments.

    Directory of Open Access Journals (Sweden)

    Rafael Nisa-Martínez

    Full Text Available Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns.

  2. Inhibition of de novo Palmitate Synthesis by Fatty Acid Synthase Induces Apoptosis in Tumor Cells by Remodeling Cell Membranes, Inhibiting Signaling Pathways, and Reprogramming Gene Expression

    Directory of Open Access Journals (Sweden)

    Richard Ventura

    2015-08-01

    Research in context: Fatty acid synthase (FASN is a vital enzyme in tumor cell biology; the over-expression of FASN is associated with diminished patient prognosis and resistance to many cancer therapies. Our data demonstrate that selective and potent FASN inhibition with TVB-3166 leads to selective death of tumor cells, without significant effect on normal cells, and inhibits in vivo xenograft tumor growth at well-tolerated doses. Candidate biomarkers for selecting tumors highly sensitive to FASN inhibition are identified. These preclinical data provide mechanistic and pharmacologic evidence that FASN inhibition presents a promising therapeutic strategy for treating a variety of cancers.

  3. Cell Cycle Reprogramming for PI3K Inhibition Overrides Relapse-Specific C481S BTK Mutation Revealed by Longitudinal Functional Genomics in Mantle Cell Lymphoma

    OpenAIRE

    Chiron, David; Di Liberto, Maurizio; Martin, Peter; Huang, Xiangao; Sharman, Jeff; Blecua, Pedro; Mathew, Susan; Vijay, Priyanka; Eng, Ken; Ali, Siraj; Johnson, Amy; Chang, Betty; Ely, Scott; Elemento, Olivier; Mason, Christopher E.

    2014-01-01

    Despite the unprecedented clinical activity of the Bruton’s tyrosine kinase inhibitor ibrutinib in MCL, acquired-resistance is common. By longitudinal integrative whole-exome and whole-transcriptome sequencing and targeted sequencing, we identified the first relapse-specific C481S mutation at the ibrutinib-binding site of BTK in MCL cells at progression following a durable response. This mutation enhanced BTK and AKT activation and tissue-specific proliferation of resistant MCL cells driven b...

  4. Nuclear microscopy of rat colon epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Ren, M., E-mail: phyrenmq@nus.edu.sg [Centre for Ion Beam Applications (CIBA), Department of Physics, National University of Singapore, Singapore 117542 (Singapore); Rajendran, Reshmi [Lab of Molecular Imaging, Singapore Bioimaging Consotium, 11 Biopolis Way, 02-02 Helios, Singapore 138667 (Singapore); Ng, Mary [Department of Pharmacology, National University of Singapore (Singapore); Udalagama, Chammika; Rodrigues, Anna E.; Watt, Frank [Centre for Ion Beam Applications (CIBA), Department of Physics, National University of Singapore, Singapore 117542 (Singapore); Jenner, Andrew Michael [Illawara Health and Medical Research Institute (IHMRI), University of Wollongong, NSW 2522 (Australia)

    2011-10-15

    Using Nuclear microscopy, we have investigated iron distributions in the colons of Sprague Dawley rats, in order to elucidate heme uptake. Four groups of five Sprague Dawley rats (mean weight 180 g) were fed different purified diets containing either heme diet (2.5% w/w hemoglobin), high fat diet (HFD) (18% w/w fat, 1% w/w cholesterol), 'western' diet (combination of hemoglobin 2.5% and 18% fat, 1% cholesterol) or control diet (7% w/w fat). After 4 weeks, animals were sacrificed by exsanguination after anaesthesia. Thin sections of frozen colon tissue were taken, freeze dried and scanned using nuclear microscopy utilising the techniques PIXE, RBS and STIM. The new data acquisition system (IonDaq) developed in CIBA was used to obtain high resolution images and line scans were used to map the iron distributions across the colon boundaries. The nuclear microscope results indicate that when HFD is given in addition to heme, the iron content of the epithelial cells that line the colon decreases, and the zinc in the smooth muscle wall increases. This implies that the level of heme and fat in diet has an important role in colon health, possibly by influencing epithelial cells directly or changing luminal composition such as bacterial flora or levels of metabolites and cytotoxins.

  5. A highly efficient method for generation of therapeutic quality human pluripotent stem cells by using naive induced pluripotent stem cells nucleus for nuclear transfer

    Directory of Open Access Journals (Sweden)

    Madhusudana Girija Sanal

    2014-09-01

    Full Text Available Even after several years since the discovery of human embryonic stem cells and induced pluripotent stem cells (iPSC, we are still unable to make any significant therapeutic benefits out of them such as cell therapy or generation of organs for transplantation. Recent success in somatic cell nuclear transfer (SCNT made it possible to generate diploid embryonic stem cells, which opens up the way to make high-quality pluripotent stem cells. However, the process is highly inefficient and hence expensive compared to the generation of iPSC. Even with the latest SCNT technology, we are not sure whether one can make therapeutic quality pluripotent stem cell from any patient’s somatic cells or by using oocytes from any donor. Combining iPSC technology with SCNT, that is, by using the nucleus of the candidate somatic cell which got reprogrammed to pluripotent state instead that of the unmodified nucleus of the candidate somatic cell, would boost the efficiency of the technique, and we would be able to generate therapeutic quality pluripotent stem cells. Induced pluripotent stem cell nuclear transfer (iPSCNT combines the efficiency of iPSC generation with the speed and natural reprogramming environment of SCNT. The new technique may be called iPSCNT. This technique could prove to have very revolutionary benefits for humankind. This could be useful in generating organs for transplantation for patients and for reproductive cloning, especially for childless men and women who cannot have children by any other techniques. When combined with advanced gene editing techniques (such as CRISPR-Cas system this technique might also prove useful to those who want to have healthy children but suffer from inherited diseases. The current code of ethics may be against reproductive cloning. However, this will change with time as it happened with most of the revolutionary scientific breakthroughs. After all, it is the right of every human to have healthy offspring and it is

  6. Statins augment the chemosensitivity of colorectal cancer cells inducing epigenetic reprogramming and reducing colorectal cancer cell 'stemness' via the bone morphogenetic protein pathway

    NARCIS (Netherlands)

    L.L. Kodach; R.J. Jacobs; P.W. Voorneveld; M.E. Wildenberg; H.W. Verspaget; T. van Wezel; H. Morreau; D.W. Hommes; M.P. Peppelenbosch; G.R. van den Brink; J.C.H. Hardwick

    2011-01-01

    Promoter hypermethylation is an important and potentially reversible mechanism of tumour suppressor gene silencing in cancer. Compounds that demethylate tumour suppressor genes and induce differentiation of cancer cells, but do not have toxic side effects, would represent an exciting option in cance

  7. From Stealing Fire to Cellular Reprogramming: A Scientific History Leading to the 2012 Nobel Prize

    Science.gov (United States)

    Lensch, M. William; Mummery, Christine L.

    2013-01-01

    Cellular reprogramming was recently “crowned” with the award of the Nobel Prize to two of its groundbreaking researchers, Sir John Gurdon and Shinya Yamanaka. The recent link between reprogramming and stem cells makes this appear almost a new field of research, but its historical roots have actually spanned more than a century. Here, the Nobel Prize in Physiology or Medicine 2012 is placed in its historical context. PMID:24052937

  8. A case of cellular alchemy: lineage reprogramming and its potential in regenerative medicine.

    Science.gov (United States)

    Asuelime, Grace E; Shi, Yanhong

    2012-08-01

    The field of regenerative medicine is rapidly gaining momentum as an increasing number of reports emerge concerning the induced conversions observed in cellular fate reprogramming. While in recent years, much attention has been focused on the conversion of fate-committed somatic cells to an embryonic-like or pluripotent state, there are still many limitations associated with the applications of induced pluripotent stem cell reprogramming, including relatively low reprogramming efficiency, the times required for the reprogramming event to take place, the epigenetic instability, and the tumorigenicity associated with the pluripotent state. On the other hand, lineage reprogramming involves the conversion from one mature cell type to another without undergoing conversion to an unstable intermediate. It provides an alternative approach in regenerative medicine that has a relatively lower risk of tumorigenesis and increased efficiency within specific cellular contexts. While lineage reprogramming provides exciting potential, there is still much to be assessed before this technology is ready to be applied in a clinical setting. PMID:22371436

  9. Animal embryonic stem (ES) cells: self-renewal, pluripotency, transgenesis and nuclear transfer.

    Science.gov (United States)

    Saito, Shigeo; Liu, Bingbing; Yokoyama, Kazunari

    2004-09-01

    Mouse embryonic stem (ES) cells can be maintained indefinitely in the presence of leukemia inhibitory factor (LIF) and they express markers of self-renewal and pluripotency, which include the transcription factor Oct 4, STAT-3, stage-specific embryonic antigen (SSEA)-1, and alkaline phosphatase (AP). Upon removal of LIF, from the culture medium they cease to express markers such as Oct 4, rapidly losing the capacity for self-renewal and differentiating into a variety of cell types. Gene targeting is feasible in murine ES cells because these cells can be maintained in an undifferentiated state long enough to allow selection of properly targeted cell colonies with a high frequency of homologous recombination. Furthermore, blastocysts cloned from cultured murine ES cells develop to term at an efficiency (10-30%) that is three to ten times higher than blastocysts cloned from the nuclei of differentiated somatic cells. It seems likely that ES cells require less extensive reprogramming than do somatic cells, perhaps because in ES cells, many genes that are essential for early development are already active and thus do not require reactivation. Recently, we succeeded in isolating immortalized equine and bovine ES cells with a normal karyotype, that exhibit features similar to those of murine ES cells and express Oct 4, STAT-3, SSEA-1 and AP. We further confirmed the pluripotential ability of these cells, which were able to undergo somatic differentiation in vitro to neural progenitors and to endothelial or hematopoietic lineages. We were able to use bovine ES cells, as a source of nuclei for nuclear transfer (NT) and we generated cloned cattle with a higher frequency of pregnancies to term than has been achieved with differentiated somatic cells. Moreover, bovine ES cells that expressed enhanced green fluorescent protein (EGFP) were incorporated into both the inner cell mass (ICM) and the trophectdermal cells of developing blastocysts. These findings should facilitate

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

    International Nuclear Information System (INIS)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  12. Technical Challenges in the Derivation of Human Pluripotent Cells

    Directory of Open Access Journals (Sweden)

    Parinya Noisa

    2011-01-01

    Full Text Available It has long been discovered that human pluripotent cells could be isolated from the blastocyst state of embryos and called human embryonic stem cells (ESCs. These cells can be adapted and propagated indefinitely in culture in an undifferentiated manner as well as differentiated into cell representing the three major germ layers: endoderm, mesoderm, and ectoderm. However, the derivation of human pluripotent cells from donated embryos is limited and restricted by ethical concerns. Therefore, various approaches have been explored and proved their success. Human pluripotent cells can also be derived experimentally by the nuclear reprogramming of somatic cells. These techniques include somatic cell nuclear transfer (SCNT, cell fusion and overexpression of pluripotent genes. In this paper, we discuss the technical challenges of these approaches for nuclear reprogramming, involving their advantages and limitations. We will also highlight the possible applications of these techniques in the study of stem cell biology.

  13. Aging, Rejuvenation, and Epigenetic Reprogramming: Resetting the Aging Clock

    OpenAIRE

    Rando, Thomas A.; Chang, Howard Y.

    2012-01-01

    The underlying cause of aging remains one of the central mysteries of biology. Recent studies in several different systems suggest that not only may the rate of aging be modified by environmental and genetic factors, but also that the aging clock can be reversed, restoring characteristics of youthfulness to aged cells and tissues. This Review focuses on the emerging biology of rejuvenation through the lens of epigenetic reprogramming. By defining youthfulness and senescence as epigenetic stat...

  14. Research of Reprogramming of Adipose Derived Stem Cells to Express Keratinocyte Phenotype Using Cell Extracts of Keratinocyte Stem Cells%表皮干细胞抽提物重编程脂肪干细胞表达表皮细胞表型的实验研究

    Institute of Scientific and Technical Information of China (English)

    孙楠; 张群

    2013-01-01

    目的 研究富集的表皮干细胞(Keratinocyte enriched with epidermal stem cells,KSC)抽提物对重编程人脂肪干细胞(Adipose derived stem cells,ASCs)表达表皮细胞表型的影响.方法 常规方法收集表皮细胞(Keratinocyte,KC)后,应用Ⅳ型胶原差速贴壁法分别收集KSC与富集后剩余的表皮细胞(Keratinocyte enriched with epidermal stem cells left,KCL),鉴定K19和P63的阳性表达率,Gimsa染色法测定KC 、KSC、KCL的克隆形成率,分别制备KC、KSC、KCL 的细胞抽提物,作用于链球菌溶血素-O(SLO)通透处理过的原代ASCs,分别应用流式细胞仪与Western-blot测定重编程后ASCs中广谱角蛋白(Pan cytokeratin,P-CK)与ASCs的BRG1表达变化.结果 KSC与KC、KCL来源的细胞抽提物重编程ASCs的CK及BRG1表达率,均有明显统计学差异(P<0.01).结论 脂肪干细胞在表皮细胞抽提物的诱导作用下能表达表皮细胞表型,且对表皮细胞优化处理后的KSC有更加显著的重编程作用.%Objective To evaluate the role of the cell extracts of keratinocyte stem cells (Keratinocyte enriched with epidermal stem cells,KSC) express keratinocytes phenotype of reprogramming of the adipose derived stem cells (Adipose derived stem cells,ASCs).Methods Keratinocyte (KC) were collected in conventional methods,then KSC were isolated from KC with differential attachment to recycled collagen IV and KCL were gathered by not attached cells in suspension culture solution.Clone forming efficiency (CFE) of KC,KSC,KCL were detected with Gimsa staining methods.Cell extracts of KC,KSC and KCL were prepared respectively.Primary cultured adipose derived stem cells were permeabilized using streptolysin O (SLO),and then incubated with cell extracts.Flow cytometry and western-blot were used to detect the expression of pan cytokeratin (Pan cytokeratin,CK) and Brahma-related Gene 1.(BRG1) of reprogrammed ASCs.Results Cell extracts of KSC comparing with that of KC and KCL,had a statistically

  15. Induced pluripotent stem cells, new tools for drug discovery and new hope for stem cell therapies

    OpenAIRE

    Shi, Yanhong

    2009-01-01

    Somatic cell nuclear transfer or therapeutic cloning has provided great hope for stem cell-based therapies. However therapeutic cloning has been experiencing both ethical and technical difficulties. Recent breakthrough studies using a combination of four factors to reprogram human somatic cells into pluripotent stem cells without using embryos or eggs led to an important revolution in stem cell research. Comparative analysis of human induced pluripotent stem cells and human embryonic stem cel...

  16. Overcoming the hurdles for a reproducible generation of human functionally mature reprogrammed neurons.

    Science.gov (United States)

    Broccoli, Vania; Rubio, Alicia; Taverna, Stefano; Yekhlef, Latefa

    2015-06-01

    The advent of cell reprogramming technologies has widely disclosed the possibility to have direct access to human neurons for experimental and biomedical applications. Human pluripotent stem cells can be instructed in vitro to generate specific neuronal cell types as well as different glial cells. Moreover, new approaches of direct neuronal cell reprogramming can strongly accelerate the generation of different neuronal lineages. However, genetic heterogeneity, reprogramming fidelity, and time in culture of the starting cells can still significantly bias their differentiation efficiency and quality of the neuronal progenies. In addition, reprogrammed human neurons exhibit a very slow pace in gaining a full spectrum of functional properties including physiological levels of membrane excitability, sustained and prolonged action potential firing, mature synaptic currents and synaptic plasticity. This delay poses serious limitations for their significance as biological experimental model and screening platform. We will discuss new approaches of neuronal cell differentiation and reprogramming as well as methods to accelerate the maturation and functional activity of the converted human neurons.

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

    Directory of Open Access Journals (Sweden)

    Han-Seop Kim

    2014-01-01

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

  18. Quantitative Proteomics of Intracellular Campylobacter jejuni Reveals Metabolic Reprogramming.

    Directory of Open Access Journals (Sweden)

    Xiaoyun Liu

    Full Text Available Campylobacter jejuni is the major cause of bacterial food-borne illness in the USA and Europe. An important virulence attribute of this bacterial pathogen is its ability to enter and survive within host cells. Here we show through a quantitative proteomic analysis that upon entry into host cells, C. jejuni undergoes a significant metabolic downshift. Furthermore, our results indicate that intracellular C. jejuni reprograms its respiration, favoring the respiration of fumarate. These results explain the poor ability of C. jejuni obtained from infected cells to grow under standard laboratory conditions and provide the bases for the development of novel anti microbial strategies that would target relevant metabolic pathways.

  19. 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.

  20. 细胞重编程改写细胞命运:细胞的返老还童——2012年诺贝尔生理学或医学奖简介%Cell Fate Changed by Reprogramming: the Rejuvenation of Cells—A Brief Introduction to the Nobel Prize in Physiology or Medicine 2012

    Institute of Scientific and Technical Information of China (English)

    王昱凯; 周琪

    2012-01-01

    The study of cell reprogramming has been carried out by many scientists for decades of years. The term cell reprogramming means "fully differentiated cells can be reprogrammed to multipo-tent stem cells". In 1962, John Gurdon proved that somatic cells can be reprogrammed in an unfertilized enucleated Xenopus egg, demonstrating that nuclei from specialized cells still held the potential to be any cell type despite its specialization. Based on Gurdon's achievement, in 2006, Shinya Yamanaka made great progress in the cell reprogramming field. He successfully performed cell reprogramming outside the body and obtained cells with multipotency, namely induced pluripotent stem cells (iPS cells ), indicating that cell fate is determined by selectively opening or closing some certain genes. Compared with embryo stem cells (ES cells), iPS has great superiority in avoiding ethical troubles caused by collecting stem cells from human embryo. Moreover, the cells derived from patients' own tissues are most likely to eliminate immune rejection problems in clinical application. The establishment of iPS technique has triggered the beginning of a brand new research field.%科学家们对细胞重编程的研究已经持续了数十年.所谓细胞重编程是指“已分化的特定细胞可以被重新编程为多功能的干细胞”.1962年,约翰·戈登(John Gurdon)在他的实验室里证明,已分化的动物体细胞在蛙卵中可以被重编程,从而具有发育成完整个体的能力,证明了细胞的分化是可逆的.2006年,山中伸弥(Shinya Yamanaka)将戈登的这一成果推进了一大步,实现了细胞在体外的重编程,诱导出了具有多能性的细胞(即诱导性多能干细胞,induced pluripotent stem cell,iPS细胞),证明了细胞命运是有选择性地打开或关闭某些基因的结果.与胚胎干细胞相比,iPS细胞的优势在于它避开了使用人体胚胎提取干细胞的伦理道德制约,使干细胞

  1. Nuclear area measurement on viable cells, using confocal microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Townsend, K.M.S.; Marsden, S.J. (Medical Research Council, Harwell (United Kingdom). Radiobiological Research Unit)

    1992-04-01

    The authors describe a rapid procedure for the accurate measurement of nuclear areas on unperturbed living cells as used in radiobiological experiments, using the confocal laser scanning microscope. The microdosimetric interpretation of radiobiological data requires precise information on the nuclear area of cells as irradiated with high-LET radiation. (author).

  2. ATM Couples Replication Stress and Metabolic Reprogramming during Cellular Senescence

    Directory of Open Access Journals (Sweden)

    Katherine M. Aird

    2015-05-01

    Full Text Available Replication stress induced by nucleotide deficiency plays an important role in cancer initiation. Replication stress in primary cells typically activates the cellular senescence tumor-suppression mechanism. Senescence bypass correlates with development of cancer, a disease characterized by metabolic reprogramming. However, the role of metabolic reprogramming in the cellular response to replication stress has been little explored. Here, we report that ataxia telangiectasia mutated (ATM plays a central role in regulating the cellular response to replication stress by shifting cellular metabolism. ATM inactivation bypasses senescence induced by replication stress triggered by nucleotide deficiency. This was due to restoration of deoxyribonucleotide triphosphate (dNTP levels through both upregulation of the pentose phosphate pathway via increased glucose-6-phosphate dehydrogenase (G6PD activity and enhanced glucose and glutamine consumption. These phenotypes were mediated by a coordinated suppression of p53 and upregulation of c-MYC downstream of ATM inactivation. Our data indicate that ATM status couples replication stress and metabolic reprogramming during senescence.

  3. Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

    International Nuclear Information System (INIS)

    Highlights: → Report for the first time that vitamin C has a beneficial effect on the development of porcine SCNT embryos. → The level of acH4K5 and Oct4 expression at blastocyst-stage was up-regulated after treatment. → A higher rate of gestation and increased number of piglets born were harvested in the treated group. -- Abstract: The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 μg/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.

  4. Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Zhou, Yang; Zhu, Jianguo; Yuan, Ting; Lai, Liangxue [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China); Pang, Daxin, E-mail: pdx@jlu.edu.cn [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China); Ouyang, Hongsheng, E-mail: ouyh@jlu.edu.cn [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China)

    2011-07-29

    Highlights: {yields} Report for the first time that vitamin C has a beneficial effect on the development of porcine SCNT embryos. {yields} The level of acH4K5 and Oct4 expression at blastocyst-stage was up-regulated after treatment. {yields} A higher rate of gestation and increased number of piglets born were harvested in the treated group. -- Abstract: The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 {mu}g/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.

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

    OpenAIRE

    Juli J. Unternaehrer; Rui Zhao; Kitai Kim; Marcella Cesana; John T. Powers; Sutheera Ratanasirintrawoot; Tamer Onder; Tsukasa Shibue; Robert A. Weinberg; George Q. Daley

    2014-01-01

    Stem Cell Reports Report The Epithelial-Mesenchymal Transition Factor SNAIL Paradoxically Enhances Reprogramming Juli J. Unternaehrer,1,2,3,7,* Rui Zhao,1,2,3,8 Kitai Kim,1,2,3,9 Marcella Cesana,1,2,3 John T. Powers,1,2,3 Sutheera Ratanasirintrawoot,1,2,3 Tamer Onder,1,2,3,10 Tsukasa Shibue,4,5 Robert A. Weinberg,4,5,6 and George Q. Daley1,2,3 1Division of Pediatric Hematology/Oncology, Stem Cell Transplantation Program, Manton Center for Orphan Disease Research, Howard H...

  6. Sox transcription factors require selective interactions with Oct4 and specific transactivation functions to mediate reprogramming.

    Science.gov (United States)

    Aksoy, Irene; Jauch, Ralf; Eras, Volker; Chng, Wen-Bin Alfred; Chen, Jiaxuan; Divakar, Ushashree; Ng, Calista Keow Leng; Kolatkar, Prasanna R; Stanton, Lawrence W

    2013-12-01

    The unique ability of Sox2 to cooperate with Oct4 at selective binding sites in the genome is critical for reprogramming somatic cells into induced pluripotent stem cells (iPSCs). We have recently demonstrated that Sox17 can be converted into a reprogramming factor by alteration of a single amino acid (Sox17EK) within its DNA binding HMG domain. Here we expanded this study by introducing analogous mutations to 10 other Sox proteins and interrogated the role of N-and C-termini on the reprogramming efficiency. We found that point-mutated Sox7 and Sox17 can convert human and mouse fibroblasts into iPSCs, but Sox4, Sox5, Sox6, Sox8, Sox9, Sox11, Sox12, Sox13, and Sox18 cannot. Next we studied regions outside the HMG domain and found that the C-terminal transactivation domain of Sox17 and Sox7 enhances the potency of Sox2 in iPSC assays and confers weak reprogramming potential to the otherwise inactive Sox4EK and Sox18EK proteins. These results suggest that the glutamate (E) to lysine (K) mutation in the HMG domain is necessary but insufficient to swap the function of Sox factors. Moreover, the HMG domain alone fused to the VP16 transactivation domain is able to induce reprogramming, albeit at low efficiency. By molecular dissection of the C-terminus of Sox17, we found that the β-catenin interaction region contributes to the enhanced reprogramming efficiency of Sox17EK. To mechanistically understand the enhanced reprogramming potential of Sox17EK, we analyzed ChIP-sequencing and expression data and identified a subset of candidate genes specifically regulated by Sox17EK and not by Sox2. PMID:23963638

  7. Epigenetic control of root and nodule development : the role of plant-specific histone deacetylases and LHP1 in root cell reprogramming

    NARCIS (Netherlands)

    Schilderink, S.

    2012-01-01

    In plants, unlike in animals, most organs develop post embryonically. These organs originate from clusters of undifferentiated dividing cells that form so-called meristems. Differentiated cells can be re-activated to enter the cell cycle and to ultimately give rise to new meristems. These differenti

  8. Embryonic stem cell as nuclear donor could promote in vitro development of the heterogeneous reconstructed embryo

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The nucleus of a somatic cell could be dedifferentiated and reprogrammed in an enucleated heterogeneous oocyte. Some reconstructed oocytes could develop into blastocysts in vitro, and a few could develop into term normally after transferred into foster mothers, but most of cloning embryos fail to develop to term. In order to evaluate the efficacy of embryonic stem cell as nucleus donor in interspecific animal cloning, we reconstructed enucleated rabbit oocytes with nuclei from mouse ES cells, and analyzed the developmental ability of reconstructed embryos in vitro. Two kinds of fibroblast cells were used as donor control, one derived from ear skin of an adult Kunming albino mouse, and the other derived from a mouse fetus. Three types of cells were transferred into perivitelline space under zona pellucida of rabbit oocytes respectively. The reconstructed oocytes were fused and activated by electric pulses, and cultured in vitro. The developmental rate of reconstructed oocytes derived from embryonic stem cells was 16.1%, which was significantly higher than that of both the adult mouse fibroblast cells (0%-3.1%, P < 0.05) and fetus mouse fibroblast cells (2.1%-3.7%, P < 0.05). Chromosome analysis confirmed that blastocyst cells were derived from ES donor cell. These observations show that reprogramming is easier in interspecific embryos reconstructed with ES cells than that reconstructed with somatic cells, and that ES cells have the higher ability to direct the reconstructed embryos development normally than fibroblast cells.

  9. Direct reprogramming of adult somatic cells towards adventitious root formation in forest tree species: the effect of the juvenile-adult transition

    Directory of Open Access Journals (Sweden)

    Carmen eDiaz-Sala

    2014-07-01

    Full Text Available Cellular plasticity refers, among others, to the capability of differentiated cells to switch the differentiation process and acquire new fates. One way by which plant cell plasticity is manifested is through de novo regeneration of organs from somatic differentiated cells in an ectopic location. However, switching the developmental program of adult cells prior to organ regeneration is difficult in many plant species, especially in forest tree species. In these species, a decline in the capacity to regenerate shoots, roots or embryos from somatic differentiated cells is associated with tree age and maturation. The decline in the ability to form adventitious roots from stem cuttings is one of the most dramatic effects of maturation, and has been the subject of investigations on the basic nature of the process. Cell fate switches, both in plants and animals, are characterized by remarkable changes in the pattern of gene expression, as cells switch from the characteristic expression pattern of a somatic cell to a new one directing a new developmental pathway. Therefore, determining the way by which cells reset their gene expression pattern is crucial to understand cellular plasticity. The presence of specific cellular signalling pathways or tissue-specific factors underlying the establishment, maintenance and redirection of gene expression patterns in the tissues involved in adventitious root formation could be crucial for cell fate switch and for the control of age-dependent cellular plasticity.

  10. Remodeling of ribosomal genes in somatic cells by Xenopus egg extract

    Energy Technology Data Exchange (ETDEWEB)

    Ostrup, Olga, E-mail: osvarcova@gmail.com [Institute of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of Copenhagen, Frederiksberg C (Denmark); Stem Cell Epigenetics Laboratory, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo (Norway); Norwegian Center for Stem Cell Research, Oslo (Norway); Hyttel, Poul; Klaerke, Dan A. [Institute of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of Copenhagen, Frederiksberg C (Denmark); Collas, Philippe, E-mail: philc@medisin.uio.no [Stem Cell Epigenetics Laboratory, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo (Norway); Norwegian Center for Stem Cell Research, Oslo (Norway)

    2011-09-02

    Highlights: {yields} Xenopus egg extract remodels nuclei and alter cell growth characteristics. {yields} Ribosomal genes are reprogrammed within 6 h after extract exposure. {yields} rDNA reprogramming involves promoter targeting of SNF2H remodeling complex. {yields} Xenopus egg extract does not initiate stress-related response in somatic cells. {yields} Aza-cytidine elicits a stress-induced response in reprogrammed cells. -- Abstract: Extracts from Xenopus eggs can reprogram gene expression in somatic nuclei, however little is known about the earliest processes associated with the switch in the transcriptional program. We show here that an early reprogramming event is the remodeling of ribosomal chromatin and gene expression. This occurs within hours of extract treatment and is distinct from a stress response. Egg extract elicits remodeling of the nuclear envelope, chromatin and nucleolus. Nucleolar remodeling involves a rapid and stable decrease in ribosomal gene transcription, and promoter targeting of the nucleolar remodeling complex component SNF2H without affecting occupancy of the transcription factor UBF and the stress silencers SUV39H1 and SIRT1. During this process, nucleolar localization of UBF and SIRT1 is not altered. On contrary, azacytidine pre-treatment has an adverse effect on rDNA remodeling induced by extract and elicits a stress-type nuclear response. Thus, an early event of Xenopus egg extract-mediated nuclear reprogramming is the remodeling of ribosomal genes involving nucleolar remodeling complex. Condition-specific and rapid silencing of ribosomal genes may serve as a sensitive marker for evaluation of various reprogramming methods.

  11. Role of the reprogramming factor KLF4 in blood formation.

    Science.gov (United States)

    Park, Chun Shik; Shen, Ye; Lewis, Andrew; Lacorazza, H Daniel

    2016-05-01

    Krüppel-like factor 4 is a zinc finger protein with dual functions that can act as a transcriptional activator and repressor of genes involved in cell proliferation, differentiation, and apoptosis. Although most studies have focused on terminally differentiated epithelial cells, evidence suggests that Krüppel-like factor 4 regulates the development and function of the myeloid and lymphoid blood lineages. The ability of Krüppel-like factor 4 to dedifferentiate from somatic cells into pluripotent stem cells in cooperation with other reprogramming factors suggests its potential function in the preservation of tissue-specific stem cells. Additionally, emerging interest in the redifferentiation of induced pluripotent stem cells into blood cells to correct hematologic deficiencies and malignancies warrants further studies on the role of Krüppel-like factor 4 in steady-state blood formation. PMID:26908828

  12. Biological computational approaches: new hopes to improve (re)programming robustness, regenerative medicine and cancer therapeutics.

    Science.gov (United States)

    Ebrahimi, Behnam

    2016-01-01

    Hundreds of transcription factors (TFs) are expressed and work in each cell type, but the identity of the cells is defined and maintained through the activity of a small number of core TFs. Existing reprogramming strategies predominantly focus on the ectopic expression of core TFs of an intended fate in a given cell type regardless of the state of native/somatic gene regulatory networks (GRNs) of the starting cells. Interestingly, an important point is that how much products of the reprogramming, transdifferentiation and differentiation (programming) are identical to their in vivo counterparts. There is evidence that shows that direct fate conversions of somatic cells are not complete, with target cell identity not fully achieved. Manipulation of core TFs provides a powerful tool for engineering cell fate in terms of extinguishment of native GRNs, the establishment of a new GRN, and preventing installation of aberrant GRNs. Conventionally, core TFs are selected to convert one cell type into another mostly based on literature and the experimental identification of genes that are differentially expressed in one cell type compared to the specific cell types. Currently, there is not a universal standard strategy for identifying candidate core TFs. Remarkably, several biological computational platforms are developed, which are capable of evaluating the fidelity of reprogramming methods and refining existing protocols. The current review discusses some deficiencies of reprogramming technologies in the production of a pure population of authentic target cells. Furthermore, it reviews the role of computational approaches (e.g. CellNet, KeyGenes, Mogrify, etc.) in improving (re)programming methods and consequently in regenerative medicine and cancer therapeutics.

  13. Biological computational approaches: new hopes to improve (re)programming robustness, regenerative medicine and cancer therapeutics.

    Science.gov (United States)

    Ebrahimi, Behnam

    2016-01-01

    Hundreds of transcription factors (TFs) are expressed and work in each cell type, but the identity of the cells is defined and maintained through the activity of a small number of core TFs. Existing reprogramming strategies predominantly focus on the ectopic expression of core TFs of an intended fate in a given cell type regardless of the state of native/somatic gene regulatory networks (GRNs) of the starting cells. Interestingly, an important point is that how much products of the reprogramming, transdifferentiation and differentiation (programming) are identical to their in vivo counterparts. There is evidence that shows that direct fate conversions of somatic cells are not complete, with target cell identity not fully achieved. Manipulation of core TFs provides a powerful tool for engineering cell fate in terms of extinguishment of native GRNs, the establishment of a new GRN, and preventing installation of aberrant GRNs. Conventionally, core TFs are selected to convert one cell type into another mostly based on literature and the experimental identification of genes that are differentially expressed in one cell type compared to the specific cell types. Currently, there is not a universal standard strategy for identifying candidate core TFs. Remarkably, several biological computational platforms are developed, which are capable of evaluating the fidelity of reprogramming methods and refining existing protocols. The current review discusses some deficiencies of reprogramming technologies in the production of a pure population of authentic target cells. Furthermore, it reviews the role of computational approaches (e.g. CellNet, KeyGenes, Mogrify, etc.) in improving (re)programming methods and consequently in regenerative medicine and cancer therapeutics. PMID:27056282

  14. Sexual Fate Change of XX Germ Cells Caused by the Deletion of SMAD4 and STRA8 Independent of Somatic Sex Reprogramming.

    Science.gov (United States)

    Wu, Quan; Fukuda, Kurumi; Kato, Yuzuru; Zhou, Zhi; Deng, Chu-Xia; Saga, Yumiko

    2016-09-01

    The differential programming of sperm and eggs in gonads is a fundamental topic in reproductive biology. Although the sexual fate of germ cells is believed to be determined by signaling factors from sexually differentiated somatic cells in fetal gonads, the molecular mechanism that determines germ cell fate is poorly understood. Herein, we show that mothers against decapentaplegic homolog 4 (SMAD4) in germ cells is required for female-type differentiation. Germ cells in Smad4-deficient ovaries respond to retinoic acid signaling but fail to undergo meiotic prophase I, which coincides with the weaker expression of genes required for follicular formation, indicating that SMAD4 signaling is essential for oocyte differentiation and meiotic progression. Intriguingly, germline-specific deletion of Smad4 in Stra8-null female germ cells resulted in the up-regulation of genes required for male gonocyte differentiation, including Nanos2 and PLZF, suggesting the initiation of male-type differentiation in ovaries. Moreover, our transcriptome analyses of mutant ovaries revealed that the sex change phenotype is achieved without global gene expression changes in somatic cells. Our results demonstrate that SMAD4 and STRA8 are essential factors that regulate the female fate of germ cells. PMID:27606421

  15. 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

  16. Simultaneous Reprogramming and Gene Correction of Patient Fibroblasts

    Directory of Open Access Journals (Sweden)

    Sara E. Howden

    2015-12-01

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

  17. Malignant transformation of CD4+ T lymphocytes mediated by oncogenic kinase NPM/ALK recapitulates IL-2-induced cell signaling and gene expression reprogramming.

    Science.gov (United States)

    Marzec, Michal; Halasa, Krzysztof; Liu, Xiaobin; Wang, Hong Y; Cheng, Mangeng; Baldwin, Donald; Tobias, John W; Schuster, Stephen J; Woetmann, Anders; Zhang, Qian; Turner, Suzanne D; Ødum, Niels; Wasik, Mariusz A

    2013-12-15

    Anaplastic lymphoma kinase (ALK), physiologically expressed only by nervous system cells, displays a remarkable capacity to transform CD4(+) T lymphocytes and other types of nonneural cells. In this study, we report that activity of nucleophosmin (NPM)/ALK chimeric protein, the dominant form of ALK expressed in T cell lymphomas (TCLs), closely resembles cell activation induced by IL-2, the key cytokine supporting growth and survival of normal CD4(+) T lymphocytes. Direct comparison of gene expression by ALK(+) TCL cells treated with an ALK inhibitor and IL-2-dependent ALK(-) TCL cells stimulated with the cytokine revealed a very similar, albeit inverse, gene-regulation pattern. Depending on the analysis method, up to 67% of the affected genes were modulated in common by NPM/ALK and IL-2. Based on the gene expression patterns, Jak/STAT- and IL-2-signaling pathways topped the list of pathways identified as affected by both IL-2 and NPM/ALK. The expression dependence on NPM/ALK and IL-2 of the five selected genes-CD25 (IL-2Rα), Egr-1, Fosl-1, SOCS3, and Irf-4-was confirmed at the protein level. In both ALK(+) TCL and IL-2-stimulated ALK(-) TCL cells, CD25, SOCS3, and Irf-4 genes were activated predominantly by the STAT5 and STAT3 transcription factors, whereas transcription of Egr-1 and Fosl-1 was induced by the MEK-ERK pathway. Finally, we found that Egr-1, a protein not associated previously with either IL-2 or ALK, contributes to the cell proliferation. These findings indicate that NPM/ALK transforms the target CD4(+) T lymphocytes, at least in part, by using the pre-existing, IL-2-dependent signaling pathways.

  18. Nuclear protein import is reduced in cells expressing nuclear envelopathy-causing lamin A mutants

    Energy Technology Data Exchange (ETDEWEB)

    Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M. [University of Wuerzburg, Institute of Anatomy and Cell Biology, Koellikerstrasse 6, 97070 Wuerzburg (Germany); Wehnert, Manfred [Institute of Human Genetics, University of Greifswald, Greifswald (Germany); Huebner, Stefan, E-mail: stefan.huebner@mail.uni-wuerzburg.de [University of Wuerzburg, Institute of Anatomy and Cell Biology, Koellikerstrasse 6, 97070 Wuerzburg (Germany)

    2009-08-15

    Lamins, which form the nuclear lamina, not only constitute an important determinant of nuclear architecture, but additionally play essential roles in many nuclear functions. Mutations in A-type lamins cause a wide range of human genetic disorders (laminopathies). The importance of lamin A (LaA) in the spatial arrangement of nuclear pore complexes (NPCs) prompted us to study the role of LaA mutants in nuclear protein transport. Two mutants, causing prenatal skin disease restrictive dermopathy (RD) and the premature aging disease Hutchinson Gilford progeria syndrome, were used for expression in HeLa cells to investigate their impact on the subcellular localization of NPC-associated proteins and nuclear protein import. Furthermore, dynamics of the LaA mutants within the nuclear lamina were studied. We observed affected localization of NPC-associated proteins, diminished lamina dynamics for both LaA mutants and reduced nuclear import of representative cargo molecules. Intriguingly, both LaA mutants displayed similar effects on nuclear morphology and functions, despite their differences in disease severity. Reduced nuclear protein import was also seen in RD fibroblasts and impaired lamina dynamics for the nucleoporin Nup153. Our data thus represent the first study of a direct link between LaA mutant expression and reduced nuclear protein import.

  19. Joint modeling of cell and nuclear shape variation.

    Science.gov (United States)

    Johnson, Gregory R; Buck, Taraz E; Sullivan, Devin P; Rohde, Gustavo K; Murphy, Robert F

    2015-11-01

    Modeling cell shape variation is critical to our understanding of cell biology. Previous work has demonstrated the utility of nonrigid image registration methods for the construction of nonparametric nuclear shape models in which pairwise deformation distances are measured between all shapes and are embedded into a low-dimensional shape space. Using these methods, we explore the relationship between cell shape and nuclear shape. We find that these are frequently dependent on each other and use this as the motivation for the development of combined cell and nuclear shape space models, extending nonparametric cell representations to multiple-component three-dimensional cellular shapes and identifying modes of joint shape variation. We learn a first-order dynamics model to predict cell and nuclear shapes, given shapes at a previous time point. We use this to determine the effects of endogenous protein tags or drugs on the shape dynamics of cell lines and show that tagged C1QBP reduces the correlation between cell and nuclear shape. To reduce the computational cost of learning these models, we demonstrate the ability to reconstruct shape spaces using a fraction of computed pairwise distances. The open-source tools provide a powerful basis for future studies of the molecular basis of cell organization. PMID:26354424

  20. Aging, rejuvenation, and epigenetic reprogramming: resetting the aging clock.

    Science.gov (United States)

    Rando, Thomas A; Chang, Howard Y

    2012-01-20

    The underlying cause of aging remains one of the central mysteries of biology. Recent studies in several different systems suggest that not only may the rate of aging be modified by environmental and genetic factors, but also that the aging clock can be reversed, restoring characteristics of youthfulness to aged cells and tissues. This Review focuses on the emerging biology of rejuvenation through the lens of epigenetic reprogramming. By defining youthfulness and senescence as epigenetic states, a framework for asking new questions about the aging process emerges. PMID:22265401

  1. EasiLIR: Lightweight Incremental Reprogramming for Sensor Networks

    OpenAIRE

    Jiefan Qiu; Dong Li; Hailong Shi; Li Cui

    2014-01-01

    Energy-efficient wireless reprogramming is key issues for long-lived sensor network. Most of wireless reprogramming approaches focus on the energy efficiency of the data transmission phase. However, the program rebuilding phase on target node is possibly as another significant part of the total reprogramming energy consumption, due to the high energy overhead of reading or writing operation on the energy-hungry nonvolatile memory. In this paper, we propose an energy-efficient reprogramming sy...

  2. Nuclear envelope rupture and repair during cancer cell migration

    Science.gov (United States)

    Denais, Celine M.; Gilbert, Rachel M.; Isermann, Philipp; McGregor, Alexandra L.; te Lindert, Mariska; Weigelin, Bettina; Davidson, Patricia M.; Friedl, Peter; Wolf, Katarina; Lammerding, Jan

    2016-01-01

    During cancer metastasis, tumor cells penetrate tissues through tight interstitial spaces, requiring extensive deformation of the cell and its nucleus. Here, we investigated tumor cell migration in confining microenvironments in vitro and in vivo. Nuclear deformation caused localized loss of nuclear envelope (NE) integrity, which led to the uncontrolled exchange of nucleo-cytoplasmic content, herniation of chromatin across the NE, and DNA damage. The incidence of NE rupture increased with cell confinement and with depletion of nuclear lamins, NE proteins that structurally support the nucleus. Cells restored NE integrity using components of the endosomal sorting complexes required for transport-III (ESCRT-III) machinery. Our findings indicate that cell migration incurs substantial physical stress on the NE and its content, requiring efficient NE and DNA damage repair for survival. PMID:27013428

  3. Transgenerational Inheritance of Increased Fat Depot Size, Stem Cell Reprogramming, and Hepatic Steatosis Elicited by Prenatal Exposure to the Obesogen Tributyltin in Mice

    OpenAIRE

    Chamorro-García, Raquel; Sahu, Margaret; Abbey, Rachelle J; Laude, Jhyme; Pham, Nhieu; Blumberg, Bruce

    2013-01-01

    Background: We have previously shown that exposure to tributyltin (TBT) modulates critical steps of adipogenesis through RXR/PPARγ and that prenatal TBT exposure predisposes multipotent mesenchymal stem cells (MSCs) to become adipocytes by epigenetic imprinting into the memory of the MSC compartment. Objective: We tested whether the effects of prenatal TBT exposure were heritable in F2 and F3 generations. Methods: We exposed C57BL/6J female mice (F0) to DMSO vehicle, the pharmaceutical obesog...

  4. Malignant transformation of CD4+ T lymphocytes mediated by oncogenic kinase NPM/ALK recapitulates IL-2-induced cell signaling and gene expression reprogramming

    DEFF Research Database (Denmark)

    Marzec, Michal; Halasa, Krzysztof; Liu, Xiaobin;

    2013-01-01

    Anaplastic lymphoma kinase (ALK), physiologically expressed only by nervous system cells, displays a remarkable capacity to transform CD4(+) T lymphocytes and other types of nonneural cells. In this study, we report that activity of nucleophosmin (NPM)/ALK chimeric protein, the dominant form of ALK...... with the cytokine revealed a very similar, albeit inverse, gene-regulation pattern. Depending on the analysis method, up to 67% of the affected genes were modulated in common by NPM/ALK and IL-2. Based on the gene expression patterns, Jak/STAT- and IL-2-signaling pathways topped the list of pathways identified...... as affected by both IL-2 and NPM/ALK. The expression dependence on NPM/ALK and IL-2 of the five selected genes-CD25 (IL-2Rα), Egr-1, Fosl-1, SOCS3, and Irf-4-was confirmed at the protein level. In both ALK(+) TCL and IL-2-stimulated ALK(-) TCL cells, CD25, SOCS3, and Irf-4 genes were activated predominantly...

  5. A simple polymeric model describes cell nuclear mechanical response

    Science.gov (United States)

    Banigan, Edward; Stephens, Andrew; Marko, John

    The cell nucleus must continually resist inter- and intracellular mechanical forces, and proper mechanical response is essential to basic cell biological functions as diverse as migration, differentiation, and gene regulation. Experiments probing nuclear mechanics reveal that the nucleus stiffens under strain, leading to two characteristic regimes of force response. This behavior depends sensitively on the intermediate filament protein lamin A, which comprises the outer layer of the nucleus, and the properties of the chromatin interior. To understand these mechanics, we study a simulation model of a polymeric shell encapsulating a semiflexible polymer. This minimalistic model qualitatively captures the typical experimental nuclear force-extension relation and observed nuclear morphologies. Using a Flory-like theory, we explain the simulation results and mathematically estimate the force-extension relation. The model and experiments suggest that chromatin organization is a dominant contributor to nuclear mechanics, while the lamina protects cell nuclei from large deformations.

  6. Integrated phosphoproteomic and metabolomic profiling reveals NPM-ALK-mediated phosphorylation of PKM2 and metabolic reprogramming in anaplastic large cell lymphoma.

    Science.gov (United States)

    McDonnell, Scott R P; Hwang, Steven R; Rolland, Delphine; Murga-Zamalloa, Carlos; Basrur, Venkatesha; Conlon, Kevin P; Fermin, Damian; Wolfe, Thomas; Raskind, Alexander; Ruan, Chunhai; Jiang, Jian-Kang; Thomas, Craig J; Hogaboam, Cory M; Burant, Charles F; Elenitoba-Johnson, Kojo S J; Lim, Megan S

    2013-08-01

    The mechanisms underlying the pathogenesis of the constitutively active tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expressing anaplastic large cell lymphoma are not completely understood. Here we show using an integrated phosphoproteomic and metabolomic strategy that NPM-ALK induces a metabolic shift toward aerobic glycolysis, increased lactate production, and biomass production. The metabolic shift is mediated through the anaplastic lymphoma kinase (ALK) phosphorylation of the tumor-specific isoform of pyruvate kinase (PKM2) at Y105, resulting in decreased enzymatic activity. Small molecule activation of PKM2 or expression of Y105F PKM2 mutant leads to reversal of the metabolic switch with increased oxidative phosphorylation and reduced lactate production coincident with increased cell death, decreased colony formation, and reduced tumor growth in an in vivo xenograft model. This study provides comprehensive profiling of the phosphoproteomic and metabolomic consequences of NPM-ALK expression and reveals a novel role of ALK in the regulation of multiple components of cellular metabolism. Our studies show that PKM2 is a novel substrate of ALK and plays a critical role in mediating the metabolic shift toward biomass production and tumorigenesis.

  7. Stress-triggered atavistic reprogramming (STAR) addiction: driving force behind head and neck cancer?

    Science.gov (United States)

    Masuda, Muneyuki; Wakasaki, Takahiro; Toh, Satoshi

    2016-01-01

    Recent results of the Cancer Genome Atlas on head and neck squamous cell carcinoma (HNSCC) revealed that HNSCC lacked predominant gain-of-function mutations in oncogenes, whereas an essential role for epigenetics in oncogenesis has become apparent. In parallel, it has gained general acceptance that cancer is considered as complex adaptive system, which evolves responding environmental selective pressures. This somatic evolution appears to proceed concurrently with the acquisition of an atavistic pluripotent state (i.e., “stemness”), which is inducible by intrinsic epigenetic reprogramming program as demonstrated by induced pluripotent stem (iPS) cells. This Nobel prize-winning discovery has markedly accelerated and expanded cancer stem cell research from the point of epigenetic reprogramming. Taken together, we hypothesize that stress-triggered atavistic reprogramming (STAR) may be the major driving force of HNSCC evolution. In this perspective, we discuss the possible mechanisms of STAR in HNSCC, focusing on recent topics of epigenetic reprogramming in developmental and cancer cell biology. PMID:27429838

  8. Genome-wide analysis of transcriptional reprogramming in mouse models of acute myeloid leukaemia.

    Directory of Open Access Journals (Sweden)

    Nicolas Bonadies

    Full Text Available Acute leukaemias are commonly caused by mutations that corrupt the transcriptional circuitry of haematopoietic stem/progenitor cells. However, the mechanisms underlying large-scale transcriptional reprogramming remain largely unknown. Here we investigated transcriptional reprogramming at genome-scale in mouse retroviral transplant models of acute myeloid leukaemia (AML using both gene-expression profiling and ChIP-sequencing. We identified several thousand candidate regulatory regions with altered levels of histone acetylation that were characterised by differential distribution of consensus motifs for key haematopoietic transcription factors including Gata2, Gfi1 and Sfpi1/Pu.1. In particular, downregulation of Gata2 expression was mirrored by abundant GATA motifs in regions of reduced histone acetylation suggesting an important role in leukaemogenic transcriptional reprogramming. Forced re-expression of Gata2 was not compatible with sustained growth of leukaemic cells thus suggesting a previously unrecognised role for Gata2 in downregulation during the development of AML. Additionally, large scale human AML datasets revealed significantly higher expression of GATA2 in CD34+ cells from healthy controls compared with AML blast cells. The integrated genome-scale analysis applied in this study represents a valuable and widely applicable approach to study the transcriptional control of both normal and aberrant haematopoiesis and to identify critical factors responsible for transcriptional reprogramming in human cancer.

  9. Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun, E-mail: yinxj33@msn.com

    2014-02-21

    Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.

  10. 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.

  11. Generation of bovine transgenics using somatic cell nuclear transfer

    Directory of Open Access Journals (Sweden)

    Stice Steven L

    2003-11-01

    Full Text Available Abstract The ability to produce transgenic animals through the introduction of exogenous DNA has existed for many years. However, past methods available to generate transgenic animals, such as pronuclear microinjection or the use of embryonic stem cells, have either been inefficient or not available in all animals, bovine included. More recently somatic cell nuclear transfer has provided a method to create transgenic animals that overcomes many deficiencies present in other methods. This review summarizes the benefits of using somatic cell nuclear transfer to create bovine transgenics as well as the possible opportunities this method creates for the future.

  12. Nuclear localization of Merkel cell polyomavirus large T antigen in Merkel cell carcinoma

    International Nuclear Information System (INIS)

    To clarify whether mutations in the large T gene encoded by Merkel cell polyomavirus affect the expression and function of large T antigen in Merkel cell carcinoma cases, we investigated the expression of large T antigen in vitro and in vivo. Immunohistochemistry using a rabbit polyclonal antibody revealed that large T antigen was expressed in the nuclei of Merkel cell carcinoma cells with Merkel cell polyomavirus infection. Deletion mutant analyses identified an Arg-Lys-Arg-Lys sequence (amino acids 277-280) as a nuclear localization signal in large T antigen. Sequence analyses revealed that there were no mutations in the nuclear localization signal in any of the eleven Merkel cell polyomavirus strains examined. Furthermore, stop codons were not observed in the upstream of the nuclear localization signal in any of the Merkel cell carcinoma cases examined. These data suggest that the nuclear localization signal is highly conserved and functional in Merkel cell carcinoma cases.

  13. Nuclear vlimata and aneuploidy in embryonic cells is caused by meiosis. Behaviour and properties of meiotic cells

    OpenAIRE

    Logothetou-Rella, H.

    1995-01-01

    This study demonstrates that human embryonic cells divide by meiosis. The use of trophoblastic tissue cells (early embryo) and amniotic cells (late embryo) exhibited the following characteristic events of meiosis: nuclear (NVs) and nucleolar (NuVs) vlimata formation; NV invasion in host cells; extrusion of chromosomes; nuclear fusion; metaphase fusion; hybrid cell formation; nuclear, nucleolar and cytoplasmic bridges, chromosomal transfer, variablesized nuc...

  14. Nuclear motility in glioma cells reveals a cell-line dependent role of various cytoskeletal components.

    Directory of Open Access Journals (Sweden)

    Alexa Kiss

    Full Text Available Nuclear migration is a general term for the movement of the nucleus towards a specific site in the cell. These movements are involved in a number of fundamental biological processes, such as fertilization, cell division, and embryonic development. Despite of its importance, the mechanism of nuclear migration is still poorly understood in mammalian cells. In order to shed light on the mechanical processes underlying nuclear movements, we adapted a micro-patterning based assay. C6 rat and U87 human glioma cells seeded on fibronectin patterns--thereby forced into a bipolar morphology--displayed oscillatory movements of the nucleus or the whole cell, respectively. We found that both the actomyosin system and microtubules are involved in the nuclear/cellular movements of both cell lines, but their contributions are cell-/migration-type specific. Dynein activity was necessary for nuclear migration of C6 cells but active myosin-II was dispensable. On the other hand, coupled nuclear and cellular movements of U87 cells were driven by actomyosin contraction. We explain these cell-line dependent effects by the intrinsic differences in the overall mechanical tension due to the various cytoskeletal elements inside the cell. Our observations showed that the movements of the nucleus and the centrosome are strongly correlated and display large variation, indicating a tight but flexible coupling between them. The data also indicate that the forces responsible for nuclear movements are not acting directly via the centrosome. Based on our observations, we propose a new model for nuclear oscillations in C6 cells in which dynein and microtubule dynamics are the main drivers of nuclear movements. This mechanism is similar to the meiotic nuclear oscillations of Schizosaccharomyces pombe and may be evolutionary conserved.

  15. Fractionation of HeLa cell nuclear extracts reveals minor small nuclear ribonucleoprotein particles.

    OpenAIRE

    Krämer, A

    1987-01-01

    Upon chromatographic fractionation of HeLa cell nuclear extracts, small RNAs of 145 and 66/65 nucleotides, respectively, were detected that are distinct from the abundant small RNAs present in the extract. These RNAs are precipitated by antibodies directed against the trimethylguanosine cap structure, characteristic for small nuclear RNAs (snRNAs) of the U type. The RNAs of 145 and 66/65 nucleotides appear to be associated with at least one of the proteins common to the major small nuclear ri...

  16. PARP activation promotes nuclear AID accumulation in lymphoma cells.

    Science.gov (United States)

    Tepper, Sandra; Jeschke, Julia; Böttcher, Katrin; Schmidt, Angelika; Davari, Kathrin; Müller, Peter; Kremmer, Elisabeth; Hemmerich, Peter; Pfeil, Ines; Jungnickel, Berit

    2016-03-15

    Activation-induced cytidine deaminase (AID) initiates immunoglobulin diversification in germinal center B cells by targeted introduction of DNA damage. As aberrant nuclear AID action contributes to the generation of B cell lymphoma, the protein's activity is tightly regulated, e.g. by nuclear/cytoplasmic shuttling and nuclear degradation. In the present study, we asked whether DNA damage may affect regulation of the AID protein. We show that exogenous DNA damage that mainly activates base excision repair leads to prevention of proteasomal degradation of AID and hence its nuclear accumulation. Inhibitor as well as knockout studies indicate that activation of poly (ADP-ribose) polymerase (PARP) by DNA damaging agents promotes both phenomena. These findings suggest that PARP inhibitors influence DNA damage dependent AID regulation, with interesting implications for the regulation of AID function and chemotherapy of lymphoma.

  17. Nuclear microscopy of sperm cell elemental structure

    International Nuclear Information System (INIS)

    Theories suggest there is a link between protamine concentrations in individual sperm and male fertility. Previously, biochemical analyses have used pooled samples containing millions of sperm to determine protamine concentrations. These methods have not been able to determine what percentage of morphologically normal sperm are biochemically defective and potentially infertile. Nuclear microscopy has been utilized to measure elemental profiles at the single sperm level. By measuring the amount of phosphorus and sulfur, the total DNA and protamine content in individual sperm from fertile bull and mouse semen have been determined. These values agree with results obtained from other biochemical analyses. Nuclear microscopy shows promise for measuring elemental profiles in the chromatin of individual sperm. The technique may be able to resolve theories regarding the importance of protamines to male fertility and identify biochemical defects responsible for certain types of male infertility. (orig.)

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

    Science.gov (United States)

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

    2014-01-01

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

  19. Recent Progress of Somatic Cell Nuclear Transfer in Pigs

    Institute of Scientific and Technical Information of China (English)

    Xu Xiaoming; Dou Zhongying

    2005-01-01

    Research in the field of somatic cell nuclear transfer (SCNT) and transgenic cloning in pigs has become a global hotspot, because porcine organs probably can be the first source of donor organs for human xenotransplantation. In recent years, though great progress has been made in porcine SCNT, the efficiency of nuclear transfer remains very low (<1% ). Thus, it is necessary to improve the procedure of nuclear transfer and to investigate some basic problems further. Recent progress and the related problems of SCNT in pigs are reviewed and analyzed so as to offer some beneficial illumination to researchers.

  20. Stochastic anomaly of methylome but persistent SRY hypermethylation in disorder of sex development in canine somatic cell nuclear transfer

    Science.gov (United States)

    Jeong, Young-Hee; Lu, Hanlin; Park, Chi-Hun; Li, Meiyan; Luo, Huijuan; Kim, Joung Joo; Liu, Siyang; Ko, Kyeong Hee; Huang, Shujia; Hwang, In Sung; Kang, Mi Na; Gong, Desheng; Park, Kang Bae; Choi, Eun Ji; Park, Jung Hyun; Jeong, Yeon Woo; Moon, Changjong; Hyun, Sang-Hwan; Kim, Nam Hyung; Jeung, Eui-Bae; Yang, Huanming; Hwang, Woo Suk; Gao, Fei

    2016-01-01

    Somatic cell nuclear transfer (SCNT) provides an excellent model for studying epigenomic reprogramming during mammalian development. We mapped the whole genome and whole methylome for potential anomalies of mutations or epimutations in SCNT-generated dogs with XY chromosomal sex but complete gonadal dysgenesis, which is classified as 78, XY disorder of sex development (DSD). Whole genome sequencing revealed no potential genomic variations that could explain the pathogenesis of DSD. However, extensive but stochastic anomalies of genome-wide DNA methylation were discovered in these SCNT DSD dogs. Persistent abnormal hypermethylation of the SRY gene was observed together with its down-regulated mRNA and protein expression. Failure of SRY expression due to hypermethylation was further correlated with silencing of a serial of testis determining genes, including SOX9, SF1, SOX8, AMH and DMRT1 in an early embryonic development stage at E34 in the XYDSD gonad, and high activation of the female specific genes, including FOXL2, RSPO1, CYP19A1, WNT4, ERα and ERβ, after one postnatal year in the ovotestis. Our results demonstrate that incomplete demethylation on the SRY gene is the driving cause of XYDSD in these XY DSD dogs, indicating a central role of epigenetic regulation in sex determination. PMID:27501986

  1. Stochastic anomaly of methylome but persistent SRY hypermethylation in disorder of sex development in canine somatic cell nuclear transfer.

    Science.gov (United States)

    Jeong, Young-Hee; Lu, Hanlin; Park, Chi-Hun; Li, Meiyan; Luo, Huijuan; Kim, Joung Joo; Liu, Siyang; Ko, Kyeong Hee; Huang, Shujia; Hwang, In Sung; Kang, Mi Na; Gong, Desheng; Park, Kang Bae; Choi, Eun Ji; Park, Jung Hyun; Jeong, Yeon Woo; Moon, Changjong; Hyun, Sang-Hwan; Kim, Nam Hyung; Jeung, Eui-Bae; Yang, Huanming; Hwang, Woo Suk; Gao, Fei

    2016-01-01

    Somatic cell nuclear transfer (SCNT) provides an excellent model for studying epigenomic reprogramming during mammalian development. We mapped the whole genome and whole methylome for potential anomalies of mutations or epimutations in SCNT-generated dogs with XY chromosomal sex but complete gonadal dysgenesis, which is classified as 78, XY disorder of sex development (DSD). Whole genome sequencing revealed no potential genomic variations that could explain the pathogenesis of DSD. However, extensive but stochastic anomalies of genome-wide DNA methylation were discovered in these SCNT DSD dogs. Persistent abnormal hypermethylation of the SRY gene was observed together with its down-regulated mRNA and protein expression. Failure of SRY expression due to hypermethylation was further correlated with silencing of a serial of testis determining genes, including SOX9, SF1, SOX8, AMH and DMRT1 in an early embryonic development stage at E34 in the XY(DSD) gonad, and high activation of the female specific genes, including FOXL2, RSPO1, CYP19A1, WNT4, ERα and ERβ, after one postnatal year in the ovotestis. Our results demonstrate that incomplete demethylation on the SRY gene is the driving cause of XY(DSD) in these XY DSD dogs, indicating a central role of epigenetic regulation in sex determination. PMID:27501986

  2. Nuclear transfer of synchronized African wild cat somatic cells into enucleated domestic cat oocytes

    Science.gov (United States)

    Gomez, M.C.; Jenkins, J.A.; Giraldo, A.; Harris, R.F.; King, A.; Dresser, B.L.; Pope, C.E.

    2003-01-01

    The African wild cat is one of the smallest wild cats and its future is threatened by hybridization with domestic cats. Nuclear transfer, a valuable tool for retaining genetic variability, offers the possibility of species continuation rather than extinction. The aim of this study was to investigate the ability of somatic cell nuclei of the African wild cat (AWC) to dedifferentiate within domestic cat (DSH) cytoplasts and to support early development after nuclear transplantation. In experiment 1, distributions of AWC and DSH fibroblasts in each cell-cycle phase were assessed by flow cytometry using cells cultured to confluency and disaggregated with pronase, trypsin, or mechanical separation. Trypsin (89.0%) and pronase (93.0%) yielded higher proportions of AWC nuclei in the G0/G1 phase than mechanical separation (82.0%). In contrast, mechanical separation yielded higher percentages of DSH nuclei in the G0/G1 phase (86.6%) than pronase (79.7%) or trypsin (74.2%) treatments. In both species, pronase induced less DNA damage than trypsin. In experiment 2, the effects of serum starvation, culture to confluency, and exposure to roscovitine on the distribution of AWC and DSH fibroblasts in various phases of the cell cycle were determined. Flow cytometry analyses revealed that the dynamics of the cell cycle varied as culture conditions were modified. Specifically, a higher percentage of AWC and DSH nuclei were in the G0/G1 phase after cells were serum starved (83% vs. 96%) than were present in cycling cells (50% vs. 64%), after contact inhibition (61% vs. 88%), or after roscovitine (56% vs. 84%) treatment, respectively. In experiment 3, we evaluated the effects of cell synchronization and oocyte maturation (in vivo vs. in vitro) on the reconstruction and development of AWC-DSH- and DSH-DSH-cloned embryos. The method of cell synchronization did not affect the fusion and cleavage rate because only a slightly higher percentage of fused couplets cleaved when donor nuclei

  3. Generation of patient-specific pluripotent stem cells and directed differentiation of embryonic stem cells for regenerative medicine

    Institute of Scientific and Technical Information of China (English)

    Minyue Ma; Jiahao Sha; Zuomin Zhou; Qi Zhou; Qingzhang Li

    2008-01-01

    Embryonic stem(ES) cells are pluripotent cells that can give rise to derivatives of all three embryonic germ layers. Due to its characteristics, the patient-specific ES cells are of great potential for transplantation therapies. Several strategies can reprogramme somatic cells back to pluripotent stem cells: nuclear transfer, fusion with ES cells, treatment with cell extract and induction by specific factors. Considering the future clinical use, the differentiation from ES to neurons, cardiomyocytes and many other types of cell scurrently provide basic cognition and experience to regenerative medicine. This article will review two courses, the reprogramming of differentiated cells and the differentiation of ES cells to specific cell types.

  4. Molecular structure and biological function of proliferating cell nuclear antigen

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Proliferating cell nuclear antigen (PCNA) is the core component of replication complex in eukaryote.As a processive factor of DNA polymerase delta, PCNA coordinates the replication process by interacting with various replication proteins. PCNA appears to play an essential role in many cell events, such as DNA damage repair, cell cycle regulation, and apoptosis, through the coordination or organization of different partners. PCNA is an essential factor in cell proliferation, and has clinical significance in tumor research. In this article we review the functional structure of PCNA, which acts as a function switch in different cell events.

  5. Hot-cell for dismantling of nuclear gauges

    CERN Document Server

    Reis, L C A

    2000-01-01

    This work objectives the design of a hot-cell that will be used for dismantling of nuclear gauges. In the hot-cell, nuclear gauges received as radioactive waste at the Centro de Desenvolvimento da Tecnologia Nuclear - CDTN will be dismantled, in order to decrease the volume of radioactive waste to be stored at the Center. Sources originally conditioned as special form radioactive material will be tested and in case do not present leakage, the respective gauges will be disposable for reusing by radioisotope users. The remaining sources will be taken off the original shielding and conditioned in special packages adequate for storage and disposal. All steps of work, the hot-cell design and methodology for conditioning are also described.

  6. Nuclear distribution of claudin-2 increases cell proliferation in human lung adenocarcinoma cells.

    Science.gov (United States)

    Ikari, Akira; Watanabe, Ryo; Sato, Tomonari; Taga, Saeko; Shimobaba, Shun; Yamaguchi, Masahiko; Yamazaki, Yasuhiro; Endo, Satoshi; Matsunaga, Toshiyuki; Sugatani, Junko

    2014-09-01

    Claudin-2 is expressed in human lung adenocarcinoma tissue and cell lines, although it is absent in normal lung tissue. However, the role of claudin-2 in cell proliferation and the regulatory mechanism of intracellular distribution remain undefined. Proliferation of human adenocarcinoma A549 cells was decreased by claudin-2 knockdown together with a decrease in the percentage of S phase cells. This knockdown decreased the expression levels of ZONAB and cell cycle regulators. Claudin-2 was distributed in the nucleus in human adenocarcinoma tissues and proliferating A549 cells. The nuclear distribution of ZONAB and percentage of S phase cells were higher in cells exogenously expressing claudin-2 with a nuclear localization signal than in cells expressing claudin-2 with a nuclear export signal. Nuclear claudin-2 formed a complex with ZO-1, ZONAB, and cyclin D1. Nuclear distribution of S208A mutant, a dephosphorylated form of claudin-2, was higher than that of wild type. We suggest that nuclear distribution of claudin-2 is up-regulated by dephosphorylation and claudin-2 serves to retain ZONAB and cyclin D1 in the nucleus, resulting in the enhancement of cell proliferation in lung adenocarcinoma cells.

  7. Analysis of cell growth and gene expression of porcine adipose tissue-derived mesenchymal stem cells as nuclear donor cell.

    Science.gov (United States)

    Oh, Hyun Ju; Park, Jung Eun; Park, Eun Jung; Kim, Min Jung; Kim, Geon A; Rhee, Sang Ho; Lim, Sang Hyun; Kang, Sung Keun; Lee, Byeong Chun

    2014-12-01

    In several laboratory animals and humans, adipose tissue-derived mesenchymal stem cells (ASC) are of considerable interest because they are easy to harvest and can generate a huge proliferation of cells from a small quantity of fat. In this study, we investigated: (i) the expression patterns of reprogramming-related genes in porcine ASC; and (ii) whether ASC can be a suitable donor cell type for generating cloned pigs. For these experiments, ASC, adult skin fibroblasts (AF) and fetal fibroblasts (FF) were derived from a 4-year-old female miniature pig. The ASC expressed cell-surface markers characteristic of stem cells, and underwent in vitro differentiation when exposed to specific differentiation-inducing conditions. Expression of DNA methyltransferase (DNMT)1 in ASC was similar to that in AF, but the highest expression of the DNMT3B gene was observed in ASC. The expression of OCT4 was significantly higher in FF and ASC than in AF (P development rate of cloned embryos derived from ASC was comparable to the development of those derived using FF. Total cell numbers of blastocysts derived using ASC and FF were significantly higher than in embryos made with AF. The results demonstrated that ASC used for SCNT have a potential comparable to those of AF and FF in terms of embryo in vitro development and blastocyst formation.

  8. Analyzing Cell Death by Nuclear Staining with Hoechst 33342.

    Science.gov (United States)

    Crowley, Lisa C; Marfell, Brooke J; Waterhouse, Nigel J

    2016-01-01

    The nuclei of healthy cells are generally spherical, and the DNA is evenly distributed. During apoptosis the DNA becomes condensed, but this process does not occur during necrosis. Nuclear condensation can therefore be used to distinguish apoptotic cells from healthy cells or necrotic cells. Dyes that bind to DNA, such as Hoechst 33342 or 4',6-diamidino-2-phenylindole (DAPI), can be used to observe nuclear condensation. These dyes fluoresce at 461 nm when excited by ultraviolet light and can therefore be visualized using conventional fluorescent microscopes equipped with light sources that emit light at ∼350 nm and filter sets that permit the transmission of light at ∼460 nm. This protocol describes staining and visualization of cells stained with Hoechst 33342, but it can be adapted for staining with DAPI or other dyes. PMID:27587774

  9. ESCRT III repairs nuclear envelope ruptures during cell migration to limit DNA damage and cell death.

    Science.gov (United States)

    Raab, M; Gentili, M; de Belly, H; Thiam, H R; Vargas, P; Jimenez, A J; Lautenschlaeger, F; Voituriez, Raphaël; Lennon-Duménil, A M; Manel, N; Piel, M

    2016-04-15

    In eukaryotic cells, the nuclear envelope separates the genomic DNA from the cytoplasmic space and regulates protein trafficking between the two compartments. This barrier is only transiently dissolved during mitosis. Here, we found that it also opened at high frequency in migrating mammalian cells during interphase, which allowed nuclear proteins to leak out and cytoplasmic proteins to leak in. This transient opening was caused by nuclear deformation and was rapidly repaired in an ESCRT (endosomal sorting complexes required for transport)-dependent manner. DNA double-strand breaks coincided with nuclear envelope opening events. As a consequence, survival of cells migrating through confining environments depended on efficient nuclear envelope and DNA repair machineries. Nuclear envelope opening in migrating leukocytes could have potentially important consequences for normal and pathological immune responses. PMID:27013426

  10. CEND1 and NEUROGENIN2 Reprogram Mouse Astrocytes and Embryonic Fibroblasts to Induced Neural Precursors and Differentiated Neurons

    Directory of Open Access Journals (Sweden)

    Katerina Aravantinou-Fatorou

    2015-09-01

    Full Text Available Recent studies demonstrate that astroglia from non-neurogenic brain regions can be reprogrammed into functional neurons through forced expression of neurogenic factors. Here we explored the effect of CEND1 and NEUROG2 on reprogramming of mouse cortical astrocytes and embryonic fibroblasts. Forced expression of CEND1, NEUROG2, or both resulted in acquisition of induced neuronal cells expressing subtype-specific markers, while long-term live-cell imaging highlighted the existence of two different modes of neuronal trans-differentiation. Of note, a subpopulation of CEND1 and NEUROG2 double-transduced astrocytes formed spheres exhibiting neural stem cell properties. mRNA and protein expression studies revealed a reciprocal feedback loop existing between the two molecules, while knockdown of endogenous CEND1 demonstrated that it is a key mediator of NEUROG2-driven neuronal reprogramming. Our data suggest that common reprogramming mechanisms exist driving the conversion of lineage-distant somatic cell types to neurons and reveal a critical role for CEND1 in NEUROG2-driven astrocytic reprogramming.

  11. EXPRESSION OF PLURIPOTENCY MARKERS IN REPROGRAMMING WITH TRANSPOSON SYSTEM MURINE FIBROBLASTS

    Directory of Open Access Journals (Sweden)

    S. V. Malysheva

    2013-10-01

    Full Text Available The search for effective and safe methods to generate induced pluripotent stem cells is especially urgent. In the paper murine embryonic fibro blasts were reprogrammed towards actively proliferating colonies with typical induced pluripotent stem cells morphology by means of Sleeping beauty transposon-based vector system. The obtained clones were checked for the expression of various pluripotency markers: alkaline phosphatase, Oct4 and Sox2 genes, SSEA-1 expression in various clones was evaluated. Also the reactivation of endogenous pluripotency factors Nanog and Rex1 was indicated. The data obtained is analyzed and compared to the established pluripotent stem cell line. It is shown that somatic cells are reprogrammed towards pluripotency by means of Sleeping beauty transposon system. Therefore, the system is a new perspective biotechnological tool to generate pluripotent cells.

  12. Induced pluripotency and direct reprogramming: a new window for treatment of neurodegenerative diseases.

    Science.gov (United States)

    Li, Rui; Bai, Ye; Liu, Tongtong; Wang, Xiaoqun; Wu, Qian

    2013-06-01

    Human embryonic stem cells (hESCs) are pluripotent cells that have the ability of unlimited self-renewal and can be differentiated into different cell lineages, including neural stem (NS) cells. Diverse regulatory signaling pathways of neural stem cells differentiation have been discovered, and this will be of great benefit to uncover the mechanisms of neuronal differentiation in vivo and in vitro. However, the limitations of hESCs resource along with the religious and ethical concerns impede the progress of ESCs application. Therefore, the induced pluripotent stem cells (iPSCs) via somatic cell reprogramming have opened up another new territory for regenerative medicine. iPSCs now can be derived from a number of lineages of cells, and are able to differentiate into certain cell types, including neurons. Patient-specifi c iPSCs are being used in human neurodegenerative disease modeling and drug screening. Furthermore, with the development of somatic direct reprogramming or lineage reprogramming technique, a more effective approach for regenerative medicine could become a complement for iPSCs.

  13. Nuclear tristetraprolin acts as a corepressor of multiple steroid nuclear receptors in breast cancer cells

    Directory of Open Access Journals (Sweden)

    Tonatiuh Barrios-García

    2016-06-01

    Full Text Available Tristetraprolin (TTP is a 34-kDa, zinc finger-containing factor that in mammalian cells acts as a tumor suppressor protein through two different mechanisms. In the cytoplasm TTP promotes the decay of hundreds of mRNAs encoding cell factors involved in inflammation, tissue invasion, and metastasis. In the cell nucleus TTP has been identified as a transcriptional corepressor of the estrogen receptor alpha (ERα, which has been associated to the development and progression of the majority of breast cancer tumors. In this work we report that nuclear TTP modulates the transactivation activity of progesterone receptor (PR, glucocorticoid receptor (GR and androgen receptor (AR. In recent years these steroid nuclear receptors have been shown to be of clinical and therapeutical relevance in breast cancer. The functional association between TTP and steroid nuclear receptors is supported by the finding that TTP physically interacts with ERα, PR, GR and AR in vivo. We also show that TTP overexpression attenuates the transactivation of all the steroid nuclear receptors tested. In contrast, siRNA-mediated reduction of endogenous TTP expression in MCF-7 cells produced an increase in the transcriptional activities of ERα, PR, GR and AR. Taken together, these results suggest that the function of nuclear TTP in breast cancer cells is to act as a corepressor of ERα, PR, GR and AR. We propose that the reduction of TTP expression observed in different types of breast cancer tumors may contribute to the development of this disease by producing a dysregulation of the transactivation activity of multiple steroid nuclear receptors.

  14. Nuclear entry of hyperbranched polylysine nanoparticles into cochlear cells

    Directory of Open Access Journals (Sweden)

    Weikai Zhang

    2011-03-01

    Full Text Available Weikai Zhang1, Ya Zhang1, Marian Löbler2, Klaus-Peter Schmitz2, Aqeel Ahmad3, Ilmari Pyykkö1, Jing Zou11Department of Otolaryngology, University of Tampere, Medical School, Tampere, Finland; 2University of Rostock, Institute for Biomedical Engineering, Rostock, Germany; 3Department of Biomedical Engineering and Computational Science, Aalto University, Espoo, FinlandBackground: Gene therapy is a potentially effective therapeutic modality for treating sensorineural hearing loss. Nonviral gene delivery vectors are expected to become extremely safe and convenient, and nanoparticles are the most promising types of vectors. However, infrequent nuclear localization in the cochlear cells limits their application for gene therapy. This study aimed to investigate the potential nuclear entry of hyperbranched polylysine nanoparticles (HPNPs for gene delivery to cochlear targets.Methods: Rat primary cochlear cells and cochlear explants generated from newborn rats were treated with different concentrations of HPNPs. For the in vivo study, HPNPs were administered to the rats' round window membranes. Subcellular distribution of HPNPs in different cell populations was observed with confocal microscope 24 hours after administration.Results: Nuclear entry was observed in various cochlear cell types in vitro and in vivo. In the primary cochlear cell culture, concentration-dependent internalization was observed. In the cochlear organotypic culture, abundant HPNPs were found in the modiolus, including the spiral ganglion, organ of Corti, and lateral wall tissues. In the in vivo study, a gradient distribution of HPNPs through different layers of the round window membrane was observed. HPNPs were also distributed in the cells of the middle ear tissue. Additionally, efficient internalization of HPNPs was observed in the organ of Corti and spiral ganglion cells. In primary cochlear cells, HPNPs induced higher transfection efficiency than did Lipofectamine

  15. Nuclear RNA sequencing of the mouse erythroid cell transcriptome.

    Directory of Open Access Journals (Sweden)

    Jennifer A Mitchell

    Full Text Available In addition to protein coding genes a substantial proportion of mammalian genomes are transcribed. However, most transcriptome studies investigate steady-state mRNA levels, ignoring a considerable fraction of the transcribed genome. In addition, steady-state mRNA levels are influenced by both transcriptional and posttranscriptional mechanisms, and thus do not provide a clear picture of transcriptional output. Here, using deep sequencing of nuclear RNAs (nucRNA-Seq in parallel with chromatin immunoprecipitation sequencing (ChIP-Seq of active RNA polymerase II, we compared the nuclear transcriptome of mouse anemic spleen erythroid cells with polymerase occupancy on a genome-wide scale. We demonstrate that unspliced transcripts quantified by nucRNA-seq correlate with primary transcript frequencies measured by RNA FISH, but differ from steady-state mRNA levels measured by poly(A-enriched RNA-seq. Highly expressed protein coding genes showed good correlation between RNAPII occupancy and transcriptional output; however, genome-wide we observed a poor correlation between transcriptional output and RNAPII association. This poor correlation is due to intergenic regions associated with RNAPII which correspond with transcription factor bound regulatory regions and a group of stable, nuclear-retained long non-coding transcripts. In conclusion, sequencing the nuclear transcriptome provides an opportunity to investigate the transcriptional landscape in a given cell type through quantification of unspliced primary transcripts and the identification of nuclear-retained long non-coding RNAs.

  16. Cell fate conversion-from the viewpoint of small molecules and lineage specifiers.

    Science.gov (United States)

    Zhao, T; Li, Y; Deng, H

    2016-09-01

    Mammalian development was generally considered a naturally unidirectional and irreversible process. However, pioneering work of recent decades has highlighted the plasticity of mammalian cells and implied the possibilities of manipulating cell fate in vitro. Pluripotent stem cells, which hold great potential for regenerative medicine, have been shown to be reprogrammed from differentiated cells either by somatic cell nuclear transfer or by ectopic expression of pluripotency factors. Nevertheless, it remained unknown whether the reprogramming could be accomplished without pluripotency genes. Recent studies show that lineage specifiers play an important role in orchestrating the process of restoring pluripotency by replacing pluripotency-associated transcription factors. Moreover, a combination of small molecules enables the acquisition of pluripotency from somatic cells without any transgenes, offering a tractable platform to precisely dissect the induction and maintenance of cell identity. Here, we will discuss recent scientific advances regarding the cell fate conversion mediated by small molecules or lineage specifiers, especially in the chemically induced somatic cell reprogramming process, and will provide new insights into the intermediate plastic state and "seesaw model" established by chemical approaches during reprogramming. PMID:27615126

  17. Generation and transplantation of reprogrammed human neurons in the brain using 3D microtopographic scaffolds.

    Science.gov (United States)

    Carlson, Aaron L; Bennett, Neal K; Francis, Nicola L; Halikere, Apoorva; Clarke, Stephen; Moore, Jennifer C; Hart, Ronald P; Paradiso, Kenneth; Wernig, Marius; Kohn, Joachim; Pang, Zhiping P; Moghe, Prabhas V

    2016-01-01

    Cell replacement therapy with human pluripotent stem cell-derived neurons has the potential to ameliorate neurodegenerative dysfunction and central nervous system injuries, but reprogrammed neurons are dissociated and spatially disorganized during transplantation, rendering poor cell survival, functionality and engraftment in vivo. Here, we present the design of three-dimensional (3D) microtopographic scaffolds, using tunable electrospun microfibrous polymeric substrates that promote in situ stem cell neuronal reprogramming, neural network establishment and support neuronal engraftment into the brain. Scaffold-supported, reprogrammed neuronal networks were successfully grafted into organotypic hippocampal brain slices, showing an ∼ 3.5-fold improvement in neurite outgrowth and increased action potential firing relative to injected isolated cells. Transplantation of scaffold-supported neuronal networks into mouse brain striatum improved survival ∼ 38-fold at the injection site relative to injected isolated cells, and allowed delivery of multiple neuronal subtypes. Thus, 3D microscale biomaterials represent a promising platform for the transplantation of therapeutic human neurons with broad neuro-regenerative relevance. PMID:26983594

  18. Role of ooplasm in nuclear and nucleolar remodeling of intergeneric somatic cell nuclear transfer embryos during the first cell cycle

    DEFF Research Database (Denmark)

    Østrup, Olga; Strejcek, Frantisek; Petrovicova, Ida;

    2011-01-01

    -cell stage embryos were processed at different points in time post activation (2 hpa, 4 hpa, 8 hpa, and 12 hpa) for detailed nuclear and nucleolar analysis by TEM, and immunofluorescence for visualization of nucleolar proteins related to transcription (UBF) and processing (fibrillarin). Bovine and porcine...

  19. Lysine-specific histone demethylase 1 inhibition promotes reprogramming by facilitating the expression of exogenous transcriptional factors and metabolic switch.

    Science.gov (United States)

    Sun, Hao; Liang, Lining; Li, Yuan; Feng, Chengqian; Li, Lingyu; Zhang, Yixin; He, Songwei; Pei, Duanqing; Guo, Yunqian; Zheng, Hui

    2016-01-01

    Lysine-specific histone demethylase 1 (LSD1) regulates histone methylation and influences the epigenetic state of cells during the generation of induced pluripotent stem cells (iPSCs). Here we reported that LSD1 inhibition via shRNA or specific inhibitor, tranylcypromine, promoted reprogramming at early stage via two mechanisms. At early stage of reprogramming, LSD1 inhibition increased the retrovirus-mediated exogenous expression of Oct4, Klf4, and Sox2 by blocking related H3K4 demethylation. Since LSD1 inhibition still promoted reprogramming even when iPSCs were induced with small-molecule compounds in a virus-free system, additional mechanisms should be involved. When RNA-seq was used for analysis, it was found that LSD1 inhibition reversed some gene expression changes induced by OKS, which subsequently promoted reprogramming. For example, by partially rescuing the decreased expression of Hif1α, LSD1 inhibition reversed the up-regulation of genes in oxidative phosphorylation pathway and the down-regulation of genes in glycolysis pathway. Such effects facilitated the metabolic switch from oxidative phosphorylation to glycolysis and subsequently promoted iPSCs induction. In addition, LSD1 inhibition also promoted the conversion from pre-iPSCs to iPSCs by facilitating the similar metabolic switch. Therefore, LSD1 inhibition promotes reprogramming by facilitating the expression of exogenous transcriptional factors and metabolic switch. PMID:27481483

  20. microRNAs and cancer metabolism reprogramming: the paradigm of metformin.

    Science.gov (United States)

    Pulito, Claudio; Donzelli, Sara; Muti, Paola; Puzzo, Luisa; Strano, Sabrina; Blandino, Giovanni

    2014-06-01

    Increasing evidence witnesses that cancer metabolism alterations represent a critical hallmark for many types of human tumors. There is a strong need to understand and dissect the molecular mechanisms underlying cancer metabolism to envisage specific biomarkers and underpin critical molecular components that might represent novel therapeutic targets. One challenge, that is the focus of this review, is the reprogramming of the altered metabolism of a cancer cell toward that of un-transformed cell. The anti-hyperglicemic agent, metformin has proven to be effective in reprogramming the metabolism of cancer cells even from those subpopulations endowed with cancer stem like features and very high chemoresistenace to conventional anticancer treatments. A functional interplay involving selective modulation of microRNAs (miRNAs) takes place along the anticancer metabolic effects exerted by metformin. The implications of this interplay will be also discussed in this review. PMID:25333033

  1. Neuroprotection Signaling of Nuclear Akt in Neuronal Cells

    OpenAIRE

    Ahn, Jee-Yin

    2014-01-01

    Akt is one of the central kinases that perform a pivotal function in mediating survival signaling in a wide range of neuronal cell types in response to growth factor stimulation. The recent findings of a number of targets for Akt suggest that it prohibits neuronal death by both impinging on the cytoplasmic cell death machinery and by regulating nuclear proteins. The presence of active Akt in the nuclei of mammalian cells is no longer debatable, and this has been corroborated by the finding of...

  2. Reprogramming with Small Molecules instead of Exogenous Transcription Factors

    Directory of Open Access Journals (Sweden)

    Tongxiang Lin

    2015-01-01

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

  3. Innate immune suppression enables frequent transfection with RNA encoding reprogramming proteins.

    Directory of Open Access Journals (Sweden)

    Matthew Angel

    Full Text Available BACKGROUND: Generating autologous pluripotent stem cells for therapeutic applications will require the development of efficient DNA-free reprogramming techniques. Transfecting cells with in vitro-transcribed, protein-encoding RNA is a straightforward method of directly expressing high levels of reprogramming proteins without genetic modification. However, long-RNA transfection triggers a potent innate immune response characterized by growth inhibition and the production of inflammatory cytokines. As a result, repeated transfection with protein-encoding RNA causes cell death. METHODOLOGY/PRINCIPAL FINDINGS: RNA viruses have evolved methods of disrupting innate immune signaling by destroying or inhibiting specific proteins to enable persistent infection. Starting from a list of known viral targets, we performed a combinatorial screen to identify siRNA cocktails that could desensitize cells to exogenous RNA. We show that combined knockdown of interferon-beta (Ifnb1, Eif2ak2, and Stat2 rescues cells from the innate immune response triggered by frequent long-RNA transfection. Using this technique, we were able to transfect primary human fibroblasts every 24 hours with RNA encoding the reprogramming proteins Oct4, Sox2, Klf4, and Utf1. We provide evidence that the encoded protein is active, and we show that expression can be maintained for many days, through multiple rounds of cell division. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that suppressing innate immunity enables frequent transfection with protein-encoding RNA. This technique represents a versatile tool for investigating expression dynamics and protein interactions by enabling precise control over levels and timing of protein expression. Our finding also opens the door for the development of reprogramming and directed-differentiation methods based on long-RNA transfection.

  4. A combined gas cooled nuclear reactor and fuel cell cycle

    Science.gov (United States)

    Palmer, David J.

    Rising oil costs, global warming, national security concerns, economic concerns and escalating energy demands are forcing the engineering communities to explore methods to address these concerns. It is the intention of this thesis to offer a proposal for a novel design of a combined cycle, an advanced nuclear helium reactor/solid oxide fuel cell (SOFC) plant that will help to mitigate some of the above concerns. Moreover, the adoption of this proposal may help to reinvigorate the Nuclear Power industry while providing a practical method to foster the development of a hydrogen economy. Specifically, this thesis concentrates on the importance of the U.S. Nuclear Navy adopting this novel design for its nuclear electric vessels of the future with discussion on efficiency and thermodynamic performance characteristics related to the combined cycle. Thus, the goals and objectives are to develop an innovative combined cycle that provides a solution to the stated concerns and show that it provides superior performance. In order to show performance, it is necessary to develop a rigorous thermodynamic model and computer program to analyze the SOFC in relation with the overall cycle. A large increase in efficiency over the conventional pressurized water reactor cycle is realized. Both sides of the cycle achieve higher efficiencies at partial loads which is extremely important as most naval vessels operate at partial loads as well as the fact that traditional gas turbines operating alone have poor performance at reduced speeds. Furthermore, each side of the cycle provides important benefits to the other side. The high temperature exhaust from the overall exothermic reaction of the fuel cell provides heat for the reheater allowing for an overall increase in power on the nuclear side of the cycle. Likewise, the high temperature helium exiting the nuclear reactor provides a controllable method to stabilize the fuel cell at an optimal temperature band even during transients helping

  5. Design of nuclear cells with re linking of trajectories

    International Nuclear Information System (INIS)

    Presently work the results obtained with the Ohtli-RT system obtained when implementing the combinatory optimization technique well-known as Trajectories re linking or Path Re linking in English. The problem to solve is the radial design of nuclear fuel, taking like base nuclear fuel assembles for boiling water reactors (BWR Boiling Water Reactor by its initials in English). To evaluate the objective function used in the system the code in two dimensions Heliums 1.5 was used, which calculates the cross sections of the proposed design. The parameters that were considered for the evaluation of the objective function are the Power peak factor of the bar that generates bigger power in the cell and the Infinite Multiplication Factor. To prove the system its were used assembles 10x10 with 2 water channels. The obtained radial designs of nuclear fuel fulfilled the restrictions imposed to the considered limits, with regard to the involved parameters. (Author)

  6. Metabolic reprogramming during neuronal differentiation.

    Science.gov (United States)

    Agostini, M; Romeo, F; Inoue, S; Niklison-Chirou, M V; Elia, A J; Dinsdale, D; Morone, N; Knight, R A; Mak, T W; Melino, G

    2016-09-01

    Newly generated neurons pass through a series of well-defined developmental stages, which allow them to integrate into existing neuronal circuits. After exit from the cell cycle, postmitotic neurons undergo neuronal migration, axonal elongation, axon pruning, dendrite morphogenesis and synaptic maturation and plasticity. Lack of a global metabolic analysis during early cortical neuronal development led us to explore the role of cellular metabolism and mitochondrial biology during ex vivo differentiation of primary cortical neurons. Unexpectedly, we observed a huge increase in mitochondrial biogenesis. Changes in mitochondrial mass, morphology and function were correlated with the upregulation of the master regulators of mitochondrial biogenesis, TFAM and PGC-1α. Concomitant with mitochondrial biogenesis, we observed an increase in glucose metabolism during neuronal differentiation, which was linked to an increase in glucose uptake and enhanced GLUT3 mRNA expression and platelet isoform of phosphofructokinase 1 (PFKp) protein expression. In addition, glutamate-glutamine metabolism was also increased during the differentiation of cortical neurons. We identified PI3K-Akt-mTOR signalling as a critical regulator role of energy metabolism in neurons. Selective pharmacological inhibition of these metabolic pathways indicate existence of metabolic checkpoint that need to be satisfied in order to allow neuronal differentiation. PMID:27058317

  7. Oligodendrocyte progenitor programming and reprogramming: Toward myelin regeneration.

    Science.gov (United States)

    Lopez Juarez, Alejandro; He, Danyang; Richard Lu, Q

    2016-05-01

    Demyelinating diseases such as multiple sclerosis (MS) are among the most disabling and cost-intensive neurological disorders. The loss of myelin in the central nervous system, produced by oligodendrocytes (OLs), impairs saltatory nerve conduction, leading to motor and cognitive deficits. Immunosuppression therapy has a limited efficacy in MS patients, arguing for a paradigm shift to strategies that target OL lineage cells to achieve myelin repair. The inhibitory microenvironment in MS lesions abrogates the expansion and differentiation of resident OL precursor cells (OPCs) into mature myelin-forming OLs. Recent studies indicate that OPCs display a highly plastic ability to differentiate into alternative cell lineages under certain circumstances. Thus, understanding the mechanisms that maintain and control OPC fate and differentiation into mature OLs in a hostile, non-permissive lesion environment may open new opportunities for regenerative therapies. In this review, we will focus on 1) the plasticity of OPCs in terms of their developmental origins, distribution, and differentiation potentials in the normal and injured brain; 2) recent discoveries of extrinsic and intrinsic factors and small molecule compounds that control OPC specification and differentiation; and 3) therapeutic potential for motivation of neural progenitor cells and reprogramming of differentiated cells into OPCs and their likely impacts on remyelination. OL-based therapies through activating regenerative potentials of OPCs or cell replacement offer exciting opportunities for innovative strategies to promote remyelination and neuroprotection in devastating demyelinating diseases like MS. This article is part of a Special Issue entitled SI:NG2-glia(Invited only). PMID:26546966

  8. Generation of cloned mice and nuclear transfer embryonic stem cell lines from urine-derived cells.

    Science.gov (United States)

    Mizutani, Eiji; Torikai, Kohei; Wakayama, Sayaka; Nagatomo, Hiroaki; Ohinata, Yasuhide; Kishigami, Satoshi; Wakayama, Teruhiko

    2016-01-01

    Cloning animals by nuclear transfer provides the opportunity to preserve endangered mammalian species. However, there are risks associated with the collection of donor cells from the body such as accidental injury to or death of the animal. Here, we report the production of cloned mice from urine-derived cells collected noninvasively. Most of the urine-derived cells survived and were available as donors for nuclear transfer without any pretreatment. After nuclear transfer, 38-77% of the reconstructed embryos developed to the morula/blastocyst, in which the cell numbers in the inner cell mass and trophectoderm were similar to those of controls. Male and female cloned mice were delivered from cloned embryos transferred to recipient females, and these cloned animals grew to adulthood and delivered pups naturally when mated with each other. The results suggest that these cloned mice had normal fertility. In additional experiments, 26 nuclear transfer embryonic stem cell lines were established from 108 cloned blastocysts derived from four mouse strains including inbreds and F1 hybrids with relatively high success rates. Thus, cells derived from urine, which can be collected noninvasively, may be used in the rescue of endangered mammalian species by using nuclear transfer without causing injury to the animal. PMID:27033801

  9. Nuclear lamins and oxidative stress in cell proliferation and longevity.

    Science.gov (United States)

    Shimi, Takeshi; Goldman, Robert D

    2014-01-01

    In mammalian cells, the nuclear lamina is composed of a complex fibrillar network associated with the inner membrane of the nuclear envelope. The lamina provides mechanical support for the nucleus and functions as the major determinant of its size and shape. At its innermost aspect it associates with peripheral components of chromatin and thereby contributes to the organization of interphase chromosomes. The A- and B-type lamins are the major structural components of the lamina, and numerous mutations in the A-type lamin gene have been shown to cause many types of human diseases collectively known as the laminopathies. These mutations have also been shown to cause a disruption in the normal interactions between the A and B lamin networks. The impact of these mutations on nuclear functions is related to the roles of lamins in regulating various essential processes including DNA synthesis and damage repair, transcription and the regulation of genes involved in the response to oxidative stress. The major cause of oxidative stress is the production of reactive oxygen species (ROS), which is critically important for cell proliferation and longevity. Moderate increases in ROS act to initiate signaling pathways involved in cell proliferation and differentiation, whereas excessive increases in ROS cause oxidative stress, which in turn induces cell death and/or senescence. In this review, we cover current findings about the role of lamins in regulating cell proliferation and longevity through oxidative stress responses and ROS signaling pathways. We also speculate on the involvement of lamins in tumor cell proliferation through the control of ROS metabolism.

  10. Statistical analysis in the design of nuclear fuel cells

    International Nuclear Information System (INIS)

    This work presents the preliminary results of a statistical analysis carried out for the design of nuclear fuel cells. The analysis consists in verifying the behavior of a cell, related with the frequency of the pines used for its design. In this preliminary study was analyzed the behavior of infinite multiplication factor and the peak factor of local power. On the other hand, the mentioned analysis was carried out using a pines group of enriched uranium previously established, for which varies the pines frequency used in the design. To carry out the study, the CASMO-IV code was used. The obtained designs are for the different axial areas of a fuel assembly. A balance cycle of the unit 1 of the nuclear power plant of Laguna Verde was used like reference. To obtain the result of the present work, systems that are already had and in which have already been implemented the heuristic techniques of ant colonies, neural networks and a hybrid between the dispersed search and the trajectories re-chaining. The results show that is possible to design nuclear fuel cells with a good performance, if is considered a statistical behavior in the frequency of the used pines, in a same way. (Author)

  11. Transcriptional reprogramming in nonhuman primate (rhesus macaque tuberculosis granulomas.

    Directory of Open Access Journals (Sweden)

    Smriti Mehra

    Full Text Available BACKGROUND: In response to Mtb infection, the host remodels the infection foci into a dense mass of cells known as the granuloma. The key objective of the granuloma is to contain the spread of Mtb into uninfected regions of the lung. However, it appears that Mtb has evolved mechanisms to resist killing in the granuloma. Profiling granuloma transcriptome will identify key immune signaling pathways active during TB infection. Such studies are not possible in human granulomas, due to various confounding factors. Nonhuman Primates (NHPs infected with Mtb accurately reflect human TB in clinical and pathological contexts. METHODOLOGY/PRINCIPAL FINDINGS: We studied transcriptomics of granuloma lesions in the lungs of NHPs exhibiting active TB, during early and late stages of infection. Early TB lesions were characterized by a highly pro-inflammatory environment, expressing high levels of immune signaling pathways involving IFNgamma, TNFalpha, JAK, STAT and C-C/C-X-C chemokines. Late TB lesions, while morphologically similar to the early ones, exhibited an overwhelming silencing of the inflammatory response. Reprogramming of the granuloma transcriptome was highly significant. The expression of approximately two-thirds of all genes induced in early lesions was later repressed. CONCLUSIONS/SIGNIFICANCE: The transcriptional characteristics of TB granulomas undergo drastic changes during the course of infection. The overwhelming reprogramming of the initial pro-inflammatory surge in late lesions may be a host strategy to limit immunopathology. We propose that these host profiles can predict changes in bacterial replication and physiology, perhaps serving as markers for latency and reactivation.

  12. Functional properties of neurons derived from in vitro reprogrammed postnatal astroglia

    OpenAIRE

    Berninger, Benedikt; Costa, Marcos R.; Koch, Ursula; Schroeder, Timm; Sutor, Bernd; Grothe, Benedikt; Götz, Magdalena

    2007-01-01

    With the exception of astroglia-like cells in the neurogenic niches of the telencephalic subependymal or hippocampal subgranular zone, astroglia in all other regions of the adult mouse brain do not normally generate neurons. Previous studies have shown, however, that early postnatal cortical astroglia in culture can be reprogrammed to adopt a neuronal fate after forced expression of Pax6, a transcription factor (TF) required for proper neuronal specification during embryonic corticogenesis. H...

  13. Fenofibrate Suppresses Oral Tumorigenesis via Reprogramming Metabolic Processes: Potential Drug Repurposing for Oral Cancer

    OpenAIRE

    Jan, Chia-Ing; Tsai, Ming-Hsui; Chiu, Chang-Fang; Huang, Yi-Ping; Liu, Chia Jen; Chang, Nai Wen

    2016-01-01

    One anticancer strategy suggests targeting mitochondrial metabolism to trigger cell death through slowing down energy production from the Warburg effect. Fenofibrate is a clinical lipid-lowering agent and an effective anticancer drug. In the present study, we demonstrate that fenofibrate provided novel mechanisms for delaying oral tumor development via the reprogramming of metabolic processes. Fenofibrate induced cytotoxicity by decreasing oxygen consumption rate (OCR) that was accompanied wi...

  14. Nuclear microprobe imaging of gallium nitrate in cancer cells

    Science.gov (United States)

    Ortega, Richard; Suda, Asami; Devès, Guillaume

    2003-09-01

    Gallium nitrate is used in clinical oncology as treatment for hypercalcemia and for cancer that has spread to the bone. Its mechanism of antitumor action has not been fully elucidated yet. The knowledge of the intracellular distribution of anticancer drugs is of particular interest in oncology to better understand their cellular pharmacology. In addition, most metal-based anticancer compounds interact with endogenous trace elements in cells, altering their metabolism. The purpose of this experiment was to examine, by use of nuclear microprobe analysis, the cellular distribution of gallium and endogenous trace elements within cancer cells exposed to gallium nitrate. In a majority of cellular analyses, gallium was found homogeneously distributed in cells following the distribution of carbon. In a smaller number of cells, however, gallium appeared concentrated together with P, Ca and Fe within round structures of about 2-5 μm diameter located in the perinuclear region. These intracellular structures are typical of lysosomial material.

  15. Nuclear microprobe imaging of gallium nitrate in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Ortega, Richard E-mail: ortega@cenbg.in2p3.fr; Suda, Asami; Deves, Guillaume

    2003-09-01

    Gallium nitrate is used in clinical oncology as treatment for hypercalcemia and for cancer that has spread to the bone. Its mechanism of antitumor action has not been fully elucidated yet. The knowledge of the intracellular distribution of anticancer drugs is of particular interest in oncology to better understand their cellular pharmacology. In addition, most metal-based anticancer compounds interact with endogenous trace elements in cells, altering their metabolism. The purpose of this experiment was to examine, by use of nuclear microprobe analysis, the cellular distribution of gallium and endogenous trace elements within cancer cells exposed to gallium nitrate. In a majority of cellular analyses, gallium was found homogeneously distributed in cells following the distribution of carbon. In a smaller number of cells, however, gallium appeared concentrated together with P, Ca and Fe within round structures of about 2-5 {mu}m diameter located in the perinuclear region. These intracellular structures are typical of lysosomial material.

  16. Cloned calves produced by nuclear transfer from cultured cumulus cells

    Institute of Scientific and Technical Information of China (English)

    AN; Xiaorong(安晓荣); GOU; Kemian(苟克勉); ZHU; Shien(朱士恩); GUAN; Hong(关宏); HOU; Jian(侯健); LIN; Aixing(林爱星); ZENG; Shenming(曾申明); TIAN; Jianhui(田见辉); CHEN; Yongfu(陈永福)

    2002-01-01

    Short-term cultured cumulus cell lines (1-5BCC) derived from 5 individual cows were used in nuclear transfer (NT) and 1188 enucleated bovine oocytes matured in vitro were used as nuclear recipients. A total of 931 (78.4%) cloned embryos were reconstructed, of which 763 (82%) cleaved, 627 (67.3%) developed to 8-cell stage, and 275 (29.5%) reached blastocyst stage. The average cell number of blastocysts was 124±24.5 (n=20). In this study, the effects of donor cell sources, serum starvation of donor cells, time interval from fusion to activation (IFA) were also tested on cloning efficiency. These results showed that blastocyst rates of embryos reconstructed from 5 different individuals cells were significantly different among them (14.1%, 45.2%, 27.3%, 34.3%, vs 1.5%, P0.05); and that blastocyst rate (20.3%) of the group with fusion/activation interval of 2-3 h, was significantly lower than that of the 3-6 h groups (31.0%), while not significantly different among 3-4 h (P < 0.05), 4-5 h, and 5-6 h groups (P ≥ 0.05). Sixty-three thawed NT blastocysts were transferred to 31 recipient cows, of which 4 pregnancies were established and two cloned calves were given birth. These results indicate that serum starvation of cumulus cells is not a key factor for successful bovine cloning, while IFA treatment and sources of donor cells have effects on cloning efficiency.

  17. White-to-brite conversion in human adipocytes promotes metabolic reprogramming towards fatty acid anabolic and catabolic pathways

    Directory of Open Access Journals (Sweden)

    V. Barquissau

    2016-05-01

    Conclusions: Conversion of human white fat cells into brite adipocytes results in a major metabolic reprogramming inducing fatty acid anabolic and catabolic pathways. PDK4 redirects glucose from oxidation towards triglyceride synthesis and favors the use of fatty acids as energy source for uncoupling mitochondria.

  18. Quantitative Phosphoproteomics Reveals Extensive Cellular Reprogramming During HIV-1 Entry

    Science.gov (United States)

    Wojcechowskyj, Jason A.; Didigu, Chuka A.; Lee, Jessica Y.; Parrish, Nicholas F.; Sinha, Rohini; Hahn, Beatrice H.; Bushman, Frederic D.; Jensen, Shane T.; Seeholzer, Steven H.; Doms, Robert W.

    2014-01-01

    SUMMARY Receptor engagement by HIV-1 during host cell entry activates signaling pathways that can reprogram the cell for optimal viral replication. To obtain a global view of the signaling events induced during HIV-1 entry, we conducted a quantitative phosphoproteomics screen of primary human CD4+ T cell after infection with an HIV-1 strain that engages the receptors CD4 and CXCR4. We quantified 1,757 phosphorylation sites with high stringency. The abundance of 239 phosphorylation sites from 175 genes, including several proteins in pathways known to be impacted by HIV-receptor binding, changed significantly within a minute after HIV-1 exposure. Several previously uncharacterized HIV-1 host factors were also identified and confirmed through RNAi depletion studies. Surprisingly, 5 serine/arginine-rich (SR)-proteins involved in mRNA splicing, including the splicing factor SRm300 (SRRM2) were differentially phosophorylated. Mechanistic studies with SRRM2 suggest that HIV-1 modulates host cell alternative splicing machinery during entry in order to facilitate virus replication and release. PMID:23684312

  19. Somatic cell nuclear transfer-derived embryonic stem cell lines in humans: pros and cons.

    Science.gov (United States)

    Langerova, Alena; Fulka, Helena; Fulka, Josef

    2013-12-01

    The recent paper, published by Mitalipov's group in Cell (Tachibana et al., 2013 ), reporting the production of human somatic cell nuclear transfer (SCNT) embryonic stem cells (ESCs), opens again the debate if, in the era of induced pluripotent stem cells (iPSCs), the production of these cells is indeed necessary and, if so, whether they are different from ESCs produced from spare embryos and iPSCs. It is our opinion that these questions are very difficult to answer because it is still unclear whether and how normal ESCs differ from iPSCs. PMID:24180743

  20. Transient p53 Suppression Increases Reprogramming of Human Fibroblasts without Affecting Apoptosis and DNA Damage

    Directory of Open Access Journals (Sweden)

    Mikkel A. Rasmussen

    2014-09-01

    Full Text Available 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, transient p53 suppression increases reprogramming efficiency without affecting genomic stability, rendering the method suitable for in vitro mechanistic studies with the possibility for future clinical translation.

  1. Metabolic reprogramming: a new relevant pathway in adult adrenocortical tumors

    Science.gov (United States)

    Longatto-Filho, Adhemar; Faria, André M.; Fragoso, Maria C. B. V.; Lovisolo, Silvana M.; Lerário, Antonio M.; Almeida, Madson Q.

    2015-01-01

    Adrenocortical carcinomas (ACCs) are complex neoplasias that may present unexpected clinical behavior, being imperative to identify new biological markers that can predict patient prognosis and provide new therapeutic options. The main aim of the present study was to evaluate the prognostic value of metabolism-related key proteins in adrenocortical carcinoma. The immunohistochemical expression of MCT1, MCT2, MCT4, CD147, CD44, GLUT1 and CAIX was evaluated in a series of 154 adult patients with adrenocortical neoplasia and associated with patients' clinicopathological parameters. A significant increase in was found for membranous expression of MCT4, GLUT1 and CAIX in carcinomas, when compared to adenomas. Importantly MCT1, GLUT1 and CAIX expressions were significantly associated with poor prognostic variables, including high nuclear grade, high mitotic index, advanced tumor staging, presence of metastasis, as well as shorter overall and disease free survival. In opposition, MCT2 membranous expression was associated with favorable prognostic parameters. Importantly, cytoplasmic expression of CD147 was identified as an independent predictor of longer overall survival and cytoplasmic expression of CAIX as an independent predictor of longer disease-free survival. We provide evidence for a metabolic reprogramming in adrenocortical malignant tumors towards the hyperglycolytic and acid-resistant phenotype, which was associated with poor prognosis. PMID:26587828

  2. Thyroid Transcription Factor 1 Reprograms Angiogenic Activities of Secretome.

    Science.gov (United States)

    Wood, Lauren W; Cox, Nicole I; Phelps, Cody A; Lai, Shao-Chiang; Poddar, Arjun; Talbot, Conover; Mu, David

    2016-01-01

    Through both gain- and loss-of-TTF-1 expression strategies, we show that TTF-1 positively regulates vascular endothelial growth factor (VEGF) and that the VEGF promoter element contains multiple TTF-1-responsive sequences. The major signaling receptor for VEGF, i.e VEGFR2, also appears to be under a direct and positive regulation of TTF-1. The TTF-1-dependent upregulation of VEGF was moderately sensitive to rapamycin, implicating a partial involvement of mammalian target of rapamycin (mTOR). However, hypoxia did not further increase the secreted VEGF level of the TTF-1(+) lung cancer cells. The TTF-1-induced VEGF upregulation occurs in both compartments (exosomes and exosome-depleted media (EDM)) of the conditioned media. Surprisingly, the EDM of TTF-1(+) lung cancer cells (designated EDM-TTF-1(+)) displayed an anti-angiogenic activity in the endothelial cell tube formation assay. Mechanistic studies suggest that the increased granulocyte-macrophage colony-stimulating factor (GM-CSF) level in the EDM-TTF-1(+) conferred the antiangiogenic activities. In human lung cancer, the expression of TTF-1 and GM-CSF exhibits a statistically significant and positive correlation. In summary, this study provides evidence that TTF-1 may reprogram lung cancer secreted proteome into an antiangiogenic state, offering a novel basis to account for the long-standing observation of favorable prognosis associated with TTF-1(+) lung adenocarcinomas. PMID:26912193

  3. Data mining in the study of nuclear fuel cells

    International Nuclear Information System (INIS)

    In this paper is presented a study of data mining application in the analysis of fuel cells and their performance within a nuclear boiling water reactor. A decision tree was used to fulfill questions of the type If (condition) and Then (conclusion) to classify if the fuel cells will have good performance. The performance is measured by compliance or not of the cold shutdown margin, the rate of linear heat generation and the average heat generation in a plane of the reactor. It is assumed that the fuel cells are simulated in the reactor under a fuel reload and rod control patterns pre designed. 18125 fuel cells were simulated according to a steady-state calculation. The decision tree works on a target variable which is one of the three mentioned before. To analyze this objective, the decision tree works with a set of attribute variables. In this case, the attributes are characteristics of the cell as number of gadolinium rods, rods number with certain uranium enrichment mixed with a concentration of gadolinium, etc. The found model was able to predict the execution or not of the shutdown margin with a precision of around 95%. However, the other two variables showed lower percentages due to few learning cases of the model in which these variables were or were not achieved. Even with this inconvenience, the model is quite reliable and can be used in way coupled in optimization systems of fuel cells. (Author)

  4. Production of transgenic calves by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    GONG Guochun; WAN Rong; HUANG Yinghua; LI Ning; DAI Yunping; FAN Baoliang; ZHU Huabing; WANG Lili; WANG Haiping; TANG Bo; LIU Ying; LI Rong

    2004-01-01

    Bovine fetal oviduct epithelial cells were transfected with constructed double marker selective vector (pCE-EGFP-IRES-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation, and a transgenic cell line was obtained. Somatic cell nuclear transfer (SCNT) was carried out using the transgenic cells as nuclei donor. A total of 424 SCNT embryos were reconstructed and 208 (49.1%) of them developed to blastocyst stage. 17 blastocysts on D 7 after reconstruction were transferred to 17 surrogate calves, and 5 (29.4%) recipients were found to be pregnant. Three of them maintained to term and delivered three cloned calves. PCR and Southern blot analysis confirmed the integration of transgene in all of the three cloned calves. In addition, expression of EGFP was detected in biopsy isolated from the transgenic cloned calves and fibroblasts derived from the biopsy. Our results suggest that transgenic calves could be efficiently produced by SCNT using transgenic cells as nuclei donor. Furthermore, all cloned animals could be ensured to be transgenic by efficiently pre-screening transgenic cells and SCNT embryos using the constructed double marker selective vector.

  5. Nuclear removal during terminal lens fiber cell differentiation requires CDK1 activity: appropriating mitosis-related nuclear disassembly.

    Science.gov (United States)

    Chaffee, Blake R; Shang, Fu; Chang, Min-Lee; Clement, Tracy M; Eddy, Edward M; Wagner, Brad D; Nakahara, Masaki; Nagata, Shigekazu; Robinson, Michael L; Taylor, Allen

    2014-09-01

    Lens epithelial cells and early lens fiber cells contain the typical complement of intracellular organelles. However, as lens fiber cells mature they must destroy their organelles, including nuclei, in a process that has remained enigmatic for over a century, but which is crucial for the formation of the organelle-free zone in the center of the lens that assures clarity and function to transmit light. Nuclear degradation in lens fiber cells requires the nuclease DNase IIβ (DLAD) but the mechanism by which DLAD gains access to nuclear DNA remains unknown. In eukaryotic cells, cyclin-dependent kinase 1 (CDK1), in combination with either activator cyclins A or B, stimulates mitotic entry, in part, by phosphorylating the nuclear lamin proteins leading to the disassembly of the nuclear lamina and subsequent nuclear envelope breakdown. Although most post-mitotic cells lack CDK1 and cyclins, lens fiber cells maintain these proteins. Here, we show that loss of CDK1 from the lens inhibited the phosphorylation of nuclear lamins A and C, prevented the entry of DLAD into the nucleus, and resulted in abnormal retention of nuclei. In the presence of CDK1, a single focus of the phosphonuclear mitotic apparatus is observed, but it is not focused in CDK1-deficient lenses. CDK1 deficiency inhibited mitosis, but did not prevent DNA replication, resulting in an overall reduction of lens epithelial cells, with the remaining cells possessing an abnormally large nucleus. These observations suggest that CDK1-dependent phosphorylations required for the initiation of nuclear membrane disassembly during mitosis are adapted for removal of nuclei during fiber cell differentiation.

  6. Characterization of Aes nuclear foci in colorectal cancer cells.

    Science.gov (United States)

    Itatani, Yoshiro; Sonoshita, Masahiro; Kakizaki, Fumihiko; Okawa, Katsuya; Stifani, Stefano; Itoh, Hideaki; Sakai, Yoshiharu; Taketo, M Mark

    2016-01-01

    Amino-terminal enhancer of split (Aes) is a member of Groucho/Transducin-like enhancer (TLE) family. Aes is a recently found metastasis suppressor of colorectal cancer (CRC) that inhibits Notch signalling, and forms nuclear foci together with TLE1. Although some Notch-associated proteins are known to form subnuclear bodies, little is known regarding the dynamics or functions of these structures. Here, we show that Aes nuclear foci in CRC observed under an electron microscope are in a rather amorphous structure, lacking surrounding membrane. Investigation of their behaviour during the cell cycle by time-lapse cinematography showed that Aes nuclear foci dissolve during mitosis and reassemble after completion of cytokinesis. We have also found that heat shock cognate 70 (HSC70) is an essential component of Aes foci. Pharmacological inhibition of the HSC70 ATPase activity with VER155008 reduces Aes focus formation. These results provide insight into the understanding of Aes-mediated inhibition of Notch signalling. PMID:26229111

  7. Reprogramming of Fibroblasts From Older Women With Pelvic Floor Disorders Alters Cellular Behavior Associated With Donor Age

    OpenAIRE

    Wen, Yan; Wani, Prachi; Zhou, Lu; Baer, Tom; Phadnis, Smruti Madan; Reijo Pera, Renee A.; Chen, Bertha

    2013-01-01

    The effect of donor age on induced pluripotent stem cell (iPSC) lines and on the cells redifferentiated from these iPSCs was examined. iPSCs were derived from vaginal fibroblasts from women with pelvic organ prolapse. Donor age did not appear to affect reprogramming and cell mitotic activity in fibroblasts redifferentiated from iPSCs, and donor age differences were not observed in the iPSCs using standard senescence markers.

  8. Tobacco mosaic virus-directed reprogramming of auxin/indole acetic acid protein transcriptional responses enhances virus phloem loading.

    Science.gov (United States)

    Collum, Tamara D; Padmanabhan, Meenu S; Hsieh, Yi-Cheng; Culver, James N

    2016-05-10

    Vascular phloem loading has long been recognized as an essential step in the establishment of a systemic virus infection. In this study, an interaction between the replication protein of tobacco mosaic virus (TMV) and phloem-specific auxin/indole acetic acid (Aux/IAA) transcriptional regulators was found to modulate virus phloem loading in an age-dependent manner. Promoter expression studies show that in mature tissues TMV 126/183-kDa-interacting Aux/IAAs predominantly express and accumulate within the nuclei of phloem companion cells (CCs). Furthermore, CC Aux/IAA nuclear localization is disrupted upon infection with an interacting virus. In situ analysis of virus spread shows that the inability to disrupt Aux/IAA CC nuclear localization correlates with a reduced ability to load into the vascular tissue. Subsequent systemic movement assays also demonstrate that a virus capable of disrupting Aux/IAA localization is significantly more competitive at moving out of older plant tissues than a noninteracting virus. Similarly, CC expression and overaccumulation of a degradation-resistant Aux/IAA-interacting protein was found to inhibit TMV accumulation and phloem loading selectively in flowering plants. Transcriptional expression studies demonstrate a role for Aux/IAA-interacting proteins in the regulation of salicylic and jasmonic acid host defense responses as well as virus-specific movement factors, including pectin methylesterase, that are involved in regulating plasmodesmata size-exclusion limits and promoting virus cell-to-cell movement. Combined, these findings indicate that TMV directs the reprogramming of auxin-regulated gene expression within the vascular phloem of mature tissues as a means to enhance phloem loading and systemic spread. PMID:27118842

  9. Differential Reprogramming Based on Constructive Interference for Wireless Sensor Network

    OpenAIRE

    Hu, Bing

    2016-01-01

    To improve the performance of reprogramming in wireless sensor network, we present a novel reprogramming structure and constructive interference-based dissemination protocol (CIDP) to transmit the patch through out the network fast and reliability. CIDP disseminates the patch, which is divided into several packets, to the network exploiting constructive interference. We evaluate our implementation of CIDP using simulation under different number of nodes. Our results show that CIDP disseminate...

  10. Preparation of materials for flexizyme reactions and genetic code reprogramming

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Yuki Goto, Takayuki Katoh & Hiroaki Suga ### Abstract Genetic code reprogramming is a method for the reassignment of arbitrary codons from proteinogenic amino acids to non-proteinogenic ones, and thus specific sequences of non-standard peptides can be ribosomally expressed according to their mRNA templates. We have developed a protocol that facilitates the genetic code reprogramming using flexizymes integrated with a custom-made in-vitro translation apparatus, referred to...

  11. A protocol for embryonic stem cell derivation by somatic cell nuclear transfer into human oocytes

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Dieter Egli & Gloryn Chia ### Abstract Here we describe detailed methods that allowed us to derive embryonic stem cell lines by nuclear transfer of fibroblasts from a newborn and from a type 1 diabetic adult. The protocol is based on the insight that 1) agents for cell fusion can act as potent mediators of oocyte activation by compromising maintaining plasma membrane integrity; minimizing the concentration at which they are used, and at least transiently remove calcium f...

  12. Chromatin remodeling system, cancer stem-like attractors, and cellular reprogramming.

    Science.gov (United States)

    Zhang, Yue; Moriguchi, Hisashi

    2011-11-01

    The cancer cell attractors theory provides a next-generation understanding of carcinogenesis and natural explanation of punctuated clonal expansions of tumor progression. The impressive notion of atavism of cancer is now updated but more evidence is awaited. Besides, the mechanisms that the ectopic expression of some germline genes result in somatic tumors such as melanoma and brain tumors are emerging but are not well understood. Cancer could be triggered by cells undergoing abnormal cell attractor transitions, and may be reversible with "cyto-education". From mammals to model organisms like Caenorhabditis elegans and Drosophila melanogaster, the versatile Mi-2β/nucleosome remodeling and histone deacetylation complexes along with their functionally related chromatin remodeling complexes (CRCs), i.e., the dREAM/Myb-MuvB complex and Polycomb group complex are likely master regulators of cell attractors. The trajectory that benign cells switch to cancerous could be the reverse of navigation of embryonic cells converging from a series of intermediate transcriptional states to a final adult state, which is supported by gene expression dynamics inspector assays and some cross-species genetic evidence. The involvement of CRCs in locking cancer attractors may help find the recipes of perturbing genes to achieve successful reprogramming such that the reprogrammed cancer cell function in the same way as the normal cells. PMID:21909785

  13. Development of buffalo (Bubalus bubalis embryonic stem cell lines from somatic cell nuclear transferred blastocysts

    Directory of Open Access Journals (Sweden)

    Syed Mohmad Shah

    2015-11-01

    Full Text Available We developed buffalo embryonic stem cell lines from somatic cell nuclear transfer derived blastocysts, produced by hand-guided cloning technique. The inner cell mass of the blastocyst was cut mechanically using a Microblade and cultured onto feeder cells in buffalo embryonic stem (ES cell culture medium at 38 °C in a 5% CO2 incubator. The stem cell colonies were characterized for alkaline phosphatase activity, karyotype, pluripotency and self-renewal markers like OCT4, NANOG, SOX2, c-Myc, FOXD3, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 and CD90. The cell lines also possessed the capability to differentiate across all the three germ layers under spontaneous differentiation conditions.

  14. 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.

  15. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells.

    Science.gov (United States)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes.

  16. 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...... 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...

  17. Equating salivary lactate dehydrogenase (LDH) with LDH-5 expression in patients with oral squamous cell carcinoma: An insight into metabolic reprogramming of cancer cell as a predictor of aggressive phenotype.

    Science.gov (United States)

    Saluja, Tajindra Singh; Spadigam, Anita; Dhupar, Anita; Syed, Shaheen

    2016-04-01

    Oral squamous cell carcinoma (OSCC) is the sixth most common human malignancy. According to World Health Organization, oral cancer has been reported to have the highest morbidity and mortality and a survival rate of approximately 50 % at 5 years from diagnosis. This is attributed to the subjectivity in TNM staging and histological grading which may result in less than optimum treatment outcomes including tumour recurrence. One of the hallmarks of cancer is aerobic glycolysis also known as the Warburg effect. This glycolytic phenotype (hypoxic state) not only confers immortality to cancer cells, but also correlates with the belligerent behaviour of various malignancies and is reflected as an increase in the expression of lactate dehydrogenase 5 (LDH-5), the main isoform of LDH catalysing the conversion of pyruvate to lactate during glycolysis. The diagnostic role of salivary LDH in assessing the metabolic phenotype of oral cancer has not been studied. Since salivary LDH is mainly sourced from oral epithelial cells, any pathological changes in the epithelium should reflect diagnostically in saliva. Thus in our current research, we made an attempt to ascertain the biological behaviour and aggressiveness of OSCC by appraising its metabolic phenotype as indirectly reflected in salivary LDH activity. We found that salivary LDH can be used to assess the aggressiveness of different histological grades of OSCC. For the first time, an evidence of differing metabolic behaviour in similar histologic tumour grade is presented. Taken together, our study examines the inclusion of salivary LDH as potential diagnostic parameter and therapeutic index in OSCC. PMID:26577856

  18. Homocysteine-induced apoptosis in endothelial cells coincides with nuclear NOX2 and peri-nuclear NOX4 activity.

    Science.gov (United States)

    Sipkens, Jessica A; Hahn, Nynke; van den Brand, Carlien S; Meischl, Christof; Cillessen, Saskia A G M; Smith, Desirée E C; Juffermans, Lynda J M; Musters, René J P; Roos, Dirk; Jakobs, Cornelis; Blom, Henk J; Smulders, Yvo M; Krijnen, Paul A J; Stehouwer, Coen D A; Rauwerda, Jan A; van Hinsbergh, Victor W M; Niessen, Hans W M

    2013-11-01

    Apoptosis of endothelial cells related to homocysteine (Hcy) has been reported in several studies. In this study, we evaluated whether reactive oxygen species (ROS)-producing signaling pathways contribute to Hcy-induced apoptosis induction, with specific emphasis on NADPH oxidases. Human umbilical vein endothelial cells were incubated with 0.01-2.5 mM Hcy. We determined the effect of Hcy on caspase-3 activity, annexin V positivity, intracellular NOX1, NOX2, NOX4, and p47(phox) expression and localization, nuclear nitrotyrosine accumulation, and mitochondrial membrane potential (ΔΨ m). Hcy induced caspase-3 activity and apoptosis; this effect was concentration dependent and maximal after 6-h exposure to 2.5 mM Hcy. It was accompanied by a significant increase in ΔΨ m. Cysteine was inactive on these parameters excluding a reactive thiol group effect. Hcy induced an increase in cellular NOX2, p47(phox), and NOX4, but not that of NOX1. 3D digital imaging microscopy followed by image deconvolution analysis showed nuclear accumulation of NOX2 and p47(phox) in endothelial cells exposed to Hcy, but not in control cells, which coincided with accumulation of nuclear nitrotyrosine residues. Furthermore, Hcy enhanced peri-nuclear localization of NOX4 coinciding with accumulation of peri-nuclear nitrotyrosine residues, a reflection of local ROS production. p47(phox) was also increased in the peri-nuclear region. The Hcy-induced increase in caspase-3 activity was prevented by DPI and apocynin, suggesting involvement of NOX activity. The data presented in this article reveal accumulation of nuclear NOX2 and peri-nuclear NOX4 accumulation as potential source of ROS production in Hcy-induced apoptosis in endothelial cells.

  19. XPO1 Inhibition Preferentially Disrupts the 3D Nuclear Organization of Telomeres in Tumor Cells.

    Science.gov (United States)

    Taylor-Kashton, Cheryl; Lichtensztejn, Daniel; Baloglu, Erkan; Senapedis, William; Shacham, Sharon; Kauffman, Michael G; Kotb, Rami; Mai, Sabine

    2016-12-01

    Previous work has shown that the three-dimensional (3D) nuclear organization of telomeres is altered in cancer cells and the degree of alterations coincides with aggressiveness of disease. Nuclear pores are essential for spatial genome organization and gene regulation and XPO1 (exportin 1/CRM1) is the key nuclear export protein. The Selective Inhibitor of Nuclear Export (SINE) compounds developed by Karyopharm Therapeutics (KPT-185, KPT-330/selinexor, and KPT-8602) inhibit XPO1 nuclear export function. In this study, we investigated whether XPO1 inhibition has downstream effects on the 3D nuclear organization of the genome. This was assessed by measuring the 3D telomeric architecture of normal and tumor cells in vitro and ex vivo. Our data demonstrate for the first time a rapid and preferential disruption of the 3D nuclear organization of telomeres in tumor cell lines and in primary cells ex vivo derived from treatment-naïve newly diagnosed multiple myeloma patients. Normal primary cells in culture as well as healthy lymphocyte control cells from the same patients were minimally affected. Using both lymphoid and non-lymphoid tumor cell lines, we found that the downstream effects on the 3D nuclear telomere structure are independent of tumor type. We conclude that the 3D nuclear organization of telomeres is a sensitive indicator of cellular response when treated with XPO1 inhibitors. J. Cell. Physiol. 231: 2711-2719, 2016. © 2016 Wiley Periodicals, Inc. PMID:26991404

  20. Frequent somatic transfer of mitochondrial DNA into the nuclear genome of human cancer cells

    NARCIS (Netherlands)

    Y.S. Ju (Young Seok); J.M.C. Tubio (Jose M.); W. Mifsud (William); B. Fu (Beiyuan); H. Davies (Helen); M. Ramakrishna (Manasa); Y. Li (Yilong); L.R. Yates (Lucy); G. Gundem (Gunes); P.S. Tarpey (Patrick); S. Behjati (Sam); E. Papaemmanuil (Elli); S. Martin; A. Fullam (Anthony); M. Gerstung (Moritz); J. Nangalia (Jyoti); A.R. Green (Anthony R.); C. Caldas (Carlos); Å. Borg (Åke); A. Tutt (Andrew); M.T. Michael Lee (M