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Sample records for adult pluripotent stem

  1. Pluripotent embryonic stem cells and multipotent adult germline stem cells reveal similar transcriptomes including pluripotency-related genes.

    PubMed

    Meyer, S; Nolte, J; Opitz, L; Salinas-Riester, G; Engel, W

    2010-11-01

    DNA microarray analysis was performed with mouse multipotent adult germline stem cells (maGSCs) and embryonic stem cells (ESCs) from different genetic backgrounds cultured under standard ESC-culture conditions and under differentiation-promoting conditions by the withdrawal of the leukemia inhibitory factor (LIF) and treatment with retinoic acid (RA). The analyzed undifferentiated cell lines are very similar based on their global gene expression pattern and show 97-99% identity dependent on the analyzed background. Only 621 genes are differentially expressed in cells derived from mouse 129SV-background and 72 genes show differences in expression in cells generated from transgenic Stra8-EGFP/Rosa26-LacZ-background. Both maGSCs and ESCs express the same genes involved in the regulation of pluripotency and even show no differences in the expression level of these genes. When comparing maGSCs with previously published signature genes of other pluripotent cell lines, we found that maGSCs shared a very similar gene expression pattern with embryonic germ cells (EGCs). Also after differentiation of maGSCs and ESCs the transcriptomes of the cell lines are nearly identical which suggests that both cell types differentiate spontaneously in a very similar way. This is the first study, at transcriptome level, to compare ESCs and a pluripotent cell line derived from an adult organism (maGSCs). PMID:20624824

  2. Multipotent (adult) and pluripotent stem cells for heart regeneration: what are the pros and cons?

    PubMed

    Liao, Song-Yan; Tse, Hung-Fat

    2013-01-01

    Heart failure after myocardial infarction is the leading cause of mortality and morbidity worldwide. Existing medical and interventional therapies can only reduce the loss of cardiomyocytes during myocardial infarction but are unable to replenish the permanent loss of cardiomyocytes after the insult, which contributes to progressive pathological left ventricular remodeling and progressive heart failure. As a result, cell-based therapies using multipotent (adult) stem cells and pluripotent stem cells (embryonic stem cells or induced pluripotent stem cells) have been explored as potential therapeutic approaches to restore cardiac function in heart failure. Nevertheless, the optimal cell type with the best therapeutic efficacy and safety for heart regeneration is still unknown. In this review, the potential pros and cons of different types of multipotent (adult) stem cells and pluripotent stem cells that have been investigated in preclinical and clinical studies are reviewed, and the future perspective of stem cell-based therapy for heart regeneration is discussed. PMID:24476362

  3. A mystery unraveled: nontumorigenic pluripotent stem cells in human adult tissues

    PubMed Central

    Simerman, Ariel A; Perone, Marcelo J; Gimeno, María L; Dumesic, Daniel A; Chazenbalk, Gregorio D

    2014-01-01

    Introduction: Embryonic stem cells and induced pluripotent stem cells have emerged as the gold standard of pluripotent stem cells and the class of stem cell with the highest potential for contribution to regenerative and therapeutic application; however, their translational use is often impeded by teratoma formation, commonly associated with pluripotency. We discuss a population of nontumorigenic pluripotent stem cells, termed Multilineage Differentiating Stress Enduring (Muse) cells, which offer an innovative and exciting avenue of exploration for the potential treatment of various human diseases. Areas covered: This review discusses the origin of Muse cells, describes in detail their various unique characteristics, and considers future avenues of their application and investigation with respect to what is currently known of adult pluripotent stem cells in scientific literature. We begin by defining cell potency, then discuss both mesenchymal and various reported populations of pluripotent stem cells, and finally delve into Muse cells and the characteristics that set them apart from their contemporaries. Expert opinion: Muse cells derived from adipose tissue (Muse-AT) are efficiently, routinely and painlessly isolated from human lipoaspirate material, exhibit tripoblastic differentiation both spontaneously and under media-specific induction, and do not form teratomas. We describe qualities specific to Muse-AT cells and their potential impact on the field of regenerative medicine and cell therapy. PMID:24745973

  4. Planarian MBD2/3 is required for adult stem cell pluripotency independently of DNA methylation☆

    PubMed Central

    Jaber-Hijazi, Farah; Lo, Priscilla J.K.P.; Mihaylova, Yuliana; Foster, Jeremy M.; Benner, Jack S.; Tejada Romero, Belen; Chen, Chen; Malla, Sunir; Solana, Jordi; Ruzov, Alexey; Aziz Aboobaker, A.

    2013-01-01

    Planarian adult stem cells (pASCs) or neoblasts represent an ideal system to study the evolution of stem cells and pluripotency as they underpin an unrivaled capacity for regeneration. We wish to understand the control of differentiation and pluripotency in pASCs and to understand how conserved, convergent or divergent these mechanisms are across the Bilateria. Here we show the planarian methyl-CpG Binding Domain 2/3 (mbd2/3) gene is required for pASC differentiation during regeneration and tissue homeostasis. The genome does not have detectable levels of 5-methylcytosine (5mC) and we find no role for a potential DNA methylase. We conclude that MBD proteins may have had an ancient role in broadly controlling animal stem cell pluripotency, but that DNA methylation is not involved in planarian stem cell differentiation. PMID:24063805

  5. Pluripotency of adult stem cells derived from human and rat pancreas

    NASA Astrophysics Data System (ADS)

    Kruse, C.; Birth, M.; Rohwedel, J.; Assmuth, K.; Goepel, A.; Wedel, T.

    Adult stem cells are undifferentiated cells found within fully developed tissues or organs of an adult individuum. Until recently, these cells have been considered to bear less self-renewal ability and differentiation potency compared to embryonic stem cells. In recent studies an undifferentiated cell type was found in primary cultures of isolated acini from exocrine pancreas termed pancreatic stellate cells. Here we show that pancreatic stellate-like cells have the capacity of extended self-renewal and are able to differentiate spontaneously into cell types of all three germ layers expressing markers for smooth muscle cells, neurons, glial cells, epithelial cells, chondrocytes and secretory cells (insulin, amylase). Differentiation and subsequent formation of three-dimensional cellular aggregates (organoid bodies) were induced by merely culturing pancreatic stellate-like cells in hanging drops. These cells were developed into stable, long-term, in vitro cultures of both primary undifferentiated cell lines as well as organoid cultures. Thus, evidence is given that cell lineages of endodermal, mesodermal, and ectodermal origin arise spontaneously from a single adult undifferentiated cell type. Based on the present findings it is assumed that pancreatic stellate-like cells are a new class of lineage uncommitted pluripotent adult stem cells with a remarkable self-renewal ability and differentiation potency. The data emphasize the versatility of adult stem cells and may lead to a reappraisal of their use for the treatment of inherited disorders or acquired degenerative diseases.

  6. Potential for a pluripotent adult stem cell treatment for acute radiation sickness

    PubMed Central

    Rodgerson, Denis O; Reidenberg, Bruce E; Harris, Alan G; Pecora, Andrew L

    2012-01-01

    Accidental radiation exposure and the threat of deliberate radiation exposure have been in the news and are a public health concern. Experience with acute radiation sickness has been gathered from atomic blast survivors of Hiroshima and Nagasaki and from civilian nuclear accidents as well as experience gained during the development of radiation therapy for cancer. This paper reviews the medical treatment reports relevant to acute radiation sickness among the survivors of atomic weapons at Hiroshima and Nagasaki, among the victims of Chernobyl, and the two cases described so far from the Fukushima Dai-Ichi disaster. The data supporting the use of hematopoietic stem cell transplantation and the new efforts to expand stem cell populations ex vivo for infusion to treat bone marrow failure are reviewed. Hematopoietic stem cells derived from bone marrow or blood have a broad ability to repair and replace radiation induced damaged blood and immune cell production and may promote blood vessel formation and tissue repair. Additionally, a constituent of bone marrow-derived, adult pluripotent stem cells, very small embryonic like stem cells, are highly resistant to ionizing radiation and appear capable of regenerating radiation damaged tissue including skin, gut and lung. PMID:24520532

  7. DNA Damage Response in Neonatal and Adult Stromal Cells Compared With Induced Pluripotent Stem Cells

    PubMed Central

    Liedtke, Stefanie; Biebernick, Sophie; Radke, Teja Falk; Stapelkamp, Daniela; Coenen, Carolin; Zaehres, Holm; Fritz, Gerhard; Kogler, Gesine

    2015-01-01

    Comprehensive analyses comparing individual DNA damage response (DDR) of induced pluripotent stem cells (iPSCs) with neonatal stromal cells with respect to their developmental age are limited. The imperative necessity of providing developmental age-matched cell sources for meaningful toxicological drug safety assessments in replacement of animal-based testing strategies is evident. Here, DDR after radiation or treatment with N-methyl-N-nitrosurea (MNU) was determined in iPSCs compared with neonatal and bone marrow stromal cells. Neonatal and adult stromal cells showed no significant morphologically detectable cytotoxicity following treatment with 1 Gy or 1 mM MNU, whereas iPSCs revealed a much higher sensitivity. Foci analyses revealed an effective DNA repair in stromal cell types and iPSCs, as reflected by a rapid formation and disappearance of phosphorylated ATM and γH2AX foci. Furthermore, quantitative polymerase chain reaction analyses revealed the highest basic expression level of DDR and repair-associated genes in iPSCs, followed by neonatal stromal cells and adult stromal cells with the lowest expression levels. In addition, the influence of genotoxic stress prior to and during osteogenic differentiation of neonatal and adult stromal cells was analyzed applying common differentiation procedures. Experiments presented here suggest a developmental age-dependent basic expression level of genes involved in the processing of DNA damage. In addition a differentiation-dependent downregulation of repair genes was observed during osteogenesis. These results strongly support the requirement to provide adequate cell sources for toxicological in vitro drug testing strategies that match to the developmental age and differentiation status of the presumptive target cell of interest. Significance The results obtained in this study advance the understanding of DNA damage processing in human neonatal stromal cells as compared with adult stromal cells and induced pluripotent

  8. Genetic regulators of a pluripotent adult stem cell system in planarians identified by RNAi and clonal analysis

    PubMed Central

    Wagner, Daniel E.; Ho, Jaclyn J.

    2012-01-01

    Summary Pluripotency is a central, well-studied feature of embryonic development, but the role of pluripotent cell regulation in somatic tissue regeneration remains poorly understood. In planarians, regeneration of entire animals from tissue fragments is promoted by the activity of adult pluripotent stem cells (cNeoblasts). We utilized transcriptional profiling to identify planarian genes expressed in adult proliferating, regenerative cells (neoblasts). We also developed quantitative clonal analysis methods for expansion and differentiation of cNeoblast descendants that, together with RNAi, revealed gene roles in stem cell biology. Genes encoding two zinc finger proteins, Vasa, a LIM domain protein, Sox and Jun-like transcription factors, two candidate RNA-binding proteins, a Setd8-like protein, and PRC2 (Polycomb) were required for proliferative expansion and/or differentiation of cNeoblast-derived clones. These findings suggest that planarian stem cells utilize molecular mechanisms found in germ cells and other pluripotent cell types, and identify novel genetic regulators of the planarian stem cell system. PMID:22385657

  9. Heterogeneity of chromatoid bodies in adult pluripotent stem cells of planarian Dugesia japonica.

    PubMed

    Kashima, Makoto; Kumagai, Nobuyoshi; Agata, Kiyokazu; Shibata, Norito

    2016-02-01

    The robust regenerative ability of planarians is known to be dependent on adult pluripotent stem cells called neoblasts. One of the morphological features of neoblasts is cytoplasmic ribonucleoprotein granules (chromatoid bodies: CBs), which resemble germ granules present in germline cells in other animals. Previously, we showed by immuno-electron microscopic analysis that DjCBC-1, a planarian Me31B/Dhh1/DDX6 homologue, which is a component of ribonucleoprotein granules, was localized in CBs in the planarian Dugesia japonica. Also, recently it was reported using another planarian species that Y12 antibody recognizing symmetrical dimethylarginine (sDMA) specifically binds to CBs in which histone mRNA is co-localized. Here, we showed by double immunostaining and RNA interference (RNAi) that DjCBC-1-containing CBs and Y12-immunoreactive CBs are distinct structures, suggesting that CBs are composed of heterogeneous populations. We also found that the Y12-immunoreactive CBs specifically contained a cytoplasmic type of planarian PIWI protein (DjPiwiC). We revealed by RNAi experiments that Y12-immunoreactive CBs may have anti-transposable element activity involving the DjPiwiC protein in the neoblasts. PMID:26857694

  10. Three-Dimensional Adult Cardiac Extracellular Matrix Promotes Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

    PubMed

    Fong, Ashley H; Romero-López, Mónica; Heylman, Christopher M; Keating, Mark; Tran, David; Sobrino, Agua; Tran, Anh Q; Pham, Hiep H; Fimbres, Cristhian; Gershon, Paul D; Botvinick, Elliot L; George, Steven C; Hughes, Christopher C W

    2016-08-01

    Pluripotent stem cell-derived cardiomyocytes (CMs) have great potential in the development of new therapies for cardiovascular disease. In particular, human induced pluripotent stem cells (iPSCs) may prove especially advantageous due to their pluripotency, their self-renewal potential, and their ability to create patient-specific cell lines. Unfortunately, pluripotent stem cell-derived CMs are immature, with characteristics more closely resembling fetal CMs than adult CMs, and this immaturity has limited their use in drug screening and cell-based therapies. Extracellular matrix (ECM) influences cellular behavior and maturation, as does the geometry of the environment-two-dimensional (2D) versus three-dimensional (3D). We therefore tested the hypothesis that native cardiac ECM and 3D cultures might enhance the maturation of iPSC-derived CMs in vitro. We demonstrate that maturation of iPSC-derived CMs was enhanced when cells were seeded into a 3D cardiac ECM scaffold, compared with 2D culture. 3D cardiac ECM promoted increased expression of calcium-handling genes, Junctin, CaV1.2, NCX1, HCN4, SERCA2a, Triadin, and CASQ2. Consistent with this, we find that iPSC-derived CMs in 3D adult cardiac ECM show increased calcium signaling (amplitude) and kinetics (maximum upstroke and downstroke) compared with cells in 2D. Cells in 3D culture were also more responsive to caffeine, likely reflecting an increased availability of calcium in the sarcoplasmic reticulum. Taken together, these studies provide novel strategies for maturing iPSC-derived CMs that may have applications in drug screening and transplantation therapies to treat heart disease. PMID:27392582

  11. Isolation of adult human pluripotent stem cells from mesenchymal cell populations and their application to liver damages.

    PubMed

    Wakao, Shohei; Kitada, Masaaki; Kuroda, Yasumasa; Dezawa, Mari

    2012-01-01

    We have found a novel type of pluripotent stem cells, Multilineage-differentiating stress enduring (Muse) cells that can be isolated from mesenchymal cell populations. Muse cells are characterized by stress tolerance, expression of pluripotency markers, self-renewal, and the ability to differentiate into endodermal-, mesodermal-, and ectodermal-lineage cells from a single cell, demonstrating that they are pluripotent stem cells. They can be isolated as cells positive for stage-specific embryonic antigen-3, a human pluripotent stem cell marker. Here, we introduce the isolation method for Muse cells and the effect of transplantation of these cells on chronic liver diseases. PMID:22167642

  12. Generation and Characterization of Leukemia Inhibitory Factor-Dependent Equine Induced Pluripotent Stem Cells from Adult Dermal Fibroblasts

    PubMed Central

    Ovchinnikov, Dmitry A.; Sun, Jane; Fortuna, Patrick R.J.; Wolvetang, Ernst J.

    2014-01-01

    In this study we have reprogrammed dermal fibroblasts from an adult female horse into equine induced pluripotent stem cells (equiPSCs). These equiPSCs are dependent only on leukemia inhibitory factor (LIF), placing them in striking contrast to previously derived equiPSCs that have been shown to be co-dependent on both LIF and basic fibroblast growth factor (bFGF). These equiPSCs have a normal karyotype and have been maintained beyond 60 passages. They possess alkaline phosphatase activity and express eqNANOG, eqOCT4, and eqTERT mRNA. Immunocytochemistry confirmed that they produce NANOG, REX1, SSEA4, TRA1-60, and TRA1-81. While our equiPSCs are LIF dependent, bFGF co-stimulates their proliferation via the PI3K/AKT pathway. EquiPSCs lack expression of eqXIST and immunostaining for H3K27me3, suggesting that during reprogramming the inactive X chromosome has likely been reactivated to generate cells that have two active X chromosomes. EquiPSCs form embryoid bodies and in vitro teratomas that contain derivatives of all three germ layers. These LIF-dependent equiPSCs likely reflect a more naive state of pluripotency than equiPSCs that are co-dependent on both LIF and bFGF and so provide a novel resource for understanding pluripotency in the horse. PMID:24555755

  13. Generation of Adult Human Induced Pluripotent Stem Cells Using Non-Viral Minicircle DNA Vectors

    PubMed Central

    Narsinh, Kazim H.; Jia, Fangjun; Robbins, Robert C.; Kay, Mark A.; Longaker, Michael T.; Wu, Joseph C.

    2013-01-01

    Human induced pluripotent stem cells (hiPSCs) derived from patient samples have tremendous potential for innovative approaches to disease pathology investigation and regenerative medicine therapies. However, most hiPSC derivation techniques utilize integrating viruses, which may leave residual transgene sequences as part of the host genome, thereby unpredictably altering cell phenotype in downstream applications. Here we describe a protocol for hiPSC derivation by transfection of a simple, nonviral minicircle DNA construct into human adipose stromal cells (hASCs). Minicircle DNA vectors are free of bacterial DNA and thereby capable of high expression in mammalian cells. Their repeated transfection into hASCs, an abundant somatic cell source that is amenable to efficient reprogramming, results in transgene-free hiPSCs. This protocol requires only readily available molecular biology reagents and expertise, and produces hiPSC colonies from an adipose tissue sample in ~4 weeks. PMID:21212777

  14. Expression of Genes Related to Germ Cell Lineage and Pluripotency in Single Cells and Colonies of Human Adult Germ Stem Cells.

    PubMed

    Conrad, Sabine; Azizi, Hossein; Hatami, Maryam; Kubista, Mikael; Bonin, Michael; Hennenlotter, Jörg; Sievert, Karl-Dietrich; Skutella, Thomas

    2016-01-01

    The aim of this study was to elucidate the molecular status of single human adult germ stem cells (haGSCs) and haGSC colonies, which spontaneously developed from the CD49f MACS and matrix- (collagen-/laminin+ binding-) selected fraction of enriched spermatogonia. Single-cell transcriptional profiling by Fluidigm BioMark system of a long-term cultured haGSCs cluster in comparison to human embryonic stem cells (hESCs) and human fibroblasts (hFibs) revealed that haGSCs showed a characteristic germ- and pluripotency-associated gene expression profile with some similarities to hESCs and with a significant distinction from somatic hFibs. Genome-wide comparisons with microarray analysis confirmed that different haGSC colonies exhibited gene expression heterogeneity with more or less pluripotency. The results of this study confirm that haGSCs are adult stem cells with a specific molecular gene expression profile in vitro, related but not identical to true pluripotent stem cells. Under ES-cell conditions haGSC colonies could be selected and maintained in a partial pluripotent state at the molecular level, which may be related to their cell plasticity and potential to differentiate into cells of all germ layers. PMID:26649052

  15. Expression of Genes Related to Germ Cell Lineage and Pluripotency in Single Cells and Colonies of Human Adult Germ Stem Cells

    PubMed Central

    Conrad, Sabine; Azizi, Hossein; Hatami, Maryam; Kubista, Mikael; Bonin, Michael; Hennenlotter, Jörg; Sievert, Karl-Dietrich; Skutella, Thomas

    2016-01-01

    The aim of this study was to elucidate the molecular status of single human adult germ stem cells (haGSCs) and haGSC colonies, which spontaneously developed from the CD49f MACS and matrix- (collagen−/laminin+ binding-) selected fraction of enriched spermatogonia. Single-cell transcriptional profiling by Fluidigm BioMark system of a long-term cultured haGSCs cluster in comparison to human embryonic stem cells (hESCs) and human fibroblasts (hFibs) revealed that haGSCs showed a characteristic germ- and pluripotency-associated gene expression profile with some similarities to hESCs and with a significant distinction from somatic hFibs. Genome-wide comparisons with microarray analysis confirmed that different haGSC colonies exhibited gene expression heterogeneity with more or less pluripotency. The results of this study confirm that haGSCs are adult stem cells with a specific molecular gene expression profile in vitro, related but not identical to true pluripotent stem cells. Under ES-cell conditions haGSC colonies could be selected and maintained in a partial pluripotent state at the molecular level, which may be related to their cell plasticity and potential to differentiate into cells of all germ layers. PMID:26649052

  16. Generation of functional endothelial-like cells from adult mouse germline-derived pluripotent stem cells.

    PubMed

    Kim, Julee; Eligehausen, Sarah; Stehling, Martin; Nikol, Sigrid; Ko, Kinarm; Waltenberger, Johannes; Klocke, Rainer

    2014-01-10

    Functional endothelial cells and their progenitors are required for vascular development, adequate vascular function, vascular repair and for cell-based therapies of ischemic diseases. Currently, cell therapy is limited by the low abundance of patient-derived cells and by the functional impairment of autologous endothelial progenitor cells (EPCs). In the present study, murine germline-derived pluripotent stem (gPS) cells were evaluated as a potential source for functional endothelial-like cells. Cells displaying an endothelial cell-like morphology were obtained from gPS cell-derived embryoid bodies using a combination of fluorescence-activated cell sorting (FACS)-based selection of CD31-positive cells and their subsequent cultivation on OP9 stromal cells in the presence of VEGF-A. Real-time reverse transcriptase polymerase chain reaction, FACS analysis and immunofluorescence staining showed that the gPS cell-derived endothelial-like cells (gPS-ECs) expressed endothelial cell-specific markers including von Willebrand Factor, Tie2, VEGFR2/Flk1, intercellular adhesion molecule 2 and vascular endothelial-cadherin. The high expression of ephrin B2, as compared to Eph B4 and VEGFR3, suggests an arterial rather than a venous or lymphatic differentiation. Their capability to take up Dil-conjugated acetylated low-density lipoprotein and to form capillary-like networks on matrigel confirmed their functionality. We conclude that gPS cells could be a novel source of endothelial cells potentially suitable for regenerative cell-based therapies for ischemic diseases. PMID:24333870

  17. Generation of leukemia inhibitory factor and basic fibroblast growth factor-dependent induced pluripotent stem cells from canine adult somatic cells.

    PubMed

    Luo, Jiesi; Suhr, Steven T; Chang, Eun Ah; Wang, Kai; Ross, Pablo J; Nelson, Laura L; Venta, Patrick J; Knott, Jason G; Cibelli, Jose B

    2011-10-01

    For more than thirty years, the dog has been used as a model for human diseases. Despite efforts made to develop canine embryonic stem cells, success has been elusive. Here, we report the generation of canine induced pluripotent stem cells (ciPSCs) from canine adult fibroblasts, which we accomplished by introducing human OCT4, SOX2, c-MYC, and KLF4. The ciPSCs expressed critical pluripotency markers and showed evidence of silencing the viral vectors and normal karyotypes. Microsatellite analysis indicated that the ciPSCs showed the same profile as the donor fibroblasts but differed from cells taken from other dogs. Under culture conditions favoring differentiation, the ciPSCs could form cell derivatives from the ectoderm, mesoderm, and endoderm. Further, the ciPSCs required leukemia inhibitory factor and basic fibroblast growth factor to survive, proliferate, and maintain pluripotency. Our results demonstrate an efficient method for deriving canine pluripotent stem cells, providing a powerful platform for the development of new models for regenerative medicine, as well as for the study of the onset, progression, and treatment of human and canine genetic diseases. PMID:21495906

  18. Generation of Leukemia Inhibitory Factor and Basic Fibroblast Growth Factor-Dependent Induced Pluripotent Stem Cells from Canine Adult Somatic Cells

    PubMed Central

    Luo, Jiesi; Suhr, Steven T.; Chang, Eun Ah; Wang, Kai; Ross, Pablo J.; Nelson, Laura L.; Venta, Patrick J.; Knott, Jason G.

    2011-01-01

    For more than thirty years, the dog has been used as a model for human diseases. Despite efforts made to develop canine embryonic stem cells, success has been elusive. Here, we report the generation of canine induced pluripotent stem cells (ciPSCs) from canine adult fibroblasts, which we accomplished by introducing human OCT4, SOX2, c-MYC, and KLF4. The ciPSCs expressed critical pluripotency markers and showed evidence of silencing the viral vectors and normal karyotypes. Microsatellite analysis indicated that the ciPSCs showed the same profile as the donor fibroblasts but differed from cells taken from other dogs. Under culture conditions favoring differentiation, the ciPSCs could form cell derivatives from the ectoderm, mesoderm, and endoderm. Further, the ciPSCs required leukemia inhibitory factor and basic fibroblast growth factor to survive, proliferate, and maintain pluripotency. Our results demonstrate an efficient method for deriving canine pluripotent stem cells, providing a powerful platform for the development of new models for regenerative medicine, as well as for the study of the onset, progression, and treatment of human and canine genetic diseases. PMID:21495906

  19. Gametogenesis from Pluripotent Stem Cells.

    PubMed

    Saitou, Mitinori; Miyauchi, Hidetaka

    2016-06-01

    The germ cell lineage originates early in development and undergoes a series of complex developmental processes that culminate in the generation of fully matured gametes, the spermatozoa and the oocytes. Remarkably, researchers have been recapitulating these developmental pathways using mouse and human pluripotent stem cells (PSCs). With further studies, including those involving non-human primate models, human gametogenesis may be fully reconstituted from PSCs, which would profoundly facilitate our understanding of human germ cell development and infertility. Here we discuss groundbreaking studies that lay the foundation for this achievement, the current state of the field, and challenges for deriving gametes from hPSCs. PMID:27257761

  20. Multipotent adult germ-line stem cells, like other pluripotent stem cells, can be killed by cytotoxic T lymphocytes despite low expression of major histocompatibility complex class I molecules

    PubMed Central

    Dressel, Ralf; Guan, Kaomei; Nolte, Jessica; Elsner, Leslie; Monecke, Sebastian; Nayernia, Karim; Hasenfuss, Gerd; Engel, Wolfgang

    2009-01-01

    Background Multipotent adult germ-line stem cells (maGSCs) represent a new pluripotent cell type that can be derived without genetic manipulation from spermatogonial stem cells (SSCs) present in adult testis. Similarly to induced pluripotent stem cells (iPSCs), they could provide a source of cellular grafts for new transplantation therapies of a broad variety of diseases. To test whether these stem cells can be rejected by the recipients, we have analyzed whether maGSCs and iPSCs can become targets for cytotoxic T lymphocytes (CTL) or whether they are protected, as previously proposed for embryonic stem cells (ESCs). Results We have observed that maGSCs can be maintained in prolonged culture with or without leukemia inhibitory factor and/or feeder cells and still retain the capacity to form teratomas in immunodeficient recipients. They were, however, rejected in immunocompetent allogeneic recipients, and the immune response controlled teratoma growth. We analyzed the susceptibility of three maGSC lines to CTL in comparison to ESCs, iPSCs, and F9 teratocarcinoma cells. Major histocompatibility complex (MHC) class I molecules were not detectable by flow cytometry on these stem cell lines, apart from low levels on one maGSC line (maGSC Stra8 SSC5). However, using a quantitative real time PCR analysis H2K and B2m transcripts were detected in all pluripotent stem cell lines. All pluripotent stem cell lines were killed in a peptide-dependent manner by activated CTLs derived from T cell receptor transgenic OT-I mice after pulsing of the targets with the SIINFEKL peptide. Conclusion Pluripotent stem cells, including maGSCs, ESCs, and iPSCs can become targets for CTLs, even if the expression level of MHC class I molecules is below the detection limit of flow cytometry. Thus they are not protected against CTL-mediated cytotoxicity. Therefore, pluripotent cells might be rejected after transplantation by this mechanism if specific antigens are presented and if specific

  1. Reprogrammed pluripotent stem cells from somatic cells.

    PubMed

    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

  2. Pluripotent Stem Cells from Domesticated Mammals.

    PubMed

    Ezashi, Toshihiko; Yuan, Ye; Roberts, R Michael

    2016-01-01

    This review deals with the latest advances in the study of embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) from domesticated species, with a focus on pigs, cattle, sheep, goats, horses, cats, and dogs. Whereas the derivation of fully pluripotent ESC from these species has proved slow, reprogramming of somatic cells to iPSC has been more straightforward. However, most of these iPSC depend on the continued expression of the introduced transgenes, a major drawback to their utility. The persistent failure in generating ESC and the dependency of iPSC on ectopic genes probably stem from an inability to maintain the stability of the endogenous gene networks necessary to maintain pluripotency. Based on work in humans and rodents, achievement of full pluripotency will likely require fine adjustments in the growth factors and signaling inhibitors provided to the cells. Finally, we discuss the future utility of these cells for biomedical and agricultural purposes. PMID:26566158

  3. Pluripotent Stem Cells and Gene Therapy

    PubMed Central

    Simara, Pavel; Motl, Jason A.; Kaufman, Dan S.

    2013-01-01

    Human pluripotent stem cells represent an accessible cell source for novel cell-based clinical research and therapies. With the realization of induced pluripotent stem cells (iPSCs), it is possible to produce almost any desired cell type from any patient's cells. Current developments in gene modification methods have opened the possibility for creating genetically corrected human iPSCs for certain genetic diseases that could be used later in autologous transplantation. Promising preclinical studies have demonstrated correction of disease-causing mutations in a number of hematological, neuronal and muscular disorders. This review aims to summarize these recent advances with a focus on iPSC generation techniques, as well as gene modification methods. We will then further discuss some of the main obstacles remaining to be overcome before successful application of human pluripotent stem cell-based therapy arrives in the clinic and what the future of stem cell research may look like. PMID:23353080

  4. Differentiation of hepatocytes from pluripotent stem cells

    PubMed Central

    Mallanna, Sunil K.

    2014-01-01

    Differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells into hepatocyte-like cells provides a platform to study the molecular basis of human hepatocyte differentiation, to develop cell culture models of liver disease, and to potentially provide hepatocytes for treatment of end-stage liver disease. Additionally, hepatocyte-like cells generated from human pluripotent stem cells could serve as platforms for drug discovery, determination of pharmaceutical induced hepatotoxicity, and evaluation of idiosyncratic drug-drug interactions. Here, we describe a step-wise protocol previously developed in our laboratory that facilitates the highly efficient and reproducible differentiation of human pluripotent stem cells into hepatocyte-like cells. Our protocol uses defined culture conditions and closely recapitulates key developmental events that are found to occur during hepatogenesis. PMID:24510789

  5. Adult Stem and Progenitor Cells

    NASA Astrophysics Data System (ADS)

    Geraerts, Martine; Verfaillie, Catherine M.

    The discovery of adult stem cells in most adult tissues is the basis of a number of clinical studies that are carried out, with therapeutic use of hematopoietic stem cells as a prime example. Intense scientific debate is still ongoing as to whether adult stem cells may have a greater plasticity than previously thought. Although cells with some features of embryonic stem cells that, among others, express Oct4, Nanog and SSEA1 are isolated from fresh tissue, it is not clear if the greater differentiation potential is acquired during cell culture. Moreover, adult more pluripotent cells do not have all pluripotent characteristics typical for embryonic stem cells. Recently, some elegant studies were published in which adult cells could be completely reprogrammed to embryonic stem cell-like cells by overexpression of some key transcription factors for pluripotency (Oct4, Sox2, Klf4 and c-Myc). It will be interesting for the future to investigate the exact mechanisms underlying this reprogramming and whether similar transcription factor pathways are present and/or can be activated in adult more pluripotent stem cells.

  6. Intrinsic Ability of Adult Stem Cell in Skeletal Muscle: An Effective and Replenishable Resource to the Establishment of Pluripotent Stem Cells

    PubMed Central

    Fujimaki, Shin; Machida, Masanao; Hidaka, Ryo; Asashima, Makoto; Takemasa, Tohru; Kuwabara, Tomoko

    2013-01-01

    Adult stem cells play an essential role in mammalian organ maintenance and repair throughout adulthood since they ensure that organs retain their ability to regenerate. The choice of cell fate by adult stem cells for cellular proliferation, self-renewal, and differentiation into multiple lineages is critically important for the homeostasis and biological function of individual organs. Responses of stem cells to stress, injury, or environmental change are precisely regulated by intercellular and intracellular signaling networks, and these molecular events cooperatively define the ability of stem cell throughout life. Skeletal muscle tissue represents an abundant, accessible, and replenishable source of adult stem cells. Skeletal muscle contains myogenic satellite cells and muscle-derived stem cells that retain multipotent differentiation abilities. These stem cell populations have the capacity for long-term proliferation and high self-renewal. The molecular mechanisms associated with deficits in skeletal muscle and stem cell function have been extensively studied. Muscle-derived stem cells are an obvious, readily available cell resource that offers promise for cell-based therapy and various applications in the field of tissue engineering. This review describes the strategies commonly used to identify and functionally characterize adult stem cells, focusing especially on satellite cells, and discusses their potential applications. PMID:23818907

  7. [Generation and application of pluripotent stem cells from spermatogonial stem cells].

    PubMed

    Zhang, Yan; Wu, Yingji

    2011-02-01

    Recent studies have confirmed that diverse adult tissue cells can be reprogrammed and induced to pluripotency, that is so-called induced pluripotent stem cells (iPS cells). But most of these dedifferentiated processes are induced by gene delivery with retroviral vectors. Some of the delivered genes are cancer causing. So, in current situation, these adult-derived embryonic stem-like cells cannot be used in clinical therapy to cure human diseases. Recently some articles that were published in the authoritative journals are receiving attentions. They show that, in mice and human, spermatogonial stem cells (SSCs) can be used for generating pluripotent stem cells without the exogenous genes and retroviruses, and they can also be used for autologous transplantation without ethical problems. These findings suggest that human SSCs may have considerable potential for cell-based, autologous organ regeneration therapy for various diseases. In this review, we describe and compare the methods that have been used to isolate, purificate and culture SSCs. We also describe the recent results in which SSCs can be transformed into pluripotent stem cells, and the pluripotent stem cells have potential applications in regenerative medicine and genetic medicine. PMID:21485215

  8. A facile method to establish human induced pluripotent stem cells from adult blood cells under feeder-free and xeno-free culture conditions: a clinically compliant approach.

    PubMed

    Chou, Bin-Kuan; Gu, Haihui; Gao, Yongxing; Dowey, Sarah N; Wang, Ying; Shi, Jun; Li, Yanxin; Ye, Zhaohui; Cheng, Tao; Cheng, Linzhao

    2015-04-01

    Reprogramming human adult blood mononuclear cells (MNCs) cells by transient plasmid expression is becoming increasingly popular as an attractive method for generating induced pluripotent stem (iPS) cells without the genomic alteration caused by genome-inserting vectors. However, its efficiency is relatively low with adult MNCs compared with cord blood MNCs and other fetal cells and is highly variable among different adult individuals. We report highly efficient iPS cell derivation under clinically compliant conditions via three major improvements. First, we revised a combination of three EBNA1/OriP episomal vectors expressing five transgenes, which increased reprogramming efficiency by ≥10-50-fold from our previous vectors. Second, human recombinant vitronectin proteins were used as cell culture substrates, alleviating the need for feeder cells or animal-sourced proteins. Finally, we eliminated the previously critical step of manually picking individual iPS cell clones by pooling newly emerged iPS cell colonies. Pooled cultures were then purified based on the presence of the TRA-1-60 pluripotency surface antigen, resulting in the ability to rapidly expand iPS cells for subsequent applications. These new improvements permit a consistent and reliable method to generate human iPS cells with minimal clonal variations from blood MNCs, including previously difficult samples such as those from patients with paroxysmal nocturnal hemoglobinuria. In addition, this method of efficiently generating iPS cells under feeder-free and xeno-free conditions allows for the establishment of clinically compliant iPS cell lines for future therapeutic applications. PMID:25742692

  9. In vitro regeneration of kidney from pluripotent stem cells

    SciTech Connect

    Osafune, Kenji

    2010-10-01

    Although renal transplantation has proved a successful treatment for the patients with end-stage renal failure, the therapy is hampered by the problem of serious shortage of donor organs. Regenerative medicine using stem cells, including cell transplantation therapy, needs to be developed to solve the problem. We previously identified the multipotent progenitor cells in the embryonic mouse kidney that can give rise to several kinds of epithelial cells found in adult kidney, such as glomerular podocytes and renal tubular epithelia. Establishing the method to generate the progenitors from human pluripotent stem cells that have the capacity to indefinitely proliferate in vitro is required for the development of kidney regeneration strategy. We review the current status of the research on the differentiation of pluripotent stem cells into renal lineages and describe cues to promote this research field.

  10. Human induced pluripotent stem cell‐derived versus adult cardiomyocytes: an in silico electrophysiological study on effects of ionic current block

    PubMed Central

    Paci, M; Hyttinen, J; Rodriguez, B

    2015-01-01

    Background and Purpose Two new technologies are likely to revolutionize cardiac safety and drug development: in vitro experiments on human‐induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) and in silico human adult ventricular cardiomyocyte (hAdultV‐CM) models. Their combination was recently proposed as a potential replacement for the present hERG‐based QT study for pharmacological safety assessments. Here, we systematically compared in silico the effects of selective ionic current block on hiPSC‐CM and hAdultV‐CM action potentials (APs), to identify similarities/differences and to illustrate the potential of computational models as supportive tools for evaluating new in vitro technologies. Experimental Approach In silico AP models of ventricular‐like and atrial‐like hiPSC‐CMs and hAdultV‐CM were used to simulate the main effects of four degrees of block of the main cardiac transmembrane currents. Key Results Qualitatively, hiPSC‐CM and hAdultV‐CM APs showed similar responses to current block, consistent with results from experiments. However, quantitatively, hiPSC‐CMs were more sensitive to block of (i) L‐type Ca2+ currents due to the overexpression of the Na+/Ca2+ exchanger (leading to shorter APs) and (ii) the inward rectifier K+ current due to reduced repolarization reserve (inducing diastolic potential depolarization and repolarization failure). Conclusions and Implications In silico hiPSC‐CMs and hAdultV‐CMs exhibit a similar response to selective current blocks. However, overall hiPSC‐CMs show greater sensitivity to block, which may facilitate in vitro identification of drug‐induced effects. Extrapolation of drug effects from hiPSC‐CM to hAdultV‐CM and pro‐arrhythmic risk assessment can be facilitated by in silico predictions using biophysically‐based computational models. PMID:26276951

  11. Induced pluripotent stem cells: advances to applications

    PubMed Central

    Nelson, Timothy J; Martinez-Fernandez, Almudena; Yamada, Satsuki; Ikeda, Yasuhiro; Perez-Terzic, Carmen; Terzic, Andre

    2010-01-01

    Induced pluripotent stem cell (iPS) technology has enriched the armamentarium of regenerative medicine by introducing autologous pluripotent progenitor pools bioengineered from ordinary somatic tissue. Through nuclear reprogramming, patient-specific iPS cells have been derived and validated. Optimizing iPS-based methodology will ensure robust applications across discovery science, offering opportunities for the development of personalized diagnostics and targeted therapeutics. Here, we highlight the process of nuclear reprogramming of somatic tissues that, when forced to ectopically express stemness factors, are converted into bona fide pluripotent stem cells. Bioengineered stem cells acquire the genuine ability to generate replacement tissues for a wide-spectrum of diseased conditions, and have so far demonstrated therapeutic benefit upon transplantation in model systems of sickle cell anemia, Parkinson’s disease, hemophilia A, and ischemic heart disease. The field of regenerative medicine is therefore primed to adopt and incorporate iPS cell-based advancements as a next generation stem cell platforms. PMID:21165156

  12. Pluripotent Stem Cells: Current Understanding and Future Directions

    PubMed Central

    Romito, Antonio

    2016-01-01

    Pluripotent stem cells have the ability to undergo self-renewal and to give rise to all cells of the tissues of the body. However, this definition has been recently complicated by the existence of distinct cellular states that display these features. Here, we provide a detailed overview of the family of pluripotent cell lines derived from early mouse and human embryos and compare them with induced pluripotent stem cells. Shared and distinct features of these cells are reported as additional hallmark of pluripotency, offering a comprehensive scenario of pluripotent stem cells. PMID:26798367

  13. Alternative sources of pluripotency: science, ethics, and stem cells.

    PubMed

    Kastenberg, Zachary J; Odorico, Jon S

    2008-07-01

    Despite many advances in human embryonic stem cell (hESC) technology the ethical dilemma involving the destruction of a human embryo is one factor that has limited the development of hESC based clinical therapies. Two recent reports describing the production of pluripotent stem cells following the in vitro reprogramming of human somatic cells with certain defined factors illustrate one potential method of bypassing the ethical debate surrounding hESCs (Yu J, Vodyanik MA, Smuga-Otto K, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007 Dec;318(5858):1917-1920; Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007 Nov;131(5): 861-872.). Other alternative methods include nuclear transfer, altered nuclear transfer, and parthenogenesis; each with its own set of advantages and disadvantages. This review discusses recent advances in these technologies with specific focus on the issues of embryo destruction, oocyte recovery, and the potential of each technology to produce large scale, patient specific cell transplantation therapies that would require little or no immunosuppression. PMID:18631882

  14. Metaboloepigenetic Regulation of Pluripotent Stem Cells

    PubMed Central

    Harvey, Alexandra J.; Gardner, David K.

    2016-01-01

    The differentiation of pluripotent stem cells is associated with extensive changes in metabolism, as well as widespread remodeling of the epigenetic landscape. Epigenetic regulation is essential for the modulation of differentiation, being responsible for cell type specific gene expression patterns through the modification of DNA and histones, thereby establishing cell identity. Each cell type has its own idiosyncratic pattern regarding the use of specific metabolic pathways. Rather than simply being perceived as a means of generating ATP and building blocks for cell growth and division, cellular metabolism can directly influence cellular regulation and the epigenome. Consequently, the significance of nutrients and metabolites as regulators of differentiation is central to understanding how cells interact with their immediate environment. This review serves to integrate studies on pluripotent stem cell metabolism, and the regulation of DNA methylation and acetylation and identifies areas in which current knowledge is limited. PMID:26839556

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

    PubMed Central

    Dey, Dilip; Evans, Gregory R. D.

    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 of iPS with defined reprogramming factors has initiated a flurry of works on stem cell in various laboratories. The pluripotent cells can be derived from various differentiated adult cells as well as from adult stem cells by nuclear reprogramming, somatic cell nuclear transfer etc. In this review article, different aspects of nuclear reprogramming are discussed. PMID:22007240

  16. Culture environment-induced pluripotency of SACK-expanded tissue stem cells.

    PubMed

    Paré, Jean-François; Sherley, James L

    2011-01-01

    Previous efforts to improve the efficiency of cellular reprogramming for the generation of induced pluripotent stem cells (iPSCs) have focused mainly on transcription factors and small molecule combinations. Here, we report the results of our focus instead on the phenotype of the cells targeted for reprogramming. We find that adult mouse pancreatic tissue stem cells derived by the method of suppression of asymmetric cell kinetics (SACK) acquire increased potency simply by culture under conditions for the production and maintenance of pluripotent stem cells. Moreover, supplementation with the SACK agent xanthine, which promotes symmetric self-renewal, significantly increases the efficiency and degree of acquisition of pluripotency properties. In transplantation analyses, clonal reprogrammed pancreatic stem cells produce slow-growing tumors with tissue derivative of all three embryonic germ layers. This acquisition of pluripotency, without transduction with exogenous transcription factors, supports the concept that tissue stem cells are predisposed to cellular reprogramming, particularly when symmetrically self-renewing. PMID:22523467

  17. Induced Pluripotent Stem Cells Meet Genome Editing.

    PubMed

    Hockemeyer, Dirk; Jaenisch, Rudolf

    2016-05-01

    It is extremely rare for a single experiment to be so impactful and timely that it shapes and forecasts the experiments of the next decade. Here, we review how two such experiments-the generation of human induced pluripotent stem cells (iPSCs) and the development of CRISPR/Cas9 technology-have fundamentally reshaped our approach to biomedical research, stem cell biology, and human genetics. We will also highlight the previous knowledge that iPSC and CRISPR/Cas9 technologies were built on as this groundwork demonstrated the need for solutions and the benefits that these technologies provided and set the stage for their success. PMID:27152442

  18. Brief Report: Isogenic Induced Pluripotent Stem Cell Lines From an Adult With Mosaic Down Syndrome Model Accelerated Neuronal Ageing and Neurodegeneration

    PubMed Central

    Murray, Aoife; Letourneau, Audrey; Canzonetta, Claudia; Stathaki, Elisavet; Gimelli, Stefania; Sloan‐Bena, Frederique; Abrehart, Robert; Goh, Pollyanna; Lim, Shuhui; Baldo, Chiara; Dagna‐Bricarelli, Franca; Hannan, Saad; Mortensen, Martin; Ballard, David; Syndercombe Court, Denise; Fusaki, Noemi; Hasegawa, Mamoru; Smart, Trevor G.; Bishop, Cleo; Antonarakis, Stylianos E.

    2015-01-01

    Abstract Trisomy 21 (T21), Down Syndrome (DS) is the most common genetic cause of dementia and intellectual disability. Modeling DS is beginning to yield pharmaceutical therapeutic interventions for amelioration of intellectual disability, which are currently being tested in clinical trials. DS is also a unique genetic system for investigation of pathological and protective mechanisms for accelerated ageing, neurodegeneration, dementia, cancer, and other important common diseases. New drugs could be identified and disease mechanisms better understood by establishment of well‐controlled cell model systems. We have developed a first nonintegration‐reprogrammed isogenic human induced pluripotent stem cell (iPSC) model of DS by reprogramming the skin fibroblasts from an adult individual with constitutional mosaicism for DS and separately cloning multiple isogenic T21 and euploid (D21) iPSC lines. Our model shows a very low number of reprogramming rearrangements as assessed by a high‐resolution whole genome CGH‐array hybridization, and it reproduces several cellular pathologies seen in primary human DS cells, as assessed by automated high‐content microscopic analysis. Early differentiation shows an imbalance of the lineage‐specific stem/progenitor cell compartments: T21 causes slower proliferation of neural and faster expansion of hematopoietic lineage. T21 iPSC‐derived neurons show increased production of amyloid peptide‐containing material, a decrease in mitochondrial membrane potential, and an increased number and abnormal appearance of mitochondria. Finally, T21‐derived neurons show significantly higher number of DNA double‐strand breaks than isogenic D21 controls. Our fully isogenic system therefore opens possibilities for modeling mechanisms of developmental, accelerated ageing, and neurodegenerative pathologies caused by T21. Stem Cells 2015;33:2077–2084 PMID:25694335

  19. Generation of Avian Induced Pluripotent Stem Cells.

    PubMed

    Lu, Yangqing; West, Franklin D; Jordan, Brian J; Beckstead, Robert B; Jordan, Erin T; Stice, Steven L

    2015-01-01

    Avian species are among the most diverse vertebrates on our planet and significantly contribute to the balance of the ecology. They are also important food source and serve as a central animal model to decipher developmental biology and disease principles. Derivation of induced pluripotent stem cells (iPSCs) from avian species would enable conservation of genetic diversity as well as offer a valuable cell source that facilitates the use of avian models in many areas of basic and applied research. In this chapter, we describe methods used to successfully reprogram quail fibroblasts into iPSCs by using human transcription factors and the techniques critical to the characterization of their pluripotency. PMID:26621592

  20. Pluripotent stem cells and livestock genetic engineering.

    PubMed

    Soto, Delia A; Ross, Pablo J

    2016-06-01

    The unlimited proliferative ability and capacity to contribute to germline chimeras make pluripotent embryonic stem cells (ESCs) perfect candidates for complex genetic engineering. The utility of ESCs is best exemplified by the numerous genetic models that have been developed in mice, for which such cells are readily available. However, the traditional systems for mouse genetic engineering may not be practical for livestock species, as it requires several generations of mating and selection in order to establish homozygous founders. Nevertheless, the self-renewal and pluripotent characteristics of ESCs could provide advantages for livestock genetic engineering such as ease of genetic manipulation and improved efficiency of cloning by nuclear transplantation. These advantages have resulted in many attempts to isolate livestock ESCs, yet it has been generally concluded that the culture conditions tested so far are not supportive of livestock ESCs self-renewal and proliferation. In contrast, there are numerous reports of derivation of livestock induced pluripotent stem cells (iPSCs), with demonstrated capacity for long term proliferation and in vivo pluripotency, as indicated by teratoma formation assay. However, to what extent these iPSCs represent fully reprogrammed PSCs remains controversial, as most livestock iPSCs depend on continuous expression of reprogramming factors. Moreover, germline chimerism has not been robustly demonstrated, with only one successful report with very low efficiency. Therefore, even 34 years after derivation of mouse ESCs and their extensive use in the generation of genetic models, the livestock genetic engineering field can stand to gain enormously from continued investigations into the derivation and application of ESCs and iPSCs. PMID:26894405

  1. Derivation, characterization and retinal differentiation of induced pluripotent stem cells.

    PubMed

    Mekala, Subba Rao; Vauhini, Vasundhara; Nagarajan, Usha; Maddileti, Savitri; Gaddipati, Subhash; Mariappan, Indumathi

    2013-03-01

    Millions of people world over suffer visual disability due to retinal dystrophies which can be age-related or a genetic disorder resulting in gradual degeneration of the retinal pigmented epithelial (RPE) cells and photoreceptors. Therefore, cell replacement therapy offers a great promise in treating such diseases. Since the adult retina does not harbour any stem cells, alternative stem cell sources like the embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer a great promise for generating different cell types of the retina. Here, we report the derivation of four iPSC lines from mouse embryonic fibroblasts (MEFs) using a cocktail of recombinant retroviruses carrying the genes for Oct4, Sox2, Klf4 and cMyc. The iPS clone MEF-4F3 was further characterized for stemness marker expression and stable reprogramming by immunocytochemistry, FACS and RT-PCR analysis. Methylation analysis of the nanog promoter confirmed the reprogrammed epigenetic state. Pluripotency was confirmed by embryoid body (EB) formation and lineage-specific marker expression. Also, upon retinal differentiation, patches of pigmented cells with typical cobble-stone phenotype similar to RPE cells are generated within 6 weeks and they expressed ZO-1 (tight junction protein), RPE65 and bestrophin (mature RPE markers) and showed phagocytic activity by the uptake of fluorescent latex beads. PMID:23385820

  2. Vascular Potential of Human Pluripotent Stem Cells

    PubMed Central

    Iacobas, Ionela; Vats, Archana; Hirschi, Karen K.

    2010-01-01

    Cardiovascular disease is the number one cause of death and disability in the US. Understanding the biological activity of stem and progenitor cells, and their ability to contribute to the repair, regeneration and remodeling of the heart and blood vessels affected by pathologic processes is an essential part of the paradigm in enabling us to achieve a reduction in related deaths. Both human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are promising sources of cells for clinical cardiovascular therapies. Additional in vitro studies are needed, however, to understand their relative phenotypes and molecular regulation toward cardiovascular cell fates. Further studies in translational animal models are also needed to gain insights into the potential and function of both human ES- and iPS-derived cardiovascular cells, and enable translation from experimental and pre-clinical studies to human trials. PMID:20453170

  3. Generating kidney tissue from pluripotent stem cells

    PubMed Central

    Little, MH

    2016-01-01

    With the isolation of human pluripotent stem cells came the possibility of generating specific cell types for regenerative medicine. This has required the development of protocols for directed differentiation into many distinct cell types. One of the more complicated tissue types to recreate is the kidney. Here we review recent progress towards the recreation of not only specific kidney cell types but complex kidney organoids, models of the developing human organ, in vitro. We will also discuss potential short and long term applications of these approaches. PMID:27551541

  4. Craniofacial Reconstruction with Induced Pluripotent Stem Cells

    PubMed Central

    Wan, Derrick C.; Wong, Victor W.; Longaker, Michael T.

    2012-01-01

    Induced pluripotent stem cells (iPSCs) hold enormous promise for the treatment of complex tissue defects throughout the entire body. The ability for iPSCs to form all tissue types makes them an ideal autogenous cellular building block for tissue engineering strategies designed to replace any combination of skin, muscle, nerve, and bone deficiencies in the craniofacial region. Several obstacles to their use remain, however, chief among which include concerns over insertional mutagenesis and tumorigenicity. As studies continue to develop strategies minimizing these risks, the potential for development of patient-specific regenerative therapies has become tantalizingly close. PMID:22627398

  5. Pursuing Self-Renewal and Pluripotency with the Stem Cell Factor Nanog

    PubMed Central

    Saunders, Arven; Faiola, Francesco; Wang, Jianlong

    2013-01-01

    Pluripotent embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) hold great promise for future use in tissue replacement therapies due to their ability to self-renew indefinitely and to differentiate into all adult cell types. Harnessing this therapeutic potential efficiently requires a much deeper understanding of the molecular processes at work within the pluripotency network. The transcription factors Nanog, Oct4, and Sox2 reside at the core of this network, where they interact and regulate their own expression as well as that of numerous other pluripotency factors. Of these core factors, Nanog is critical for blocking the differentiation of pluripotent cells, and more importantly, for establishing the pluripotent ground state during somatic cell reprogramming. Both mouse and human Nanog are able to form dimers in vivo, allowing them to preferentially interact with certain factors and perform unique functions. Recent studies have identified an evolutionary functional conservation among vertebrate Nanog orthologs from chick, zebrafish, and the axolotl salamander, adding an additional layer of complexity to Nanog function. Here we present a detailed overview of published work focusing on Nanog structure, function, dimerization, and regulation at the genetic and post-translational levels with regard to the establishment and maintenance of pluripotency. The full spectrum of Nanog function in pluripotent stem cells and in cancer is only beginning to be revealed. We therefore use this evidence to advocate for more comprehensive analysis of Nanog in the context of disease, development, and regeneration. PMID:23653415

  6. Induced pluripotent stem cells in cartilage repair

    PubMed Central

    Lietman, Steven A

    2016-01-01

    Articular cartilage repair techniques are challenging. Human embryonic stem cells and induced pluripotent stem cells (iPSCs) theoretically provide an unlimited number of specialized cells which could be used in articular cartilage repair. However thus far chondrocytes from iPSCs have been created primarily by viral transfection and with the use of cocultured feeder cells. In addition chondrocytes derived from iPSCs have usually been formed in condensed cell bodies (resembling embryoid bodies) that then require dissolution with consequent substantial loss of cell viability and phenotype. All of these current techniques used to derive chondrocytes from iPSCs are problematic but solutions to these problems are on the horizon. These solutions will make iPSCs a viable alternative for articular cartilage repair in the near future. PMID:27004161

  7. Induced pluripotent stem cells in cartilage repair.

    PubMed

    Lietman, Steven A

    2016-03-18

    Articular cartilage repair techniques are challenging. Human embryonic stem cells and induced pluripotent stem cells (iPSCs) theoretically provide an unlimited number of specialized cells which could be used in articular cartilage repair. However thus far chondrocytes from iPSCs have been created primarily by viral transfection and with the use of cocultured feeder cells. In addition chondrocytes derived from iPSCs have usually been formed in condensed cell bodies (resembling embryoid bodies) that then require dissolution with consequent substantial loss of cell viability and phenotype. All of these current techniques used to derive chondrocytes from iPSCs are problematic but solutions to these problems are on the horizon. These solutions will make iPSCs a viable alternative for articular cartilage repair in the near future. PMID:27004161

  8. Generation of induced pluripotent stem cells from human blood.

    PubMed

    Loh, Yuin-Han; Agarwal, Suneet; Park, In-Hyun; Urbach, Achia; Huo, Hongguang; Heffner, Garrett C; Kim, Kitai; Miller, Justine D; Ng, Kitwa; Daley, George Q

    2009-05-28

    Human dermal fibroblasts obtained by skin biopsy can be reprogrammed directly to pluripotency by the ectopic expression of defined transcription factors. Here, we describe the derivation of induced pluripotent stem cells from CD34+ mobilized human peripheral blood cells using retroviral transduction of OCT4/SOX2/KLF4/MYC. Blood-derived human induced pluripotent stem cells are indistinguishable from human embryonic stem cells with respect to morphology, expression of surface antigens, and pluripotency-associated transcription factors, DNA methylation status at pluripotent cell-specific genes, and the capacity to differentiate in vitro and in teratomas. The ability to reprogram cells from human blood will allow the generation of patient-specific stem cells for diseases in which the disease-causing somatic mutations are restricted to cells of the hematopoietic lineage. PMID:19299331

  9. Isolation and Characterization of Pluripotent Human Spermatogonial Stem Cell-Derived Cells

    PubMed Central

    Kossack, Nina; Meneses, Juanito; Shefi, Shai; Nguyen, Ha Nam; Chavez, Shawn; Nicholas, Cory; Gromoll, Joerg; Turek, Paul J; Reijo-Pera, Renee A

    2009-01-01

    Several reports have documented the derivation of pluripotent cells (multipotent germline stem cells) from spermatogonial stem cells obtained from the adult mouse testis. These spermatogonia-derived stem cells express embryonic stem cell markers and differentiate to the three primary germ layers, as well as the germline. Data indicate that derivation may involve reprogramming of endogenous spermatogonia in culture. Here, we report the derivation of human multipotent germline stem cells (hMGSCs) from a testis biopsy. The cells express distinct markers of pluripotency, form embryoid bodies that contain derivatives of all three germ layers, maintain a normal XY karyotype, are hypomethylated at the H19 locus, and express high levels of telomerase. Teratoma assays indicate the presence of human cells 8 weeks post-transplantation but limited teratoma formation. Thus, these data suggest the potential to derive pluripotent cells from human testis biopsies but indicate a need for novel strategies to optimize hMGSC culture conditions and reprogramming. PMID:18927477

  10. Mitochondria in human pluripotent stem cell apoptosis.

    PubMed

    TeSlaa, Tara; Setoguchi, Kiyoko; Teitell, Michael A

    2016-04-01

    Human pluripotent stem cells (hPSCs) have great potential in regenerative medicine because they can differentiate into any cell type in the body. Genome integrity is vital for human development and for high fidelity passage of genetic information across generations through the germ line. To ensure genome stability, hPSCs maintain a lower rate of mutation than somatic cells and undergo rapid apoptosis in response to DNA damage and additional cell stresses. Furthermore, cellular metabolism and the cell cycle are also differentially regulated between cells in pluripotent and differentiated states and can aid in protecting hPSCs against DNA damage and damaged cell propagation. Despite these safeguards, clinical use of hPSC derivatives could be compromised by tumorigenic potential and possible malignant transformation from failed to differentiate cells. Since hPSCs and mature cells differentially respond to cell stress, it may be possible to specifically target undifferentiated cells for rapid apoptosis in mixed cell populations to enable safer use of hPSC-differentiated cells in patients. PMID:26828436

  11. Induced pluripotent stem cell-derived neural stem cell therapies for spinal cord injury.

    PubMed

    Lee-Kubli, Corinne A; Lu, Paul

    2015-01-01

    The greatest challenge to successful treatment of spinal cord injury is the limited regenerative capacity of the central nervous system and its inability to replace lost neurons and severed axons following injury. Neural stem cell grafts derived from fetal central nervous system tissue or embryonic stem cells have shown therapeutic promise by differentiation into neurons and glia that have the potential to form functional neuronal relays across injured spinal cord segments. However, implementation of fetal-derived or embryonic stem cell-derived neural stem cell therapies for patients with spinal cord injury raises ethical concerns. Induced pluripotent stem cells can be generated from adult somatic cells and differentiated into neural stem cells suitable for therapeutic use, thereby providing an ethical source of implantable cells that can be made in an autologous fashion to avoid problems of immune rejection. This review discusses the therapeutic potential of human induced pluripotent stem cell-derived neural stem cell transplantation for treatment of spinal cord injury, as well as addressing potential mechanisms, future perspectives and challenges. PMID:25788906

  12. Induced Pluripotent Stem Cells in Dermatology: Potentials, Advances, and Limitations

    PubMed Central

    Bilousova, Ganna; Roop, Dennis R.

    2015-01-01

    The discovery of methods for reprogramming adult somatic cells into induced pluripotent stem cells (iPSCs) has raised the possibility of producing truly personalized treatment options for numerous diseases. Similar to embryonic stem cells (ESCs), iPSCs can give rise to any cell type in the body and they are amenable to genetic correction by homologous recombination. These ESC properties of iPSCs allow for the development of permanent corrective therapies for many currently incurable disorders, including inherited skin diseases, without using embryonic tissues or oocytes. Here, we review recent progress and limitations of iPSC research with a focus on clinical applications of iPSCs, and using iPSCs to model human diseases for drug discovery in the field of dermatology. PMID:25368014

  13. Development of pluripotent stem cells for vascular therapy

    PubMed Central

    Volz, Katharina S.; Miljan, Erik; Khoo, Amanda; Cooke, John P.

    2013-01-01

    Peripheral arterial disease (PAD) is characterized by reduced limb blood flow due to arterial obstruction. Current treatment includes surgical or endovascular procedures, the failure of which may result in amputation of the affected limb. An emerging therapeutic approach is cell therapy to enhance angiogenesis and tissue survival. Small clinical trials of adult progenitor cell therapies have generated promising results, although large randomized clinical trials using well-defined cells have not been performed. Intriguing pre-clinical studies have been performed using vascular cells derived from human embryonic stem cells (hESC) or human induced pluripotent stem cells (hiPSCs). In particular, hiPSC-derived vascular cells may be a superior approach for vascular regeneration. The regulatory roadmap to the clinic will be arduous, but achievable with further understanding of the reprogramming and differentiation processes; with meticulous attention to quality control; and perseverance. PMID:22387745

  14. Generation of induced pluripotent stem cells.

    PubMed

    Deyle, David R

    2015-01-01

    Induced pluripotent stem cells (iPSCs) are generated from somatic cells that have been reprogrammed by the ectopic expression of defined embryonic transcription factors. This technology has provided investigators with a powerful tool for modelling disease and developing treatments for human disorders. This chapter provides the researcher with some background on iPSCs and details on how to produce MEF-conditioned medium, prepare mitotically arrested mouse embryonic fibroblasts (MEFs), create iPSCs using viral vectors, passage iPSCs, and cryopreserve iPSCs. The methods offered here have been used in many laboratories around the world and the reader can initially follow these methods. However, not all cell types are easily transduced using viral vectors and other methods of delivering the reprogramming transcription factors may need to be tested. PMID:25331042

  15. Induced pluripotent stem cells from goat fibroblasts.

    PubMed

    Song, Hui; Li, Hui; Huang, Mingrui; Xu, Dan; Gu, Chenghao; Wang, Ziyu; Dong, Fulu; Wang, Feng

    2013-12-01

    Embryonic stem cells (ESCs) are a powerful model for genetic engineering, studying developmental biology, and modeling disease. To date, ESCs have been established from the mouse (Evans and Kaufman, 1981, Nature 292:154-156), non-human primates (Thomson et al., , Proc Nat Acad Sci USA 92:7844-7848), humans (Thomson et al., 1998, Science 282:1145-1147), and rats (Buehr et al., , Cell 135:1287-1298); however, the derivation of ESCs from domesticated ungulates such as goats, sheep, cattle, and pigs have not been successful. Alternatively, induced pluripotent stem cells (iPSCs) can be generated by reprogramming somatic cells with several combinations of genes encoding transcription factors (OCT3/4, SOX2, KLF4, cMYC, LIN28, and NANOG). To date, iPSCs have been isolated from various species, but only limited information is available regarding goat iPSCs (Ren et al., 2011, Cell Res 21:849-853). The objectives of this study were to generate goat iPSCs from fetal goat primary ear fibroblasts using lentiviral transduction of four human transcription factors: OCT4, SOX2, KLF4, and cMYC. The goat iPSCs were successfully generated by co-culture with mitomycin C-treated mouse embryonic fibroblasts using medium supplemented with knockout serum replacement and human basic fibroblast growth factor. The goat iPSCs colonies are flat, compact, and closely resemble human iPSCs. They have a normal karyotype; stain positive for alkaline phosphatase, OCT4, and NANOG; express endogenous pluripotency genes (OCT4, SOX2, cMYC, and NANOG); and can spontaneously differentiate into three germ layers in vitro and in vivo. PMID:24123501

  16. Maintenance and neuronal differentiation of chicken induced pluripotent stem-like cells.

    PubMed

    Dai, Rui; Rossello, Ricardo; Chen, Chun-Chun; Kessler, Joeran; Davison, Ian; Hochgeschwender, Ute; Jarvis, Erich D

    2014-01-01

    Pluripotent stem cells have the potential to become any cell in the adult body, including neurons and glia. Avian stem cells could be used to study questions, like vocal learning, that would be difficult to examine with traditional mouse models. Induced pluripotent stem cells (iPSCs) are differentiated cells that have been reprogrammed to a pluripotent stem cell state, usually using inducing genes or other molecules. We recently succeeded in generating avian iPSC-like cells using mammalian genes, overcoming a limitation in the generation and use of iPSCs in nonmammalian species (Rosselló et al., 2013). However, there were no established optimal cell culture conditions for avian iPSCs to establish long-term cell lines and thus to study neuronal differentiation in vitro. Here we present an efficient method of maintaining chicken iPSC-like cells and for differentiating them into action potential generating neurons. PMID:25610469

  17. Maintenance and Neuronal Differentiation of Chicken Induced Pluripotent Stem-Like Cells

    PubMed Central

    Rossello, Ricardo; Chen, Chun-chun; Kessler, Joeran; Davison, Ian; Jarvis, Erich D.

    2014-01-01

    Pluripotent stem cells have the potential to become any cell in the adult body, including neurons and glia. Avian stem cells could be used to study questions, like vocal learning, that would be difficult to examine with traditional mouse models. Induced pluripotent stem cells (iPSCs) are differentiated cells that have been reprogrammed to a pluripotent stem cell state, usually using inducing genes or other molecules. We recently succeeded in generating avian iPSC-like cells using mammalian genes, overcoming a limitation in the generation and use of iPSCs in nonmammalian species (Rosselló et al., 2013). However, there were no established optimal cell culture conditions for avian iPSCs to establish long-term cell lines and thus to study neuronal differentiation in vitro. Here we present an efficient method of maintaining chicken iPSC-like cells and for differentiating them into action potential generating neurons. PMID:25610469

  18. Hydrodynamic modulation of pluripotent stem cells

    PubMed Central

    2012-01-01

    Controlled expansion and differentiation of pluripotent stem cells (PSCs) using reproducible, high-throughput methods could accelerate stem cell research for clinical therapies. Hydrodynamic culture systems for PSCs are increasingly being used for high-throughput studies and scale-up purposes; however, hydrodynamic cultures expose PSCs to complex physical and chemical environments that include spatially and temporally modulated fluid shear stresses and heterogeneous mass transport. Furthermore, the effects of fluid flow on PSCs cannot easily be attributed to any single environmental parameter since the cellular processes regulating self-renewal and differentiation are interconnected and the complex physical and chemical parameters associated with fluid flow are thus difficult to independently isolate. Regardless of the challenges posed by characterizing fluid dynamic properties, hydrodynamic culture systems offer several advantages over traditional static culture, including increased mass transfer and reduced cell handling. This article discusses the challenges and opportunities of hydrodynamic culture environments for the expansion and differentiation of PSCs in microfluidic systems and larger-volume suspension bioreactors. Ultimately, an improved understanding of the effects of hydrodynamics on the self-renewal and differentiation of PSCs could yield improved bioprocessing technologies to attain scalable PSC culture strategies that will probably be requisite for the development of therapeutic and diagnostic applications. PMID:23168068

  19. Genome Editing in Human Pluripotent Stem Cells.

    PubMed

    Smith, Cory; Ye, Zhaohui; Cheng, Linzhao

    2016-01-01

    Pluripotent stem cells (PSCs), defined by their capacity for self-renewal and differentiation into all cell types, are an integral tool for basic biological research and disease modeling. However, full use of PSCs for research and regenerative medicine requires the ability to precisely edit their DNA to correct disease-causing mutations and for functional analysis of genetic variations. Recent advances in DNA editing of human stem cells (including PSCs) have benefited from the use of designer nucleases capable of making double-strand breaks (DSBs) at specific sequences that stimulate endogenous DNA repair. The clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has become the preferred designer nuclease for genome editing in human PSCs and other cell types. Here we describe the principles for designing a single guide RNA to uniquely target a gene of interest and describe strategies for disrupting, inserting, or replacing a specific DNA sequence in human PSCs. The improvements in efficiency and ease provided by these techniques allow individuals to precisely engineer PSCs in a way previously limited to large institutes and core facilities. PMID:27037079

  20. Regulatory Insight into the European Human Pluripotent Stem Cell Registry

    PubMed Central

    Kurtz, Andreas; Stacey, Glyn; Kidane, Luam; Seriola, Anna; Stachelscheid, Harald; Veiga, Anna

    2014-01-01

    Abstract The European pluripotent stem cell registry aims at listing qualified pluripotent stem cell (PSC) lines that are available globally together with relevant information for each cell line. Specific emphasis is being put on documenting ethical procurement of the cells and providing evidence of pluripotency. The report discusses the tasks and challenges for a global PSC registry as an instrument to develop collaboration, to access cells from diverse resources and banks, and to implement standards, and as a means to follow up usage of cells and support adherence to regulatory and scientific standards and transparency for stakeholders. PMID:25457963

  1. Regulatory insight into the European human pluripotent stem cell registry.

    PubMed

    Kurtz, Andreas; Stacey, Glyn; Kidane, Luam; Seriola, Anna; Stachelscheid, Harald; Veiga, Anna

    2014-12-01

    The European pluripotent stem cell registry aims at listing qualified pluripotent stem cell (PSC) lines that are available globally together with relevant information for each cell line. Specific emphasis is being put on documenting ethical procurement of the cells and providing evidence of pluripotency. The report discusses the tasks and challenges for a global PSC registry as an instrument to develop collaboration, to access cells from diverse resources and banks, and to implement standards, and as a means to follow up usage of cells and support adherence to regulatory and scientific standards and transparency for stakeholders. PMID:25457963

  2. Glycosyltransferase ST6GAL1 contributes to the regulation of pluripotency in human pluripotent stem cells

    PubMed Central

    Wang, Yu-Chieh; Stein, Jason W.; Lynch, Candace L.; Tran, Ha T.; Lee, Chia-Yao; Coleman, Ronald; Hatch, Adam; Antontsev, Victor G.; Chy, Hun S.; O’Brien, Carmel M.; Murthy, Shashi K.; Laslett, Andrew L.; Peterson, Suzanne E.; Loring, Jeanne F.

    2015-01-01

    Many studies have suggested the significance of glycosyltransferase-mediated macromolecule glycosylation in the regulation of pluripotent states in human pluripotent stem cells (hPSCs). Here, we observed that the sialyltransferase ST6GAL1 was preferentially expressed in undifferentiated hPSCs compared to non-pluripotent cells. A lectin which preferentially recognizes α-2,6 sialylated galactosides showed strong binding reactivity with undifferentiated hPSCs and their glycoproteins, and did so to a much lesser extent with differentiated cells. In addition, downregulation of ST6GAL1 in undifferentiated hPSCs led to a decrease in POU5F1 (also known as OCT4) protein and significantly altered the expression of many genes that orchestrate cell morphogenesis during differentiation. The induction of cellular pluripotency in somatic cells was substantially impeded by the shRNA-mediated suppression of ST6GAL1, partially through interference with the expression of endogenous POU5F1 and SOX2. Targeting ST6GAL1 activity with a sialyltransferase inhibitor during cell reprogramming resulted in a dose-dependent reduction in the generation of human induced pluripotent stem cells (hiPSCs). Collectively, our data indicate that ST6GAL1 plays an important role in the regulation of pluripotency and differentiation in hPSCs, and the pluripotent state in human cells can be modulated using pharmacological tools to target sialyltransferase activity. PMID:26304831

  3. 28. Embryonic and adult stem cell therapy.

    PubMed

    Henningson, Carl T; Stanislaus, Marisha A; Gewirtz, Alan M

    2003-02-01

    Stem cells are characterized by the ability to remain undifferentiated and to self-renew. Embryonic stem cells derived from blastocysts are pluripotent (able to differentiate into many cell types). Adult stem cells, which were traditionally thought to be monopotent multipotent, or tissue restricted, have recently also been shown to have pluripotent properties. Adult bone marrow stem cells have been shown to be capable of differentiating into skeletal muscle, brain microglia and astroglia, and hepatocytes. Stem cell lines derived from both embryonic stem and embryonic germ cells (from the embryonic gonadal ridge) are pluripotent and capable of self-renewal for long periods. Therefore embryonic stem and germ cells have been widely investigated for their potential to cure diseases by repairing or replacing damaged cells and tissues. Studies in animal models have shown that transplantation of fetal, embryonic stem, or embryonic germ cells may be able to treat some chronic diseases. In this review, we highlight recent developments in the use of stem cells as therapeutic agents for three such diseases: Diabetes, Parkinson disease, and congestive heart failure. We also discuss the potential use of stem cells as gene therapy delivery cells and the scientific and ethical issues that arise with the use of human stem cells. PMID:12592319

  4. Cell therapy using induced pluripotent stem cells or somatic stem cells: this is the question.

    PubMed

    Somoza, Rodrigo A; Rubio, Francisco J

    2012-05-01

    A lot of effort has been developed to bypass the use of embryonic stem cells (ES) in human therapies, because of several concerns and ethical issues. Some unsolved problems of using stem cells for human therapies, excluding the human embryonic origin, are: how to regulate cell plasticity and proliferation, immunological compatibility, potential adverse side-effects when stem cells are systemically administrated, and the in vivo signals to rule out a specific cell fate after transplantation. Currently, it is known that almost all tissues of an adult organism have somatic stem cells (SSC). Whereas ES are primary involved in the genesis of new tissues and organs, SSC are involved in regeneration processes, immuno-regulatory and homeostasis mechanisms. Although the differentiating potential of ES is higher than SSC, several studies suggest that some types of SSC, such as mesenchymal stem cells (MSC), can be induced epigenetically to differentiate into tissue-specific cells of different lineages. This unexpected pluripotency and the variety of sources that they come from, can make MSC-like cells suitable for the treatment of diverse pathologies and injuries. New hopes for cell therapy came from somatic/mature cells and the discovery that could be reprogrammed to a pluripotent stage similar to ES, thus generating induced pluripotent stem cells (iPS). For this, it is necessary to overexpress four main reprogramming factors, Sox2, Oct4, Klf4 and c-Myc. The aim of this review is to analyze the potential and requirements of cellular based tools in human therapy strategies, focusing on the advantage of using MSC over iPS. PMID:22329581

  5. Mesenchymal and induced pluripotent stem cells: general insights and clinical perspectives

    PubMed Central

    Zomer, Helena D; Vidane, Atanásio S; Gonçalves, Natalia N; Ambrósio, Carlos E

    2015-01-01

    Mesenchymal stem cells have awakened a great deal of interest in regenerative medicine due to their plasticity, and immunomodulatory and anti-inflammatory properties. They are high-yield and can be acquired through noninvasive methods from adult tissues. Moreover, they are nontumorigenic and are the most widely studied. On the other hand, induced pluripotent stem (iPS) cells can be derived directly from adult cells through gene reprogramming. The new iPS technology avoids the embryo destruction or manipulation to generate pluripotent cells, therefore, are exempt from ethical implication surrounding embryonic stem cell use. The pre-differentiation of iPS cells ensures the safety of future approaches. Both mesenchymal stem cells and iPS cells can be used for autologous cell transplantations without the risk of immune rejection and represent a great opportunity for future alternative therapies. In this review we discussed the therapeutic perspectives using mesenchymal and iPS cells. PMID:26451119

  6. Mechanobiology of Human Pluripotent Stem Cells

    PubMed Central

    Earls, Jonathan K.; Jin, Sha

    2013-01-01

    Human pluripotent stem cells (hPSCs) are self-renewing and have the potential to differentiate into any cell type in the body, making them attractive cell sources for applications in tissue engineering and regenerative medicine. However, in order for hPSCs to find use in the clinic, the mechanisms underlying their self-renewal and lineage commitment must be better understood. Many technologies that have been developed for the maintenance and directed differentiation of hPSCs involve the use of soluble growth factors, but recent studies suggest that other elements of the hPSC microenvironment also influence the growth and differentiation of hPSCs. This includes the influences of cell–cell interactions, substrate mechanics, cellular interactions with extracellular matrix, as well as the nanotopography of the substrate and physical forces such as shear stress, cyclic mechanical strain, and compression. In this review, we highlight the recent progress of this area of research and discuss ways in which the mechanical cues may be incorporated into hPSC culture regimes to improve methods for expanding and differentiating hPSCs. PMID:23472616

  7. Trichostatin A-Mediated Epigenetic Transformation of Adult Bone Marrow-Derived Mesenchymal Stem Cells Biases the In Vitro Developmental Capability, Quality, and Pluripotency Extent of Porcine Cloned Embryos

    PubMed Central

    Samiec, Marcin; Opiela, Jolanta; Lipiński, Daniel

    2015-01-01

    The current research was conducted to explore the in vitro developmental outcome and cytological/molecular quality of porcine nuclear-transferred (NT) embryos reconstituted with adult bone marrow-derived mesenchymal stem cells (ABM-MSCs) that were epigenetically transformed by treatment with nonspecific inhibitor of histone deacetylases, known as trichostatin A (TSA). The cytological quality of cloned blastocysts was assessed by estimation of the total cells number (TCN) and apoptotic index. Their molecular quality was evaluated by real-time PCR-mediated quantification of gene transcripts for pluripotency- and multipotent stemness-related markers (Oct4, Nanog, and Nestin). The morula and blastocyst formation rates of NT embryos derived from ABM-MSCs undergoing TSA treatment were significantly higher than in the TSA-unexposed group. Moreover, the NT blastocysts generated using TSA-treated ABM-MSCs exhibited significantly higher TCN and increased pluripotency extent measured with relative abundance of Oct4 and Nanog mRNAs as compared to the TSA-untreated group. Altogether, the improvements in morula/blastocyst yields and quality of cloned pig embryos seem to arise from enhanced abilities for promotion of correct epigenetic reprogramming of TSA-exposed ABM-MSC nuclei in a cytoplasm of reconstructed oocytes. To our knowledge, we are the first to report the successful production of mammalian high-quality NT blastocysts using TSA-dependent epigenomic modulation of ABM-MSCs. PMID:25866813

  8. Induced pluripotent stem cells generated without viral integration.

    PubMed

    Stadtfeld, Matthias; Nagaya, Masaki; Utikal, Jochen; Weir, Gordon; Hochedlinger, Konrad

    2008-11-01

    Pluripotent stem cells have been generated from mouse and human somatic cells by viral expression of the transcription factors Oct4, Sox2, Klf4, and c-Myc. A major limitation of this technology is the use of potentially harmful genome-integrating viruses. We generated mouse induced pluripotent stem (iPS) cells from fibroblasts and liver cells by using nonintegrating adenoviruses transiently expressing Oct4, Sox2, Klf4, and c-Myc. These adenoviral iPS (adeno-iPS) cells show DNA demethylation characteristic of reprogrammed cells, express endogenous pluripotency genes, form teratomas, and contribute to multiple tissues, including the germ line, in chimeric mice. Our results provide strong evidence that insertional mutagenesis is not required for in vitro reprogramming. Adenoviral reprogramming may provide an improved method for generating and studying patient-specific stem cells and for comparing embryonic stem cells and iPS cells. PMID:18818365

  9. CCL2 enhances pluripotency of human induced pluripotent stem cells by activating hypoxia related genes

    PubMed Central

    Hasegawa, Yuki; Tang, Dave; Takahashi, Naoko; Hayashizaki, Yoshihide; Forrest, Alistair R. R.; the FANTOM consortium; Suzuki, Harukazu

    2014-01-01

    Standard culture of human induced pluripotent stem cells (hiPSCs) requires basic Fibroblast Growth Factor (bFGF) to maintain the pluripotent state, whereas hiPSC more closely resemble epiblast stem cells than true naïve state ES which requires LIF to maintain pluripotency. Here we show that chemokine (C-C motif) ligand 2 (CCL2) enhances the expression of pluripotent marker genes through the phosphorylation of the signal transducer and activator of transcription 3 (STAT3) protein. Moreover, comparison of transcriptomes between hiPSCs cultured with CCL2 versus with bFGF, we found that CCL2 activates hypoxia related genes, suggesting that CCL2 enhanced pluripotency by inducing a hypoxic-like response. Further, we show that hiPSCs cultured with CCL2 can differentiate at a higher efficiency than culturing with just bFGF and we show CCL2 can be used in feeder-free conditions in the absence of LIF. Taken together, our finding indicates the novel functions of CCL2 in enhancing its pluripotency in hiPSCs. PMID:24957798

  10. Concise Review: In Vitro T-Cell Generation from Adult, Embryonic, and Induced Pluripotent Stem Cells: Many Roads to One Destination

    PubMed Central

    Smith, Michelle J.; Webber, Beau R.; Mohtashami, Mahmood; Stefanski, Heather E.; Zúñiga-Pflücker, Juan Carlos; Blazar, Bruce R.

    2016-01-01

    T lymphocytes are critical mediators of the adaptive immune system and have the capacity to serve as therapeutic agents in the areas of transplant and cancer immunotherapy. While T cells can be isolated and expanded from patients, T cells derived in vitro from both hematopoietic stem/progenitor cells (HSPCs) and human pluripotent stem cells (hPSCs) offer great potential advantages in generating a self-renewing source of T cells that can be readily genetically modified. T-cell differentiation in vivo is a complex process requiring tightly regulated signals; providing the correct signals in vitro to induce T-cell lineage commitment followed by their development into mature, functional, single positive T cells, is similarly complex. In this review, we discuss current methods for the in vitro derivation of T cells from murine and human HSPCs and hPSCs that use feeder-cell and feeder-cell-free systems. Furthermore, we explore their potential for adoption for use in T-cell-based therapies. PMID:26227158

  11. Derivation of porcine pluripotent stem cells for biomedical research.

    PubMed

    Shiue, Yow-Ling; Yang, Jenn-Rong; Liao, Yu-Jing; Kuo, Ting-Yung; Liao, Chia-Hsin; Kang, Ching-Hsun; Tai, Chein; Anderson, Gary B; Chen, Lih-Ren

    2016-07-01

    Pluripotent stem cells including embryonic stem cells (ESCs), embryonic germ cells (EGCs), and induced pluripotent stem cells (iPSCs) are capable of self-renew and limitlessly proliferating in vitro with undifferentiated characteristics. They are able to differentiate in vitro, spontaneously or responding to suitable signals, into cells of all three primary germ layers. Consequently, these pluripotent stem cells will be valuable sources for cell replacement therapy in numerous disorders. However, the promise of human ESCs and EGCs is cramped by the ethical argument about destroying embryos and fetuses for cell line creation. Moreover, there are still carcinogenic risks existing toward the goal of clinical application for human ESCs, EGCs, and iPSCs. Therefore, a suitable animal model for stem cell research will benefit the further development of human stem cell technology. The pigs, on the basis of their similarity in anatomy, immunology, physiology, and biochemical properties, have been wide used as model animals in the study of various human diseases. The development of porcine pluripotent stem cell lines will hold the opportunity to provide an excellent material for human counterpart to the transplantation in biomedical research and further development of cell-based therapeutic strategy. PMID:27158128

  12. Pluripotent stem cells and DNA damage response to ionizing radiations

    PubMed Central

    Mujoo, Kalpana; Butler, E. Brian; Pandita, Raj K.; Hunt, Clayton R.; Pandita, Tej K.

    2016-01-01

    Pluripotent stem cells (PSCs) hold great promise in regenerative medicine, disease modeling, functional genomics, toxicological studies and cell-based therapeutics due to their unique characteristics of self-renewal and pluripotency. Novel methods for generation of pluripotent stem cells and their differentiation to the specialized cell types such as neuronal cells, myocardial cells, hepatocytes, and beta cells of the pancreas and many other cells of the body are constantly being refined. Pluripotent stem cell derived differentiated cells, including neuronal cells or cardiac cells are ideal for stem cell transplantation as autologous or allogeneic cells from healthy donors due to their minimum risks of rejection. DNA damage induced by ionizing radiation (IR), ultraviolet (UV) light, genotoxic stress, and other intrinsic and extrinsic factors trigger a series of biochemical reactions termed as DNA damage response (DDR). In order to maintain genomic stability, and avoid transmission of mutations into progenitors cells, stem cells have robust DNA damage response signaling – a contrast to somatic cells. Stem cell transplantation may over come the late effects related to radiation. This review will particularly focus on differential DNA damage response between stem cells and derived differentiated cells and the possible pathways that determine such differences. PMID:27332952

  13. Pluripotent Stem Cells and DNA Damage Response to Ionizing Radiations.

    PubMed

    Mujoo, Kalpana; Butler, E Brian; Pandita, Raj K; Hunt, Clayton R; Pandita, Tej K

    2016-07-01

    Pluripotent stem cells (PSCs) hold great promise in regenerative medicine, disease modeling, functional genomics, toxicological studies and cell-based therapeutics due to their unique characteristics of self-renewal and pluripotency. Novel methods for generation of pluripotent stem cells and their differentiation to the specialized cell types such as neuronal cells, myocardial cells, hepatocytes and beta cells of the pancreas and many other cells of the body are constantly being refined. Pluripotent stem cell derived differentiated cells, including neuronal cells or cardiac cells, are ideal for stem cell transplantation as autologous or allogeneic cells from healthy donors due to their minimal risk of rejection. Radiation-induced DNA damage, ultraviolet light, genotoxic stress and other intrinsic and extrinsic factors triggers a series of biochemical reactions known as DNA damage response. To maintain genomic stability and avoid transmission of mutations into progenitors cells, stem cells have robust DNA damage response signaling, a contrast to somatic cells. Stem cell transplantation may protect against radiation-induced late effects. In particular, this review focuses on differential DNA damage response between stem cells and derived differentiated cells and the possible pathways that determine such differences. PMID:27332952

  14. Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells

    PubMed Central

    Patsch, Christoph; Challet-Meylan, Ludivine; Thoma, Eva C.; Urich, Eduard; Heckel, Tobias; O’Sullivan, John F; Grainger, Stephanie J; Kapp, Friedrich G.; Sun, Lin; Christensen, Klaus; Xia, Yulei; Florido, Mary H. C.; He, Wei; Pan, Wei; Prummer, Michael; Warren, Curtis R.; Jakob-Roetne, Roland; Certa, Ulrich; Jagasia, Ravi; Freskgård, Per-Ola; Adatto, Isaac; Kling, Dorothee; Huang, Paul; Zon, Leonard I; Chaikof, Elliot L.; Gerszten, Robert E.; Graf, Martin; Iacone, Roberto; Cowan, Chad A.

    2015-01-01

    The use of human pluripotent stem cells for in vitro disease modeling and clinical applications requires protocols that convert these cells into relevant adult cell types. Here, we report the rapid and efficient differentiation of human pluripotent stem cells into vascular endothelial and smooth muscle cells. We found that GSK3 inhibition and BMP4 treatment rapidly committed pluripotent cells to a mesodermal fate and subsequent exposure to VEGF or PDGF-BB resulted in the differentiation of either endothelial or vascular smooth muscle cells, respectively. Both protocols produced mature cells with efficiencies over 80% within six days. Upon purification to 99% via surface markers, endothelial cells maintained their identity, as assessed by marker gene expression, and showed relevant in vitro and in vivo functionality. Global transcriptional and metabolomic analyses confirmed that the cells closely resembled their in vivo counterparts. Our results suggest that these cells could be used to faithfully model human disease. PMID:26214132

  15. Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells.

    PubMed

    Patsch, Christoph; Challet-Meylan, Ludivine; Thoma, Eva C; Urich, Eduard; Heckel, Tobias; O'Sullivan, John F; Grainger, Stephanie J; Kapp, Friedrich G; Sun, Lin; Christensen, Klaus; Xia, Yulei; Florido, Mary H C; He, Wei; Pan, Wei; Prummer, Michael; Warren, Curtis R; Jakob-Roetne, Roland; Certa, Ulrich; Jagasia, Ravi; Freskgård, Per-Ola; Adatto, Isaac; Kling, Dorothee; Huang, Paul; Zon, Leonard I; Chaikof, Elliot L; Gerszten, Robert E; Graf, Martin; Iacone, Roberto; Cowan, Chad A

    2015-08-01

    The use of human pluripotent stem cells for in vitro disease modelling and clinical applications requires protocols that convert these cells into relevant adult cell types. Here, we report the rapid and efficient differentiation of human pluripotent stem cells into vascular endothelial and smooth muscle cells. We found that GSK3 inhibition and BMP4 treatment rapidly committed pluripotent cells to a mesodermal fate and subsequent exposure to VEGF-A or PDGF-BB resulted in the differentiation of either endothelial or vascular smooth muscle cells, respectively. Both protocols produced mature cells with efficiencies exceeding 80% within six days. On purification to 99% via surface markers, endothelial cells maintained their identity, as assessed by marker gene expression, and showed relevant in vitro and in vivo functionality. Global transcriptional and metabolomic analyses confirmed that the cells closely resembled their in vivo counterparts. Our results suggest that these cells could be used to faithfully model human disease. PMID:26214132

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

    PubMed

    Bose, Bipasha; Shenoy P, Sudheer

    2016-02-01

    Muscle derived stem cells (MDSCs) are multipotent stem cells that can differentiate into several lineages including skeletal muscle precursor cells. Here, we show that MDSCs from myostatin null mice (Mstn (-/-) ) can be readily induced into pluripotent stem cells without using reprogramming factors. Microarray studies revealed a strong upregulation of markers like Leukemia Inhibitory factor (LIF) and Leukemia Inhibitory factor receptor (LIFR) in Mstn (-/-) MDSCs as compared to wild type MDSCs (WT-MDSCs). Furthermore when cultured in mouse embryonic stem cell media with LIF for 95 days, Mstn (-/-) MDSCs formed embryonic stem cell (ES) like colonies. We termed such ES like cells as the culture-induced pluripotent stem cells (CiPSC). CiPSCs from Mstn (-/-) MDSCs were phenotypically similar to ESCs, expressed high levels of Oct4, Nanog, Sox2 and SSEA-1, maintained a normal karyotype. Furthermore, CiPSCs formed embryoid bodies and teratomas when injected into immunocompromised mice. In addition, CiPSCs differentiated into somatic cells of all three lineages. We further show that culturing in ES cell media, resulted in hypermethylation and downregulation of BMP2 in Mstn(-/-) MDSCs. Western blot further confirmed a down regulation of BMP2 signaling in Mstn (-/-) MDSCs in supportive of pluripotent reprogramming. Given that down regulation of BMP2 has been shown to induce pluripotency in cells, we propose that lack of myostatin epigenetically reprograms the MDSCs to become pluripotent stem cells. Thus, here we report the successful establishment of ES-like cells from adult stem cells of the non-germline origin under culture-induced conditions without introducing reprogramming genes. PMID:26358783

  17. Generation of Induced Pluripotent Stem Cells from Conjunctiva

    PubMed Central

    Yang, Jin; Li, Yao; Erol, Deniz; Wu, Wen-Hsuan; Tsai, Yi-Ting; Li, Xiao-Rong; Davis, Richard J.; Tsang, Stephen H.

    2014-01-01

    Purpose The objective of this study was to determine whether cells from the conjunctiva could be reprogrammed into induced pluripotent stem (iPS) cells, providing an alternative source of stem cells. Methods We employed a doxycycline induced reprogrammable mouse strain to generate iPS cells from conjunctiva. The identity of the stem cells was confirmed by Reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence assays. Immunocytochemistry and teratoma assays are established means for scoring stem cell pluripotency. The reprogramming efficiencies of conjunctiva cells and ear fibroblasts were compared. Results We confirmed the identity of the stem cells and demonstrated expression of pluripotency markers (OCT4, SOX2, NANOG, and SSEA1), as tested by RT-PCR and immunofluorescence assays. In addition, derived iPS cells differentiated successfully into embryoid bodies and showed teratoma formation when injected into immunodeficient mice. Reprogramming conjunctival tissue is as efficient as reprogramming ear fibroblasts. Conjunctiva-iPS exhibited classic features of embryonic stem (ES) cells with respect to morphology, expression of surface antigens and pluripotency-associated transcription factors, capacity to differentiate in vitro, and the ability to form all three germ layers in vivo. Conclusion The present study demonstrated that conjunctival cells, which are readily obtained during the course of many routine conjunctival biopsies and ophthalmic procedures, can be another reliable source of iPS cells. PMID:24492934

  18. Back to the future: how human induced pluripotent stem cells will transform regenerative medicine

    PubMed Central

    Svendsen, Clive N.

    2013-01-01

    Based on cloning studies in mammals, all adult human cells theoretically contain DNA that is capable of creating a whole new person. Cells are maintained in their differentiated state by selectively activating some genes and silencing. The dogma until recently was that cell differentiation was largely fixed unless exposed to the environment of an activated oocyte. However, it is now possible to activate primitive pluripotent genes within adult human cells that take them back in time to a pluripotent state (termed induced pluripotent stem cells). This technology has grown at an exponential rate over the past few years, culminating in the Nobel Prize in medicine. Discussed here are recent developments in the field as they relate to regenerative medicine, with an emphasis on creating functional cells, editing their genome, autologous transplantation and how this ground-breaking field may eventually impact human aging. PMID:23945396

  19. Canine Pluripotent Stem Cells: Are They Ready for Clinical Applications?

    PubMed

    Betts, Dean H; Tobias, Ian C

    2015-01-01

    The derivation of canine embryonic stem cells and generation of canine-induced pluripotent stem cells are significant achievements that have unlocked the potential for developing novel cell-based disease models, drug discovery platforms, and transplantation therapies in the dog. A progression from concept to cure in this clinically relevant companion animal will not only help our canine patients but also help advance human regenerative medicine. Nevertheless, many issues remain to be resolved before pluripotent cells can be used clinically in a safe and reproducible manner. PMID:26664969

  20. A Novel Feeder-Free Culture System for Human Pluripotent Stem Cell Culture and Induced Pluripotent Stem Cell Derivation

    PubMed Central

    Vuoristo, Sanna; Toivonen, Sanna; Weltner, Jere; Mikkola, Milla; Ustinov, Jarkko; Trokovic, Ras; Palgi, Jaan; Lund, Riikka; Tuuri, Timo; Otonkoski, Timo

    2013-01-01

    Correct interactions with extracellular matrix are essential to human pluripotent stem cells (hPSC) to maintain their pluripotent self-renewal capacity during in vitro culture. hPSCs secrete laminin 511/521, one of the most important functional basement membrane components, and they can be maintained on human laminin 511 and 521 in defined culture conditions. However, large-scale production of purified or recombinant laminin 511 and 521 is difficult and expensive. Here we have tested whether a commonly available human choriocarcinoma cell line, JAR, which produces high quantities of laminins, supports the growth of undifferentiated hPSCs. We were able to maintain several human pluripotent stem cell lines on decellularized matrix produced by JAR cells using a defined culture medium. The JAR matrix also supported targeted differentiation of the cells into neuronal and hepatic directions. Importantly, we were able to derive new human induced pluripotent stem cell (hiPSC) lines on JAR matrix and show that adhesion of the early hiPSC colonies to JAR matrix is more efficient than to matrigel. In summary, JAR matrix provides a cost-effective and easy-to-prepare alternative for human pluripotent stem cell culture and differentiation. In addition, this matrix is ideal for the efficient generation of new hiPSC lines. PMID:24098444

  1. Gene expression of pluripotency determinants is conserved between mammalian and planarian stem cells

    PubMed Central

    Önal, Pinar; Grün, Dominic; Adamidi, Catherine; Rybak, Agnieszka; Solana, Jordi; Mastrobuoni, Guido; Wang, Yongbo; Rahn, Hans-Peter; Chen, Wei; Kempa, Stefan; Ziebold, Ulrike; Rajewsky, Nikolaus

    2012-01-01

    Freshwater planaria possess extreme regeneration capabilities mediated by abundant, pluripotent stem cells (neoblasts) in adult animals. Although planaria emerged as an attractive in vivo model system for stem cell biology, gene expression in neoblasts has not been profiled comprehensively and it is unknown how molecular mechanisms for pluripotency in neoblasts relate to those in mammalian embryonic stem cells (ESCs). We purified neoblasts and quantified mRNA and protein expression by sequencing and shotgun proteomics. We identified ∼4000 genes specifically expressed in neoblasts, including all ∼30 known neoblast markers. Genes important for pluripotency in ESCs, including regulators as well as targets of OCT4, were well conserved and upregulated in neoblasts. We found conserved expression of epigenetic regulators and demonstrated their requirement for planarian regeneration by knockdown experiments. Post-transcriptional regulatory genes characteristic for germ cells were also enriched in neoblasts, suggesting the existence of a common ancestral state of germ cells and ESCs. We conclude that molecular determinants of pluripotency are conserved throughout evolution and that planaria are an informative model system for human stem cell biology. PMID:22543868

  2. Dynamic and social behaviors of human pluripotent stem cells

    PubMed Central

    Phadnis, Smruti M.; Loewke, Nathan O.; Dimov, Ivan K.; Pai, Sunil; Amwake, Christine E.; Solgaard, Olav; Baer, Thomas M.; Chen, Bertha; Pera, Renee A. Reijo

    2015-01-01

    Human pluripotent stem cells (hPSCs) can self-renew or differentiate to diverse cell types, thus providing a platform for basic and clinical applications. However, pluripotent stem cell populations are heterogeneous and functional properties at the single cell level are poorly documented leading to inefficiencies in differentiation and concerns regarding reproducibility and safety. Here, we use non-invasive time-lapse imaging to continuously examine hPSC maintenance and differentiation and to predict cell viability and fate. We document dynamic behaviors and social interactions that prospectively distinguish hPSC survival, self-renewal, and differentiation. Results highlight the molecular role of E-cadherin not only for cell-cell contact but also for clonal propagation of hPSCs. Results indicate that use of continuous time-lapse imaging can distinguish cellular heterogeneity with respect to pluripotency as well as a subset of karyotypic abnormalities whose dynamic properties were monitored. PMID:26381699

  3. Equine Induced Pluripotent Stem Cells have a Reduced Tendon Differentiation Capacity Compared to Embryonic Stem Cells

    PubMed Central

    Bavin, Emma P.; Smith, Olivia; Baird, Arabella E. G.; Smith, Lawrence C.; Guest, Deborah J.

    2015-01-01

    Tendon injuries occur commonly in horses and their repair through scar tissue formation predisposes horses to a high rate of re-injury. Pluripotent stem cells may provide a cell replacement therapy to improve tendon tissue regeneration and lower the frequency of re-injury. We have previously demonstrated that equine embryonic stem cells (ESCs) differentiate into the tendon cell lineage upon injection into the damaged horse tendon and can differentiate into functional tendon cells in vitro to generate artificial tendons. Induced pluripotent stem cells (iPSCs) have now been derived from horses but, to date, there are no reports on their ability to differentiate into tendon cells. As iPSCs can be produced from adult cell types, they provide a more accessible source of cells than ESCs, which require the use of horse embryos. The aim of this study was to compare tendon differentiation by ESCs and iPSCs produced through two independent methods. In two-dimensional differentiation assays, the iPSCs expressed tendon-associated genes and proteins, which were enhanced by the presence of transforming growth factor-β3. However, in three-dimensional (3D) differentiation assays, the iPSCs failed to differentiate into functional tendon cells and generate artificial tendons. These results demonstrate the utility of the 3D in vitro tendon assay for measuring tendon differentiation and the need for more detailed studies to be performed on equine iPSCs to identify and understand their epigenetic differences from pluripotent ESCs prior to their clinical application. PMID:26664982

  4. Biomedical Application of Dental Tissue-Derived Induced Pluripotent Stem Cells

    PubMed Central

    Lee, Jung-Hwan; Seo, Seog-Jin

    2016-01-01

    The academic researches and clinical applications in recent years found interest in induced pluripotent stem cells (iPSCs-) based regenerative medicine due to their pluripotency able to differentiate into any cell types in the body without using embryo. However, it is limited in generating iPSCs from adult somatic cells and use of these cells due to the low stem cell potency and donor site morbidity. In biomedical applications, particularly, dental tissue-derived iPSCs have been getting attention as a type of alternative sources for regenerating damaged tissues due to high potential of stem cell characteristics, easy accessibility and attainment, and their ectomesenchymal origin, which allow them to have potential for nerve, vessel, and dental tissue regeneration. This paper will cover the overview of dental tissue-derived iPSCs and their application with their advantages and drawbacks. PMID:26989423

  5. Reverse engineering human neurodegenerative disease using pluripotent stem cell technology.

    PubMed

    Liu, Ying; Deng, Wenbin

    2016-05-01

    With the technology of reprogramming somatic cells by introducing defined transcription factors that enables the generation of "induced pluripotent stem cells (iPSCs)" with pluripotency comparable to that of embryonic stem cells (ESCs), it has become possible to use this technology to produce various cells and tissues that have been difficult to obtain from living bodies. This advancement is bringing forth rapid progress in iPSC-based disease modeling, drug screening, and regenerative medicine. More and more studies have demonstrated that phenotypes of adult-onset neurodegenerative disorders could be rather faithfully recapitulated in iPSC-derived neural cell cultures. Moreover, despite the adult-onset nature of the diseases, pathogenic phenotypes and cellular abnormalities often exist in early developmental stages, providing new "windows of opportunity" for understanding mechanisms underlying neurodegenerative disorders and for discovering new medicines. The cell reprogramming technology enables a reverse engineering approach for modeling the cellular degenerative phenotypes of a wide range of human disorders. An excellent example is the study of the human neurodegenerative disease amyotrophic lateral sclerosis (ALS) using iPSCs. ALS is a progressive neurodegenerative disease characterized by the loss of upper and lower motor neurons (MNs), culminating in muscle wasting and death from respiratory failure. The iPSC approach provides innovative cell culture platforms to serve as ALS patient-derived model systems. Researchers have converted iPSCs derived from ALS patients into MNs and various types of glial cells, all of which are involved in ALS, to study the disease. The iPSC technology could be used to determine the role of specific genetic factors to track down what׳s wrong in the neurodegenerative disease process in the "disease-in-a-dish" model. Meanwhile, parallel experiments of targeting the same specific genes in human ESCs could also be performed to

  6. Lost in translation: pluripotent stem cell-derived hematopoiesis

    PubMed Central

    Ackermann, Mania; Liebhaber, Steffi; Klusmann, Jan-Henning; Lachmann, Nico

    2015-01-01

    Pluripotent stem cells (PSCs) such as embryonic stem cells or induced pluripotent stem cells represent a promising cell type to gain novel insights into human biology. Understanding the differentiation process of PSCs in vitro may allow for the identification of cell extrinsic/intrinsic factors, driving the specification process toward all cell types of the three germ layers, which may be similar to the human in vivo scenario. This would not only lay the ground for an improved understanding of human embryonic development but would also contribute toward the generation of novel cell types used in cell replacement therapies. In this line, especially the developmental process of mesodermal cells toward the hematopoietic lineage is of great interest. Therefore, this review highlights recent progress in the field of hematopoietic specification of pluripotent stem cell sources. In addition, we would like to shed light on emerging factors controlling primitive and definitive hematopoietic development and to highlight recent approaches to improve the differentiation potential of PSC sources toward hematopoietic stem/progenitor cells. While the generation of fully defined hematopoietic stem cells from PSCs remains challenging in vitro, we here underline the instructive role of cell extrinsic factors such as cytokines for the generation of PSC-derived mature hematopoietic cells. Thus, we have comprehensively examined the role of cytokines for the derivation of mature hematopoietic cell types such as macrophages, granulocytes, megakaryocytes, erythrocytes, dendritic cells, and cells of the B- and T-cell lineage. PMID:26174486

  7. Advances in Culture and Manipulation of Human Pluripotent Stem Cells

    PubMed Central

    Qian, X.; Villa-Diaz, L.G.; Krebsbach, P.H.

    2013-01-01

    Recent advances in the understanding of pluripotent stem cell biology and emerging technologies to reprogram somatic cells to a stem cell–like state are helping bring stem cell therapies for a range of human disorders closer to clinical reality. Human pluripotent stem cells (hPSCs) have become a promising resource for regenerative medicine and research into early development because these cells are able to self-renew indefinitely and are capable of differentiation into specialized cell types of all 3 germ layers and trophoectoderm. Human PSCs include embryonic stem cells (hESCs) derived from the inner cell mass of blastocyst-stage embryos and induced pluripotent stem cells (hiPSCs) generated via the reprogramming of somatic cells by the overexpression of key transcription factors. The application of hiPSCs and the finding that somatic cells can be directly reprogrammed into different cell types will likely have a significant impact on regenerative medicine. However, a major limitation for successful therapeutic application of hPSCs and their derivatives is the potential xenogeneic contamination and instability of current culture conditions. This review summarizes recent advances in hPSC culture and methods to induce controlled lineage differentiation through regulation of cell-signaling pathways and manipulation of gene expression as well as new trends in direct reprogramming of somatic cells. PMID:23934156

  8. Banking of pluripotent adult stem cells as an unlimited source for red blood cell production: potential applications for alloimmunized patients and rare blood challenges.

    PubMed

    Peyrard, Thierry; Bardiaux, Laurent; Krause, Claire; Kobari, Ladan; Lapillonne, Hélène; Andreu, Georges; Douay, Luc

    2011-07-01

    The transfusion of red blood cells (RBCs) is now considered a well-settled and essential therapy. However, some difficulties and constraints still occur, such as long-term blood product shortage, blood donor population aging, known and yet unknown transfusion-transmitted infectious agents, growing cost of the transfusion supply chain management, and the inescapable blood group polymorphism barrier. Red blood cells can be now cultured in vitro from human hematopoietic, human embryonic, or human-induced pluripotent stem cells (hiPSCs). The highly promising hiPSC technology represents a potentially unlimited source of RBCs and opens the door to the revolutionary development of a new generation of allogeneic transfusion products. Assuming that in vitro large-scale cultured RBC production efficiently operates in the near future, we draw here some futuristic but realistic scenarios regarding potential applications for alloimmunized patients and those with a rare blood group. We retrospectively studied a cohort of 16,486 consecutive alloimmunized patients (10-year period), showing 1 to 7 alloantibodies with 361 different antibody combinations. We showed that only 3 hiPSC clones would be sufficient to match more than 99% of the 16,486 patients in need of RBC transfusions. The study of the French National Registry of People with a Rare Blood Phenotype/Genotype (10-year period) shows that 15 hiPSC clones would cover 100% of the needs in patients of white ancestry. In addition, one single hiPSC clone would meet 73% of the needs in alloimmunized patients with sickle cell disease for whom rare cryopreserved RBC units were required. As a result, we consider that a very limited number of RBC clones would be able to not only provide for the need for most alloimmunized patients and those with a rare blood group but also efficiently allow for a policy for alloimmunization prevention in multiply transfused patients. PMID:21377319

  9. Induced pluripotent stem cells--alchemist's tale or clinical reality?

    PubMed

    Rashid, S Tamir; Vallier, Ludovic

    2010-01-01

    Following Shinya Yamanaka's first report describing the reprogramming of fibroblasts into stem cells over three years ago, some sceptics initially drew analogies between this new field of research and the quasi-mystical practice of 'alchemy'. Unlike the alchemist, however, stem cell researchers have rigorously tested and repeated experiments, proving their very own brand of cellular 'alchemy' to be a reality, with potentially massive implications for the study of human biology and clinical medicine. These investigations have resulted in an explosion of related publications and initiated the field of stem cell research known as 'induced pluripotency'. In this review, we give an account of the historical development, current technologies and potential clinical applications of induced pluripotency and conclude with a perspective on the possible future directions for this dynamic field. PMID:20707936

  10. Induced Pluripotent Stem Cells: Development in the Ophthalmologic Field

    PubMed Central

    2016-01-01

    Human induced pluripotent stem cells (iPSCs) are a type of stem cells that can be derived from human somatic cells by introducing certain transcription factors. Induced pluripotent stem cells can divide indefinitely and are able to differentiate into every cell type, which make them viable for transplantation and individual disease modeling. Recently, various ocular cells, including corneal epithelial-like cells, retinal pigment epithelium (RPE) cells displaying functions similar to native RPE, photoreceptors, and retinal ganglion cells, have all been successfully derived from iPSCs. Transplantation of these cells in animal models showed great promise for reversing blindness, and the first clinical trial on humans started in 2013. Despite these promising results, more research is in demand for preventing inadvertent tumor growth, developing precise functionality of the cells, and promoting integration into the host tissue. PMID:27594887

  11. Induced Pluripotent Stem Cells: Development in the Ophthalmologic Field.

    PubMed

    Wu, Nan; Doorenbos, Marianne; Chen, Dong Feng

    2016-01-01

    Human induced pluripotent stem cells (iPSCs) are a type of stem cells that can be derived from human somatic cells by introducing certain transcription factors. Induced pluripotent stem cells can divide indefinitely and are able to differentiate into every cell type, which make them viable for transplantation and individual disease modeling. Recently, various ocular cells, including corneal epithelial-like cells, retinal pigment epithelium (RPE) cells displaying functions similar to native RPE, photoreceptors, and retinal ganglion cells, have all been successfully derived from iPSCs. Transplantation of these cells in animal models showed great promise for reversing blindness, and the first clinical trial on humans started in 2013. Despite these promising results, more research is in demand for preventing inadvertent tumor growth, developing precise functionality of the cells, and promoting integration into the host tissue. PMID:27594887

  12. Transgene Reactivation in Induced Pluripotent Stem Cell Derivatives and Reversion to Pluripotency of Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells.

    PubMed

    Galat, Vasiliy; Galat, Yekaterina; Perepitchka, Mariana; Jennings, Lawrence J; Iannaccone, Philip M; Hendrix, Mary J C

    2016-07-15

    Induced pluripotent stem cells (iPSCs) have enormous potential in regenerative medicine and disease modeling. It is now felt that clinical trials should be performed with iPSCs derived with nonintegrative constructs. Numerous studies, however, including those describing disease models, are still being published using cells derived from iPSCs generated with integrative constructs. Our experimental work presents the first evidence of spontaneous transgene reactivation in vitro in several cellular types. Our results show that the transgenes were predominantly silent in parent iPSCs, but in mesenchymal and endothelial iPSC derivatives, the transgenes experienced random upregulation of Nanog and c-Myc. Additionally, we provide evidence of spontaneous secondary reprogramming and reversion to pluripotency in mesenchymal stem cells derived from iPSCs. These findings strongly suggest that the studies, which use cellular products derived from iPSCs generated with retro- or lentiviruses, should be evaluated with consideration of the possibility of transgene reactivation. The in vitro model described here provides insight into the earliest events of culture transformation and suggests the hypothesis that reversion to pluripotency may be responsible for the development of tumors in cell replacement experiments. The main goal of this work, however, is to communicate the possibility of transgene reactivation in retro- or lenti-iPSC derivatives and the associated loss of cellular fidelity in vitro, which may impact the outcomes of disease modeling and related experimentation. PMID:27193052

  13. Transgene Reactivation in Induced Pluripotent Stem Cell Derivatives and Reversion to Pluripotency of Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells

    PubMed Central

    Galat, Yekaterina; Perepitchka, Mariana; Jennings, Lawrence J.; Iannaccone, Philip M.; Hendrix, Mary J.C.

    2016-01-01

    Induced pluripotent stem cells (iPSCs) have enormous potential in regenerative medicine and disease modeling. It is now felt that clinical trials should be performed with iPSCs derived with nonintegrative constructs. Numerous studies, however, including those describing disease models, are still being published using cells derived from iPSCs generated with integrative constructs. Our experimental work presents the first evidence of spontaneous transgene reactivation in vitro in several cellular types. Our results show that the transgenes were predominantly silent in parent iPSCs, but in mesenchymal and endothelial iPSC derivatives, the transgenes experienced random upregulation of Nanog and c-Myc. Additionally, we provide evidence of spontaneous secondary reprogramming and reversion to pluripotency in mesenchymal stem cells derived from iPSCs. These findings strongly suggest that the studies, which use cellular products derived from iPSCs generated with retro- or lentiviruses, should be evaluated with consideration of the possibility of transgene reactivation. The in vitro model described here provides insight into the earliest events of culture transformation and suggests the hypothesis that reversion to pluripotency may be responsible for the development of tumors in cell replacement experiments. The main goal of this work, however, is to communicate the possibility of transgene reactivation in retro- or lenti-iPSC derivatives and the associated loss of cellular fidelity in vitro, which may impact the outcomes of disease modeling and related experimentation. PMID:27193052

  14. Fate by RNA methylation: m6A steers stem cell pluripotency.

    PubMed

    Zhao, Boxuan Simen; He, Chuan

    2015-01-01

    The N 6-methyladenosine (m6A) modification of mRNA has a crucial function in regulating pluripotency in murine stem cells: it facilitates resolution of naïve pluripotency towards differentiation. PMID:25723450

  15. "Mouse Clone Model" for evaluating the immunogenicity and tumorigenicity of pluripotent stem cells.

    PubMed

    Zhang, Gang; Zhang, Yi

    2015-01-01

    To investigate the immune-rejection and tumor-formation potentials of induced pluripotent stem cells and other stem cells, we devised a model-designated the "Mouse Clone Model"-which combined the theory of somatic animal cloning, tetraploid complementation, and induced pluripotent stem cells to demonstrate the applicability of stem cells for transplantation therapy. PMID:26687081

  16. Reprogramming T cell Lymphocytes to Induced Pluripotent Stem Cells

    NASA Astrophysics Data System (ADS)

    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.

  17. Induced pluripotent stem cells as a source of hepatocytes

    PubMed Central

    Sauer, Vanessa; Roy-Chowdhury, Namita; Guha, Chandan; Roy-Chowdhury, Jayanta

    2014-01-01

    During the past decade, a series of discoveries has established the potential of the so called terminally differentiated cells to transition to more primitive progenitor cells. The dramatic demonstration of the ability to reprogram differentiated somatic cells to induced pluripotent stem cells (iPSC) that can then give rise to cells of all three germ layers has opened the possibility of generating virtually any cell type in culture, from any given individual. Taking advantage of these concepts, researchers have generated iPSCs by reprogramming a wide variety of somatic cells. In addition to their practical implications, these studies have provided crucial insights into the mechanism of cell plasticity that underlies the transition from one cell type to another. Using concepts derived from research on embryological development, investigators have differentiated iPSCs to cells resembling hepatocytes in many ways. Such hepatocyte-like cells could be of enormous value in disease modeling, drug discovery and regenerative medicine. However, the currently available methods do not yield cells that fully reproduce the characteristics of adult primary hepatocytes. Thus generating hepatocytes from iPSCs is very much a work in progress. In addition to chronicling these exciting developments, this review will discuss the emergent new approaches to generating iPSCs, improving their differentiation to hepatocyte-like cells and maintaining the hepatocyte-like cells in culture for longer survival and better function. PMID:25650171

  18. Making gametes from pluripotent stem cells--a promising role for very small embryonic-like stem cells.

    PubMed

    Bhartiya, Deepa; Hinduja, Indira; Patel, Hiren; Bhilawadikar, Rashmi

    2014-01-01

    The urge to have one's own biological child supersedes any desire in life. Several options have been used to obtain gametes including pluripotent stem cells (embryonic ES and induced pluripotent iPS stem cells); gonadal stem cells (spermatogonial SSCs, ovarian OSCs stem cells), bone marrow, mesenchymal cells and fetal skin. However, the field poses a huge challenge including inefficient existing protocols for differentiation, epigenetic and genetic changes associated with extensive in vitro manipulation and also ethical/regulatory constraints. A tremendous leap in the field occurred using mouse ES and iPS cells wherein they were first differentiated into epiblast-like cells and then primordial germ cell-like cells. These on further development produced sperm, oocytes and live offspring (had associated genetic problems). Evidently differentiating pluripotent stem cells into primordial germ cells (PGCs) remains a major bottleneck. Against this backdrop, we propose that a novel population of pluripotent stem cells termed very small embryonic-like stem cells (VSELs) may serve as an alternative, potential source of autologus gametes, keeping in mind that they are indeed PGCs surviving in adult mammalian ovaries and testes. Both VSELs and PGCs are pluripotent, relatively quiescent because of epigenetic modifications of parentally imprinted genes loci like Igf2-H19 and KCNQ1p57, share several markers like Stella, Fragilis, Mvh, Dppa2, Dppa4, Sall4, Blimp1 and functional receptors. VSELs are localized in the basement membrane of seminiferous tubules in testis and in the ovary surface epithelium. Ovarian stem cells from mouse, rabbit, sheep, marmoset and humans (menopausal women and those with premature ovarian failure) spontaneously differentiate into oocyte-like structures in vitro with no additional requirement of growth factors. Thus a more pragmatic option to obtain autologus gametes may be the pluripotent VSELs and if we could manipulate them in vivo - existing

  19. Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin

    PubMed Central

    Dorn, Isabel; Klich, Katharina; Arauzo-Bravo, Marcos J.; Radstaak, Martina; Santourlidis, Simeon; Ghanjati, Foued; Radke, Teja F.; Psathaki, Olympia E.; Hargus, Gunnar; Kramer, Jan; Einhaus, Martin; Kim, Jeong Beom; Kögler, Gesine; Wernet, Peter; Schöler, Hans R.; Schlenke, Peter; Zaehres, Holm

    2015-01-01

    Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34+ hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34+ hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34+ hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34+ cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential. PMID:25326431

  20. Pluripotent Stem Cells and Other Innovative Strategies for the Treatment of Ocular Surface Diseases.

    PubMed

    Erbani, Johanna; Aberdam, Daniel; Larghero, Jerome; Vanneaux, Valérie

    2016-04-01

    The cornea provides two thirds of the refractive power of the eye and protection against insults such as infection and injury. The outermost tissue of the cornea is renewed by stem cells located in the limbus. Depletion or destruction of these stem cells may lead to blinding limbal stem cell deficiency (LSCD) that concerns millions of patients around the world. Innovative strategies based on adult stem cell therapies have been developed in the recent years but they are still facing numerous unresolved issues, and the long term results can be deceiving. Today there is a clear need to improve these therapies, and/or to develop new approaches for the treatment of LSCD. Here, we review the current cell-based therapies used for the treatment of ocular diseases, and discuss the potential of pluripotent stem cells (embryonic and induced pluripotent stem cells) in corneal repair. As the secretion of paracrine factors is known to have a crucial role in maintaining stem cell homeostasis and in wound repair, we also consider the therapeutic potential of a promising novel pathway, the exosomes. Exosomes are nano-sized vesicles that have the ability to transfer RNAs and proteins to recipient cells, and several studies demonstrated their role in cell protection and wound healing. Exosomes could circumvent the hurdles of stem-cell based approaches, and they could become a strong candidate as an alternative therapy for ocular surface diseases. PMID:26779895

  1. Livestock Models for Exploiting the Promise of Pluripotent Stem Cells

    PubMed Central

    Roberts, R. Michael; Yuan, Ye; Genovese, Nicholas; Ezashi, Toshihiko

    2015-01-01

    Livestock species are widely used as biomedical models. Pigs, in particular, are beginning to have a significant role in regenerative medicine for testing the applicability, success, and safety of grafts derived from induced pluripotent stem cells. Animal testing must always be performed before any clinical trials are performed in humans, and pigs may sometimes be the species of choice because of their physiological and anatomical similarities to humans. Induced pluripotent stem cells (iPSC) have been generated with some success from livestock species by a variety of reprogramming procedures, but authenticated embryonic stem cells (ESC) have not. There are now several studies in which porcine iPSC have been tested for their ability to provide functional grafts in pigs. Pigs have also served as recipients for grafts derived from human iPSC. There have also been recent advances in creating pigs with severe combined immunodeficiency (SCID). Like SCID mice, these pigs are expected to be graft tolerant. Additionally, chimeric, partially humanized pigs could be sources of human organs. Another potential application of pluripotent stem cells from livestock is for the purpose of differentiating the cells into skeletal muscle, which, in turn, could be used either to produce cultured meat or to engraft into damaged muscle. None of these technologies has advanced to a stage that they have become mainstream, however. Despite the value of livestock models in regenerative medicine, only a limited number of institutions are able to use these animals. PMID:25991700

  2. Livestock models for exploiting the promise of pluripotent stem cells.

    PubMed

    Roberts, R Michael; Yuan, Ye; Genovese, Nicholas; Ezashi, Toshihiko

    2015-01-01

    Livestock species are widely used as biomedical models. Pigs, in particular, are beginning to have a significant role in regenerative medicine for testing the applicability, success, and safety of grafts derived from induced pluripotent stem cells. Animal testing must always be performed before any clinical trials are performed in humans, and pigs may sometimes be the species of choice because of their physiological and anatomical similarities to humans. Induced pluripotent stem cells (iPSC) have been generated with some success from livestock species by a variety of reprogramming procedures, but authenticated embryonic stem cells (ESC) have not. There are now several studies in which porcine iPSC have been tested for their ability to provide functional grafts in pigs. Pigs have also served as recipients for grafts derived from human iPSC. There have also been recent advances in creating pigs with severe combined immunodeficiency (SCID). Like SCID mice, these pigs are expected to be graft tolerant. Additionally, chimeric, partially humanized pigs could be sources of human organs. Another potential application of pluripotent stem cells from livestock is for the purpose of differentiating the cells into skeletal muscle, which, in turn, could be used either to produce cultured meat or to engraft into damaged muscle. None of these technologies has advanced to a stage that they have become mainstream, however. Despite the value of livestock models in regenerative medicine, only a limited number of institutions are able to use these animals. PMID:25991700

  3. Challenges of stem cell therapy for spinal cord injury: human embryonic stem cells, endogenous neural stem cells, or induced pluripotent stem cells?

    PubMed

    Ronaghi, Mohammad; Erceg, Slaven; Moreno-Manzano, Victoria; Stojkovic, Miodrag

    2010-01-01

    Spinal cord injury (SCI) causes myelopathy, damage to white matter, and myelinated fiber tracts that carry sensation and motor signals to and from the brain. The gray matter damage causes segmental losses of interneurons and motoneurons and restricts therapeutic options. Recent advances in stem cell biology, neural injury, and repair, and the progress toward development of neuroprotective and regenerative interventions are the basis for increased optimism. This review summarizes the pathophysiological mechanisms following SCI and compares human embryonic, adult neural, and the induced pluripotent stem cell-based therapeutic strategies for SCI. PMID:19904738

  4. The use of pluripotent stem cell for personalized cell therapies against neurological disorders.

    PubMed

    Ha, Hye-Yeong; Jang, Si-Hyong; Jung, Ji-Won

    2011-01-01

    Although there are a number of weaknesses for clinical use, pluripotent stem cells are valuable sources for patient-specific cell therapies against various diseases. Backed-up by a huge number of basic researches, neuronal differentiation mechanism is well established and pluripotent stem cell therapies against neurological disorders are getting closer to clinical application. However, there are increasing needs for standardization of the sourcing pluripotent stem cells by establishing stem cell registries and banking. Global harmonization will accelerate practical use of personalized therapies using pluripotent stem cells. PMID:22203784

  5. Induced Pluripotent Stem Cells and Their Use in Cardiac and Neural Regenerative Medicine

    PubMed Central

    Skalova, Stepanka; Svadlakova, Tereza; Qureshi, Wasay Mohiuddin Shaikh; Dev, Kapil; Mokry, Jaroslav

    2015-01-01

    Stem cells are unique pools of cells that are crucial for embryonic development and maintenance of adult tissue homeostasis. The landmark Nobel Prize winning research by Yamanaka and colleagues to induce pluripotency in somatic cells has reshaped the field of stem cell research. The complications related to the usage of pluripotent embryonic stem cells (ESCs) in human medicine, particularly ESC isolation and histoincompatibility were bypassed with induced pluripotent stem cell (iPSC) technology. The human iPSCs can be used for studying embryogenesis, disease modeling, drug testing and regenerative medicine. iPSCs can be diverted to different cell lineages using small molecules and growth factors. In this review we have focused on iPSC differentiation towards cardiac and neuronal lineages. Moreover, we deal with the use of iPSCs in regenerative medicine and modeling diseases like myocardial infarction, Timothy syndrome, dilated cardiomyopathy, Parkinson’s, Alzheimer’s and Huntington’s disease. Despite the promising potential of iPSCs, genome contamination and low efficacy of cell reprogramming remain significant challenges. PMID:25689424

  6. Site-Specific Genome Engineering in Human Pluripotent Stem Cells.

    PubMed

    Merkert, Sylvia; Martin, Ulrich

    2016-01-01

    The possibility to generate patient-specific induced pluripotent stem cells (iPSCs) offers an unprecedented potential of applications in clinical therapy and medical research. Human iPSCs and their differentiated derivatives are tools for diseases modelling, drug discovery, safety pharmacology, and toxicology. Moreover, they allow for the engineering of bioartificial tissue and are promising candidates for cellular therapies. For many of these applications, the ability to genetically modify pluripotent stem cells (PSCs) is indispensable, but efficient site-specific and safe technologies for genetic engineering of PSCs were developed only recently. By now, customized engineered nucleases provide excellent tools for targeted genome editing, opening new perspectives for biomedical research and cellular therapies. PMID:27347935

  7. Site-Specific Genome Engineering in Human Pluripotent Stem Cells

    PubMed Central

    Merkert, Sylvia; Martin, Ulrich

    2016-01-01

    The possibility to generate patient-specific induced pluripotent stem cells (iPSCs) offers an unprecedented potential of applications in clinical therapy and medical research. Human iPSCs and their differentiated derivatives are tools for diseases modelling, drug discovery, safety pharmacology, and toxicology. Moreover, they allow for the engineering of bioartificial tissue and are promising candidates for cellular therapies. For many of these applications, the ability to genetically modify pluripotent stem cells (PSCs) is indispensable, but efficient site-specific and safe technologies for genetic engineering of PSCs were developed only recently. By now, customized engineered nucleases provide excellent tools for targeted genome editing, opening new perspectives for biomedical research and cellular therapies. PMID:27347935

  8. Generation of Megakaryocytes and Platelets from Human Pluripotent Stem Cells.

    PubMed

    Pick, Marjorie

    2016-01-01

    Human pluripotent stem cells (hPSC) have the potential to produce any tissue type in the body and thus represent a source of cells for regenerative medicine. Here we have shown that human platelets can be produced from embryonic or induced pluripotent stem cells in a defined culture system. We describe a serum- and feeder-free culture system that enabled the generation of megakaryocyte (Mk) progenitors and functional platelets from hPSCs. After 13 days the differentiated population included precursor cells that formed colonies containing differentiated Mks, and after 20 days these Mks were able to fragment into platelet-like particles that were functional. This protocol represents an important step towards the generation of human platelets for therapeutic use. PMID:24297316

  9. Human Induced Pluripotent Stem Cell Models of Inherited Cardiovascular Diseases.

    PubMed

    Jiang, Wenjian; Lan, Feng; Zhang, Hongjia

    2014-10-16

    Cardiovascular cells derived from patient specific induced Pluripotent Stem Cell (iPSC) harbor gene mutations associated with the pathogenesis of inherited cardiac diseases and congenital heart diseases (CHD). Numerous reports have demonstrated the utilization of human induced Pluripotent Stem Cell (hiPSC) to model cardiac diseases as a means of investigating their underlying mechanisms. So far, they have been shown to investigate the molecular mechanisms of many cardiac disorders, such as long-QT syndrome (LQT), catecholaminergic polymorphic ventricular tachycardia (CPVT), dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), LEOPARD syndrome (LS), arrhythmogenic cardiomyopathy (ACM), Friedreich ataxia (FRDA), Barth syndrome (BTHS), hypoplastic left heart syndrome (HLHS), Marfan syndrome (MFS) and other CHD. This article summarizes the growing body of research related to modeling various cardiac diseases using hiPSCs. Moreover, by reviewing the methods used in previous studies, we propose multiple novel applications of hiPSCs to investigate comprehensive cardiovascular disorders and facilitate drug discovery. PMID:25322695

  10. Myogenic Progenitors from Mouse Pluripotent Stem Cells for Muscle Regeneration.

    PubMed

    Magli, Alessandro; Incitti, Tania; Perlingeiro, Rita C R

    2016-01-01

    Muscle homeostasis is maintained by resident stem cells which, in both pathologic and non-pathologic conditions, are able to repair or generate new muscle fibers. Although muscle stem cells have tremendous regenerative potential, their application in cell therapy protocols is prevented by several restrictions, including the limited ability to grow ex vivo. Since pluripotent stem cells have the unique potential to both self-renew and expand almost indefinitely, they have become an attractive source of progenitors for regenerative medicine studies. Our lab has demonstrated that embryonic stem cell (ES)-derived myogenic progenitors retain the ability to repair existing muscle fibers and contribute to the pool of resident stem cells. Because of their relevance in both cell therapy and disease modeling, in this chapter we describe the protocol to derive myogenic progenitors from murine ES cells followed by their intramuscular delivery in a murine muscular dystrophy model. PMID:27492174

  11. Single-Cell Gene Expression Profiles Define Self-Renewing, Pluripotent, and Lineage Primed States of Human Pluripotent Stem Cells

    PubMed Central

    Hough, Shelley R.; Thornton, Matthew; Mason, Elizabeth; Mar, Jessica C.; Wells, Christine A.; Pera, Martin F.

    2014-01-01

    Summary Pluripotent stem cells display significant heterogeneity in gene expression, but whether this diversity is an inherent feature of the pluripotent state remains unknown. Single-cell gene expression analysis in cell subsets defined by surface antigen expression revealed that human embryonic stem cell cultures exist as a continuum of cell states, even under defined conditions that drive self-renewal. The majority of the population expressed canonical pluripotency transcription factors and could differentiate into derivatives of all three germ layers. A minority subpopulation of cells displayed high self-renewal capacity, consistently high transcripts for all pluripotency-related genes studied, and no lineage priming. This subpopulation was characterized by its expression of a particular set of intercellular signaling molecules whose genes shared common regulatory features. Our data support a model of an inherently metastable self-renewing population that gives rise to a continuum of intermediate pluripotent states, which ultimately become primed for lineage specification. PMID:24936473

  12. Regeneration of tracheal epithelium using mouse induced pluripotent stem cells.

    PubMed

    Ikeda, Masakazu; Imaizumi, Mitsuyoshi; Yoshie, Susumu; Otsuki, Koshi; Miyake, Masao; Hazama, Akihiro; Wada, Ikuo; Omori, Koichi

    2016-04-01

    Conclusion The findings demonstrated the potential use of induced pluripotent stem cells for regeneration of tracheal epithelium. Objective Autologous tissue implantation techniques using skin or cartilage are often applied in cases of tracheal defects with laryngeal inflammatory lesions and malignant tumor invasion. However, these techniques are invasive with an unstable clinical outcome. The purpose of this study was to investigate regeneration in a tracheal defect site of nude rats after implantation of ciliated epithelium that was differentiated from induced pluripotent stem cells. Method Embryoid bodies were formed from mouse induced pluripotent stem cells. They were cultured with growth factors for 5 days, and then cultured at the air-liquid interface. The degree of differentiation achieved prior to implantation was determined by histological findings and the results of real-time polymerase chain reaction. Embryoid bodies including ciliated epithelium were embedded into collagen gel that served as an artificial scaffold, and then implanted into nude rats, creating an 'air-liquid interface model'. Histological evaluation was performed 7 days after implantation. Results The ciliated epithelial structure survived on the lumen side of regenerated tissue. It was demonstrated histologically that the structure was composed of ciliated epithelial cells. PMID:26755348

  13. The safety of human pluripotent stem cells in clinical treatment.

    PubMed

    Simonson, Oscar E; Domogatskaya, Anna; Volchkov, Pavel; Rodin, Sergey

    2015-01-01

    Human pluripotent stem cells (hPSCs) have practically unlimited proliferation potential and a capability to differentiate into any cell type in the human body. Since the first derivation in 1998, they have been an attractive source of cells for regenerative medicine. Numerous ethical, technological, and regulatory complications have been hampering hPSC use in clinical applications. Human embryonic stem cells (ESCs), parthenogenetic human ESCs, human nuclear transfer ESCs, and induced pluripotent stem cells are four types of hPSCs that are different in many clinically relevant features such as propensity to epigenetic abnormalities, generation methods, and ability for development of autologous cell lines. Propensity to genetic mutations and tumorigenicity are common features of all pluripotent cells that complicate hPSC-based therapies. Several recent advances in methods of derivation, culturing, and monitoring of hPSCs have addressed many ethical concerns and technological challenges in development of clinical-grade hPSC lines. Generation of banks of such lines may be useful to minimize immune rejection of hPSC-derived allografts. In this review, we discuss different sources of hPSCs available at the moment, various safety risks associated with them, and possible solutions for successful use of hPSCs in the clinic. We also discuss ongoing clinical trials of hPSC-based treatments. PMID:26140342

  14. Induced pluripotent stem (iPS) cells offer a powerful new tool for the life sciences.

    PubMed

    Nakamura, Y

    2010-01-01

    Stem cell biology started with the analysis of somatic stem cells that function to maintain the adult body. We now know that the body is maintained by regeneration of a wide range of cell types, such as skin cells, blood cells and gastrointestinal mucous cells, from somatic stem cells. This regenerative activity is essential for survival. Regenerative medicine was initiated to identify therapies that support and/or accelerate this natural regenerative ability. For example, bone marrow transplantation is a therapy for reconstituting hematopoiesis from the hematopoietic stem cells present in the donor bone marrow. The successful development of a protocol for obtaining human embryonic stem (ES) cells prompted medical scientists to utilize human ES cells for regenerative medicine. However, use of these cells raises ethical issues as they are derived from human embryos. An alternative approach using ES-like pluripotent stem cells has the considerable advantage that it does not necessitate use of human embryos. Pluripotent stem cells can be induced from terminally differentiated somatic cells by the introduction of only four defined factors. The products of this method are termed "induced pluripotent stem (iPS)" cells. iPS cells have considerable promise as a substitute for ES cells not only for regenerative medicine but also in many other fields. For example, liver and heart cells derived from iPS cells can be used in pharmaceutical research. In addition, iPS cell technology opens new avenues of disease research, for example, by construction of so-called "disease-specific iPS cells" from a patient's somatic cells. PMID:24693054

  15. Efficient induction of pluripotent stem cells from menstrual blood.

    PubMed

    Li, Yang; Li, Xiaoni; Zhao, Hongxi; Feng, Ruopeng; Zhang, Xiaoyan; Tai, Dapeng; An, Guangyu; Wen, Jinhua; Tan, Jichun

    2013-04-01

    The technology to reprogram human somatic cells back to pluripotency allows the production of patient-specific induced pluripotent stem cells (iPSCs) and holds a great promise for regenerative medicine. Choosing the most suitable cell type for induction and reducing the risk of viral transgene activation, especially oncogene activation, are important for iPSC research. To date, human dermal fibroblasts (HDFs) are the most frequent cell source used for iPSC generation, but they have several limitations. An invasive skin biopsy must be performed to obtain HDFs, and HDFs must be cultured for a prolonged period before they can be used for experiments. Thus, in an effort to develop a suitable source for iPSC studies to avoid the limitations mentioned above, we have here identified stromal cells derived from menstrual blood (MenSCs) as suitable candidates. In the present study, we found that MenSCs can be reprogrammed to pluripotent status by doxycycline-inducible lentiviral transduction of OCT4, SOX2, and KLF4. Additionally, we found that MenSCs have a significantly higher reprogramming efficiency than HDFs. The combination of OCT4 and SOX2 is sufficient to reprogram MenSCs into iPSCs without the use of c-MYC or KLF4. The resulting MenSC-iPSCs showed the same characteristics as human embryonic stem cells with regard to morphology, pluripotent markers, gene expression, and the epigenetic status of pluripotent-cell-specific genes. These cells were able to differentiate into various cell types of all 3 germ layers both in vitro and in vivo. Therefore, MenSCs may be a preferred candidate for generation of iPSCs. PMID:23151296

  16. Adult stem-like cells in kidney.

    PubMed

    Hishikawa, Keiichi; Takase, Osamu; Yoshikawa, Masahiro; Tsujimura, Taro; Nangaku, Masaomi; Takato, Tsuyoshi

    2015-03-26

    Human pluripotent cells are promising for treatment for kidney diseases, but the protocols for derivation of kidney cell types are still controversial. Kidney tissue regeneration is well confirmed in several lower vertebrates such as fish, and the repair of nephrons after tubular damages is commonly observed after renal injury. Even in adult mammal kidney, renal progenitor cell or system is reportedly presents suggesting that adult stem-like cells in kidney can be practical clinical targets for kidney diseases. However, it is still unclear if kidney stem cells or stem-like cells exist or not. In general, stemness is defined by several factors such as self-renewal capacity, multi-lineage potency and characteristic gene expression profiles. The definite use of stemness may be obstacle to understand kidney regeneration, and here we describe the recent broad findings of kidney regeneration and the cells that contribute regeneration. PMID:25815133

  17. OSKM Induce Extraembryonic Endoderm Stem Cells in Parallel to Induced Pluripotent Stem Cells

    PubMed Central

    Parenti, Anthony; Halbisen, Michael A.; Wang, Kai; Latham, Keith; Ralston, Amy

    2016-01-01

    Summary The reprogramming factors OCT4, SOX2, KLF4, and MYC (OSKM) can reactivate the pluripotency network in terminally differentiated cells, but also regulate expression of non-pluripotency genes in other contexts, such as the mouse primitive endoderm. The primitive endoderm is an extraembryonic lineage established in parallel to the pluripotent epiblast in the blastocyst, and is the progenitor pool for extraembryonic endoderm stem (XEN) cells. We show that OSKM induce expression of endodermal genes, leading to formation of induced XEN (iXEN) cells, which possess key properties of blastocyst-derived XEN cells, including morphology, transcription profile, self-renewal, and multipotency. Our data show that iXEN cells arise in parallel to induced pluripotent stem cells, indicating that OSKM drive cells to two distinct cell fates during reprogramming. PMID:26947975

  18. Human pluripotent stem cell models of Fragile X syndrome.

    PubMed

    Bhattacharyya, Anita; Zhao, Xinyu

    2016-06-01

    Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability and autism. The causal mutation in FXS is a trinucleotide CGG repeat expansion in the FMR1 gene that leads to human specific epigenetic silencing and loss of Fragile X Mental Retardation Protein (FMRP) expression. Human pluripotent stem cells (PSCs), including human embryonic stem cells (ESCs) and particularly induced PSCs (iPSCs), offer a model system to reveal cellular and molecular events underlying human neuronal development and function in FXS. Human FXS PSCs have been established and have provided insight into the epigenetic silencing of the FMR1 gene as well as aspects of neuronal development. PMID:26640241

  19. Current state of the opportunities for derivation of germ-like cells from pluripotent stem cells: are you a man, or a mouse?

    PubMed Central

    Petkova, Rumena; Arabadjiev, Borislav; Chakarov, Stoyan; Pankov, Roumen

    2014-01-01

    The concept of pluripotency as a prerogative of cells of early mammal embryos and cultured embryonic stem cells (ESC) has been invalidated with the advent of induced pluripotent stem cells. Later, it became clear that the ability to generate all cell types of the adult organism is also a questionable aspect of pluripotency, as there are cell types, such as germ cells, which are difficult to produce from pluripotent stem cells. Recently it has been proposed that there are at least two different states of pluripotency; namely, the naïve, or ground state, and the primed state, which may differ radically in terms of timeline of existence, signalling mechanisms, cell properties, capacity for differentiation into different cell types, etc. Germ-like male and female rodent cells have been successfully produced in vitro from ESC and induced pluripotent stem cells. The attempts to derive primate primordial germ cells (PGC) and germ cells in vitro from pluripotent stem cells, however, still have a low success rate, especially with the female germline. The paper reviews the properties of rodent and primate ESC with regard to their capacity for differentiation in vitro to germ-like cells, outlining the possible caveats to derivation of PGC and germ cells from primate and human pluripotent cells. PMID:26019504

  20. Big Animal Cloning Using Transgenic Induced Pluripotent Stem Cells: A Case Study of Goat Transgenic Induced Pluripotent Stem Cells.

    PubMed

    Song, Hui; Li, Hui; Huang, Mingrui; Xu, Dan; Wang, Ziyu; Wang, Feng

    2016-02-01

    Using of embryonic stem cells (ESCs) could improve production traits and disease resistance by improving the efficiency of somatic cell nuclear transfer (SCNT) technology. However, robust ESCs have not been established from domestic ungulates. In the present study, we generated goat induced pluripotent stem cells (giPSCs) and transgenic cloned dairy goat induced pluripotent stem cells (tgiPSCs) from dairy goat fibroblasts (gFs) and transgenic cloned dairy goat fibroblasts (tgFs), respectively, using lentiviruses that contained hOCT4, hSOX2, hMYC, and hKLF4 without chemical compounds. The giPSCs and tgiPSCs expressed endogenous pluripotent markers, including OCT4, SOX2, MYC, KLF4, and NANOG. Moreover, they were able to maintain a normal karyotype and differentiate into derivatives from all three germ layers in vitro and in vivo. Using SCNT, tgFs and tgiPSCs were used as donor cells to produce embryos, which were named tgF-Embryos and tgiPSC-Embryos. The fusion rates and cleavage rates had no significant differences between tgF-Embryos and tgiPSC-Embryos. However, the expression of IGF-2, which is an important gene associated with embryonic development, was significantly lower in tgiPSC-Embryos than in tgF-Embryos and was not significantly different from vivo-Embryos. PMID:26836033

  1. The application of induced pluripotent stem cells for bone regeneration: current progress and prospects.

    PubMed

    Teng, Songsong; Liu, Chaoxu; Krettek, Christian; Jagodzinski, Michael

    2014-08-01

    Loss of healthy bone tissue and dysosteogenesis are still common and significant problems in clinics. Cell-based therapy using mesenchymal stem cells (MSCs) has been performed in patients for quite some time, but the inherent drawbacks of these cells, such as the reductions in proliferation rate and osteogenic differentiation potential that occur with aging, greatly limit their further application. Moreover, embryonic stem cells (ESCs) have brought new hope to osteoregenerative medicine because of their full pluripotent differentiation potential and excellent performance in bone regeneration. However, the ethical issues involved in destroying human embryos and the immune reactions that occur after transplantation are two major stumbling blocks impeding the clinical application of ESCs. Instead, induced pluripotent stem cells (iPSCs), which are ESC-like pluripotent cells that are reprogrammed from adult somatic cells using defined transcription factors, are considered a more promising source of cells for regenerative medicine because they present no ethical or immunological issues. Here, we summarize the primary technologies for generating iPSCs and the biological properties of these cells, review the current advances in iPSC-based bone regeneration and, finally, discuss the remaining challenges associated with these cells, particularly safety issues and their potential application for osteoregenerative medicine. PMID:24102431

  2. Vascular potential of human pluripotent stem cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cardiovascular disease is the number one cause of death and disability in the US. Understanding the biological activity of stem and progenitor cells, and their ability to contribute to the repair, regeneration and remodeling of the heart and blood vessels affected by pathological processes is an ess...

  3. Human-induced pluripotent stem cells produced under xeno-free conditions.

    PubMed

    Ross, Pablo Juan; Suhr, Steven Thomas; Rodriguez, Ramon Maria; Chang, Eun-Ah; Wang, Kai; Siripattarapravat, Kannika; Ko, Tak; Cibelli, Jose Bernardo

    2010-08-01

    Induced pluripotent stem cells (iPSCs) have radically advanced the field of regenerative medicine by making possible the production of patient-specific pluripotent stem cells from adult individuals. While cell differentiation protocols have been successfully developed, and animal models of human disease have proved that these cells have the potential to treat human diseases and conditions produced as a consequence of aging, degeneration, injury, and birth defects, logistical issues still remain unsolved and hamper the possibility of testing these cells in human clinical trials. Among them is the widely spread use of animal products for the generation and culture of iPSCs. We report here a xeno-free iPSC generation system that addresses all the steps of iPSCs production including the isolation and culture of adult skin fibroblasts, and iPSCs generation, expansion, and maintenance. iPSCs generated with a polycistronic lentiviral vector under xeno-free conditions displayed markers of pluripotency and gave rise to embryoid bodies (EBs) displaying indicators of the 3 primary germ layers. Xeno-free iPSCs injected into nude mice produced classic teratomas, and teratoma explants cultured under conditions favoring fibroblastic cells gave rise to cells morphologically indistinguishable from input cells. Protocols here described will facilitate the implementation of new cellular therapies for preclinical and clinical studies, potentially reducing the regulatory burden without compromising the differentiation potential of the cells. PMID:20030562

  4. Genome editing in pluripotent stem cells: research and therapeutic applications.

    PubMed

    Deleidi, Michela; Yu, Cong

    2016-05-01

    Recent progress in human pluripotent stem cell (hPSC) and genome editing technologies has opened up new avenues for the investigation of human biology in health and disease as well as the development of therapeutic applications. Gene editing approaches with programmable nucleases have been successfully established in hPSCs and applied to study gene function, develop novel animal models and perform genetic and chemical screens. Several studies now show the successful editing of disease-linked alleles in somatic and patient-derived induced pluripotent stem cells (iPSCs) as well as in animal models. Importantly, initial clinical trials have shown the safety of programmable nucleases for ex vivo somatic gene therapy. In this context, the unlimited proliferation potential and the pluripotent properties of iPSCs may offer advantages for gene targeting approaches. However, many technical and safety issues still need to be addressed before genome-edited iPSCs are translated into the clinical setting. Here, we provide an overview of the available genome editing systems and discuss opportunities and perspectives for their application in basic research and clinical practice, with a particular focus on hPSC based research and gene therapy approaches. Finally, we discuss recent research on human germline genome editing and its social and ethical implications. PMID:26930470

  5. Characterization of Induced Pluripotent Stem Cell Microvesicle Genesis, Morphology and Pluripotent Content

    PubMed Central

    Zhou, Jing; Ghoroghi, Shima; Benito-Martin, Alberto; Wu, Hao; Unachukwu, Uchenna John; Einbond, Linda Saxe; Guariglia, Sara; Peinado, Hector; Redenti, Stephen

    2016-01-01

    Microvesicles (MVs) are lipid bilayer-covered cell fragments that range in diameter from 30 nm–1uM and are released from all cell types. An increasing number of studies reveal that MVs contain microRNA, mRNA and protein that can be detected in the extracellular space. In this study, we characterized induced pluripotent stem cell (iPSC) MV genesis, content and fusion to retinal progenitor cells (RPCs) in vitro. Nanoparticle tracking revealed that iPSCs released approximately 2200 MVs cell/hour in the first 12 hrs with an average diameter of 122 nm. Electron and light microscopic analysis of iPSCs showed MV release via lipid bilayer budding. The mRNA content of iPSC MVs was characterized and revealed the presence of the transcription factors Oct-3/4, Nanog, Klf4, and C-Myc. The protein content of iPSCs MVs, detected by immunogold electron microscopy, revealed the presence of the Oct-3/4 and Nanog. Isolated iPSC MVs were shown to fuse with RPCs in vitro at multiple points along the plasma membrane. These findings demonstrate that the mRNA and protein cargo in iPSC MVs have established roles in maintenance of pluripotency. Building on this work, iPSC derived MVs may be shown to be involved in maintaining cellular pluripotency and may have application in regenerative strategies for neural tissue. PMID:26797168

  6. Derivation of endodermal progenitors from pluripotent stem cells†

    PubMed Central

    Ikonomou, Laertis; Kotton, Darrell N.

    2014-01-01

    Stem and progenitor cells play important roles in organogenesis during development and in tissue homeostasis and response to injury postnatally. As the regenerative capacity of many human tissues is limited, cell replacement therapies hold great promise for human disease management. Pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are prime candidates for the derivation of unlimited quantities of clinically relevant cell types through development of directed differentiation protocols, i.e. the recapitulation of developmental milestones in in vitro cell culture. Tissue-specific progenitors, including progenitors of endodermal origin, are important intermediates in such protocols since they give rise to all mature parenchymal cells. In this review, we focus on the in vivo biology of embryonic endodermal progenitors in terms of key transcription factors and signaling pathways. We critically review the emerging literature aiming to apply this basic knowledge to achieve the efficient and reproducible in vitro derivation of endodermal progenitors such as pancreas, liver and lung precursor cells. PMID:25160562

  7. Nanoengineered Platforms to Guide Pluripotent Stem Cell Fate

    PubMed Central

    Rutledge, Katy; Jabbarzadeh, Ehsan

    2016-01-01

    Tissue engineering utilizes cells, signaling molecules, and scaffolds towards creating functional tissue to repair damaged organs. Pluripotent stem cells (PSCs) are a promising cell source due to their ability to self-renewal indefinitely and their potential to differentiate into almost any cell type. Great strides have been taken to parse the physiological mechanisms by which PSCs respond to their microenvironment and commit to a specific lineage. The combination of physical cues and chemical factors is thought to have the most profound influence on stem cell behavior, therefore a major focus of tissue engineering strategies is scaffold design to incorporate these signals. One overlooked component of the in vivo microenvironment researchers attempt to recapitulate with three dimensional (3D) substrates is the nanoarchitecture formed by the fibrillar network of extracellular matrix (ECM) proteins. These nanoscale features have the ability to impact cell adhesion, migration, proliferation, and lineage commitment. Significant advances have been made in deciphering how these nanoscale cues interact with stem cells to determine phenotype, but much is still unknown as to how the interplay between physical and chemical signals regulate in vitro and in vivo cellular fate. This review dives deeper to investigate nanoscale platforms for engineering tissue, as well use the use of these nanotechnologies to drive pluripotent stem cell lineage determination. PMID:26918198

  8. Generation of induced pluripotent stem cells from human mesenchymal stem cells of parotid gland origin

    PubMed Central

    Yan, Xing; Xu, Nuo; Meng, Cen; Wang, Bianhong; Yuan, Jinghong; Wang, Caiyun; Li, Yang

    2016-01-01

    The technology to reprogram human somatic cells to pluripotent state allows the generation of patient-specific induced pluripotent stem cells (iPSCs) and holds a great promise for regenerative medicine and autologous transplantation. Here we, for the first time, identified mesenchymal stem cells isolated from parotid gland (hPMSCs) as a suitable candidate for iPSC production. In the present study, hPMSCs were isolated from parotid gland specimens in patients with squamous cell carcinoma of the oral cavity. The mesenchymal stem cell properties of cultured hPMSCs were confirmed by expression of surface markers and induced differentiation into osteogenic, chondrogenic and adipogenic cell lineages. hPMSCs were then reprogrammed to pluripotent cells by episomal vector-mediated transduction of reprogramming factors (OCT3/4, SOX2, KLF4, c-MYC, LIN28 and TP53 shRNA). The resulting hPMSC-iPSCs showed similar characteristics as human embryonic stem cells (ESCs) with regard to morphology, pluripotent markers, global gene expression, and methylation status of pluripotent cell-specific genes OCT4 and NANOG. These hPMSC-iPSCs were able to differentiate into cells of all three germ layers both in vitro and in vivo. Our results indicate that hPMSCs could be an alternative cell source for generation of iPSCs and have the potential to be used in cell-based regenerative medicine. PMID:27158336

  9. Cell signalling pathways underlying induced pluripotent stem cell reprogramming

    PubMed Central

    Hawkins, Kate; Joy, Shona; McKay, Tristan

    2014-01-01

    Induced pluripotent stem (iPS) 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, PI3K, 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 cMyc, 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-iPS 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, iPS 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 iPS 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. PMID:25426259

  10. Peripheral-Nerve and Spinal-Cord Regeneration in Mice Using Hair-Follicle-Associated Pluripotent (HAP) Stem Cells.

    PubMed

    Amoh, Yasuyuki; Katsuoka, Kensei; Hoffman, Robert M

    2016-01-01

    Nestin, a neural stem cell marker protein, is expressed in hair follicle cells above the bulge area. These nestin-positive hair follicle-associated-pluripotent (HAP) stem cells are negative for the keratinocyte marker K15 and can differentiate into neurons, glia, keratinocytes, smooth muscle cells, cardiac muscle cells, and melanocytes in vitro. HAP stem cells are positive for the stem cell marker CD34, as well as K15-negative, suggesting their relatively undifferentiated state. HAP stem cells promoted the functional recovery of injured peripheral nerves and the spinal cord. HAP stem cells differentiated into glial fibrillary acidic protein (GFAP)-positive Schwann cells when implanted in severed sciatic nerves and spinal cords in mice. These results suggest that HAP stem cells provide an important accessible, autologous source of adult stem cells for regenerative medicine, that have critical advantages over ES and iPS stem cells. PMID:27431243

  11. Hurdles to clinical translation of human induced pluripotent stem cells.

    PubMed

    Neofytou, Evgenios; O'Brien, Connor Galen; Couture, Larry A; Wu, Joseph C

    2015-07-01

    Human pluripotent stem cells are known to have the capacity to renew indefinitely, being intrinsically able to differentiate into many different cell types. These characteristics have generated tremendous enthusiasm about the potential applications of these cells in regenerative medicine. However, major challenges remain with the development and testing of novel experimental stem cell therapeutics in the field. In this Review, we focus on the nature of the preclinical challenges and discuss potential solutions that could help overcome them. Furthermore, we discuss the use of allogeneic versus autologous stem cell products, including a review of their respective advantages and disadvantages, major clinical requirements, quality standards, time lines, and costs of clinical grade development. PMID:26132109

  12. Hurdles to clinical translation of human induced pluripotent stem cells

    PubMed Central

    Neofytou, Evgenios; O’Brien, Connor Galen; Couture, Larry A.; Wu, Joseph C.

    2015-01-01

    Human pluripotent stem cells are known to have the capacity to renew indefinitely, being intrinsically able to differentiate into many different cell types. These characteristics have generated tremendous enthusiasm about the potential applications of these cells in regenerative medicine. However, major challenges remain with the development and testing of novel experimental stem cell therapeutics in the field. In this Review, we focus on the nature of the preclinical challenges and discuss potential solutions that could help overcome them. Furthermore, we discuss the use of allogeneic versus autologous stem cell products, including a review of their respective advantages and disadvantages, major clinical requirements, quality standards, time lines, and costs of clinical grade development. PMID:26132109

  13. Utilizing FUCCI reporters to understand pluripotent stem cell biology.

    PubMed

    Singh, Amar M; Trost, Robert; Boward, Benjamin; Dalton, Stephen

    2016-05-15

    The fluorescence ubiquitination cell cycle indicator (FUCCI) system provides a powerful method to evaluate cell cycle mechanisms associated with stem cell self-renewal and cell fate specification. By integrating the FUCCI system into human pluripotent stem cells (hPSCs) it is possible to isolate homogeneous fractions of viable cells representative of all cell cycle phases. This method avoids problems associated with traditional tools used for cell cycle analysis such as synchronizing drugs, elutriation and temperature sensitive mutants. Importantly, FUCCI reporters allow cell cycle events in dynamic systems, such as differentiation, to be evaluated. Initial reports on the FUCCI system focused on its strengths in reporting spatio-temporal aspects of cell cycle events in living cells and developmental models. In this report, we describe approaches that broaden the application of FUCCI reporters in PSCs through incorporation of FACS. This approach allows molecular analysis of the cell cycle in stem cell systems that were not previously possible. PMID:26404921

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

    PubMed

    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

  15. hPSCreg—the human pluripotent stem cell registry

    PubMed Central

    Seltmann, Stefanie; Lekschas, Fritz; Müller, Robert; Stachelscheid, Harald; Bittner, Marie-Sophie; Zhang, Weiping; Kidane, Luam; Seriola, Anna; Veiga, Anna; Stacey, Glyn; Kurtz, Andreas

    2016-01-01

    The human pluripotent stem cell registry (hPSCreg), accessible at http://hpscreg.eu, is a public registry and data portal for human embryonic and induced pluripotent stem cell lines (hESC and hiPSC). Since their first isolation the number of hESC lines has steadily increased to over 3000 and new iPSC lines are generated in a rapidly growing number of laboratories as a result of their potentially broad applicability in biomedicine and drug testing. Many of these lines are deposited in stem cell banks, which are globally established to store tens of thousands of lines from healthy and diseased donors. The Registry provides comprehensive and standardized biological and legal information as well as tools to search and compare information from multiple hPSC sources and hence addresses a translational research need. To facilitate unambiguous identification over different resources, hPSCreg automatically creates a unique standardized name for each cell line registered. In addition to biological information, hPSCreg stores extensive data about ethical standards regarding cell sourcing and conditions for application and privacy protection. hPSCreg is the first global registry that holds both, manually validated scientific and ethical information on hPSC lines, and provides access by means of a user-friendly, mobile-ready web application. PMID:26400179

  16. hPSCreg--the human pluripotent stem cell registry.

    PubMed

    Seltmann, Stefanie; Lekschas, Fritz; Müller, Robert; Stachelscheid, Harald; Bittner, Marie-Sophie; Zhang, Weiping; Kidane, Luam; Seriola, Anna; Veiga, Anna; Stacey, Glyn; Kurtz, Andreas

    2016-01-01

    The human pluripotent stem cell registry (hPSCreg), accessible at http://hpscreg.eu, is a public registry and data portal for human embryonic and induced pluripotent stem cell lines (hESC and hiPSC). Since their first isolation the number of hESC lines has steadily increased to over 3000 and new iPSC lines are generated in a rapidly growing number of laboratories as a result of their potentially broad applicability in biomedicine and drug testing. Many of these lines are deposited in stem cell banks, which are globally established to store tens of thousands of lines from healthy and diseased donors. The Registry provides comprehensive and standardized biological and legal information as well as tools to search and compare information from multiple hPSC sources and hence addresses a translational research need. To facilitate unambiguous identification over different resources, hPSCreg automatically creates a unique standardized name for each cell line registered. In addition to biological information, hPSCreg stores extensive data about ethical standards regarding cell sourcing and conditions for application and privacy protection. hPSCreg is the first global registry that holds both, manually validated scientific and ethical information on hPSC lines, and provides access by means of a user-friendly, mobile-ready web application. PMID:26400179

  17. Developing High-Fidelity Hepatotoxicity Models From Pluripotent Stem Cells

    PubMed Central

    Medine, Claire N.; Lucendo-Villarin, Baltasar; Storck, Christopher; Wang, Faye; Szkolnicka, Dagmara; Khan, Ferdous; Pernagallo, Salvatore; Black, James R.; Marriage, Howard M.; Ross, James A.; Bradley, Mark; Iredale, John P.; Flint, Oliver

    2013-01-01

    Faithfully recapitulating human physiology “in a dish” from a renewable source remains a holy grail for medicine and pharma. Many procedures have been described that, to a limited extent, exhibit human tissue-specific function in vitro. In particular, incomplete cellular differentiation and/or the loss of cell phenotype postdifferentiation play a major part in this void. We have developed an interdisciplinary approach to address this problem, using skill sets in cell biology, materials chemistry, and pharmacology. Pluripotent stem cells were differentiated to hepatocytes before being replated onto a synthetic surface. Our approach yielded metabolically active hepatocyte populations that displayed stable function for more than 2 weeks in vitro. Although metabolic activity was an important indication of cell utility, the accurate prediction of cellular toxicity in response to specific pharmacological compounds represented our goal. Therefore, detailed analysis of hepatocellular toxicity was performed in response to a custom-built and well-defined compound set and compared with primary human hepatocytes. Importantly, stem cell-derived hepatocytes displayed equivalence to primary human material. Moreover, we demonstrated that our approach was capable of modeling metabolic differences observed in the population. In conclusion, we report that pluripotent stem cell-derived hepatocytes will model toxicity predictably and in a manner comparable to current gold standard assays, representing a major advance in the field. PMID:23757504

  18. Induced Pluripotent Stem Cell Technology in Regenerative Medicine and Biology

    NASA Astrophysics Data System (ADS)

    Pei, Duanqing; Xu, Jianyong; Zhuang, Qiang; Tse, Hung-Fat; Esteban, Miguel A.

    The potential of human embryonic stem cells (ESCs) for regenerative medicine is unquestionable, but practical and ethical considerations have hampered clinical application and research. In an attempt to overcome these issues, the conversion of somatic cells into pluripotent stem cells similar to ESCs, commonly termed nuclear reprogramming, has been a top objective of contemporary biology. More than 40 years ago, King, Briggs, and Gurdon pioneered somatic cell nuclear reprogramming in frogs, and in 1981 Evans successfully isolated mouse ESCs. In 1997 Wilmut and collaborators produced the first cloned mammal using nuclear transfer, and then Thomson obtained human ESCs from in vitro fertilized blastocysts in 1998. Over the last 2 decades we have also seen remarkable findings regarding how ESC behavior is controlled, the importance of which should not be underestimated. This knowledge allowed the laboratory of Shinya Yamanaka to overcome brilliantly conceptual and technical barriers in 2006 and generate induced pluripotent stem cells (iPSCs) from mouse fibroblasts by overexpressing defined combinations of ESC-enriched transcription factors. Here, we discuss some important implications of human iPSCs for biology and medicine and also point to possible future directions.

  19. Concise review: programming human pluripotent stem cells into blood.

    PubMed

    Easterbrook, Jennifer; Fidanza, Antonella; Forrester, Lesley M

    2016-06-01

    Blood disorders are treated with cell therapies including haematopoietic stem cell (HSC) transplantation as well as platelet and red blood cell transfusions. However the source of cells is entirely dependent on donors, procedures are susceptible to transfusion-transmitted infections and serious complications can arise in recipients due to immunological incompatibility. These problems could be alleviated if it was possible to produce haematopoietic cells in vitro from an autologous and renewable cell source. The production of haematopoietic cells in the laboratory from human induced pluripotent stem cells (iPSCs) may provide a route to realize this goal but it has proven challenging to generate long-term reconstituting HSCs. To date, the optimization of differentiation protocols has mostly relied on the manipulation of extrinsic signals to mimic the in vivo environment. We review studies that have taken an alternative approach to modulate intrinsic signals by enforced expression of transcription factors. Single and combinations of multiple transcription factors have been used in a variety of contexts to enhance the production of haematopoietic cells from human pluripotent stem cells. This programming approach, together with the recent advances in the production and use of synthetic transcription factors, holds great promise for the production of fully functional HSCs in the future. PMID:26996518

  20. Embryonic stem cells or induced pluripotent stem cells? A DNA integrity perspective

    PubMed Central

    Bai, Qiang; Desprat, Romain; Klein, Bernard; Lemaitre, Jean-Marc; De Vos, John

    2013-01-01

    Induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) are two types of pluripotent stem cells that hold great promise for biomedical research and medical applications. iPSCs were initially favorably compared to ESCs. This view was first based on ethical arguments (the generation of iPSCs does not require the destruction of an embryo) and on immunological reasons (it is easier to derive patient HLA-matched iPSCs than ESCs). However, several reports suggest that iPSCs might be characterized by higher occurrence of epigenetic and genetic aberrations than ESCs as a consequence of the reprogramming process. We focus here on the DNA integrity of pluripotent stem cells and examine the three main sources of genomic abnormalities in iPSCs: (1) genomic variety of the parental cells, (2) cell reprogramming, and (3) in vitro cell culture. Recent reports claim that it is possible to generate mouse or human iPSC lines with a mutation level similar to that of the parental cells, suggesting that “genome-friendly” reprogramming techniques can be developed. The issue of iPSC DNA integrity clearly highlights the crucial need of guidelines to define the acceptable level of genomic integrity of pluripotent stem cells for biomedical applications. We discuss here the main issues that such guidelines should address. PMID:23317057

  1. Human Pluripotent Stem Cells: Applications and Challenges in Neurological Diseases

    PubMed Central

    Hibaoui, Youssef; Feki, Anis

    2012-01-01

    The ability to generate human pluripotent stem cells (hPSCs) holds great promise for the understanding and the treatment of human neurological diseases in modern medicine. The hPSCs are considered for their in vitro use as research tools to provide relevant cellular model for human diseases, drug discovery, and toxicity assays and for their in vivo use in regenerative medicine applications. In this review, we highlight recent progress, promises, and challenges of hPSC applications in human neurological disease modeling and therapies. PMID:22934023

  2. Fluorescence lifetime imaging of induced pluripotent stem cells

    NASA Astrophysics Data System (ADS)

    Uchugonova, Aisada; Batista, Ana; König, Karsten

    2014-02-01

    The multiphoton FLIM tomograph MPTflex with its flexible scan head, articulated arm, and the tunable femtosecond laser source was employed to study cell monolayers and 3D cell clusters. FLIM was performed with 250 ps temporal resolution and submicron special resolution using time-correlated single photon counting. The autofluorescence based on NAD(P)H and flavins/flavoproteins has been measured in mouse embryonic fibroblasts, induced pluripotent stem cells (iPS cells) originated from mouse embryonic fibroblasts and non-proliferative mouse embryonic fibroblasts.

  3. Using human induced pluripotent stem cells to treat retinal disease☆

    PubMed Central

    Borooah, S.; Phillips, M.J.; Bilican, B.; Wright, A.F.; Wilmut, I.; Chandran, S.; Gamm, D.; Dhillon, B.

    2013-01-01

    The eye is an ideal target for exploiting the potential of human induced pluripotent stem cell (hiPSC) technology in order to understand disease pathways and explore novel therapeutic strategies for inherited retinal disease. The aim of this article is to map the pathway from state-of-the art laboratory-based discoveries to realising the translational potential of this emerging technique. We describe the relevance and routes to establishing hiPSCs in selected models of human retinal disease. Additionally, we define pathways for applying hiPSC technology in treating currently incurable, progressive and blinding retinal disease. PMID:24104210

  4. Maintaining embryonic stem cell pluripotency with Wnt signaling.

    PubMed

    Sokol, Sergei Y

    2011-10-01

    Wnt signaling pathways control lineage specification in vertebrate embryos and regulate pluripotency in embryonic stem (ES) cells, but how the balance between progenitor self-renewal and differentiation is achieved during axis specification and tissue patterning remains highly controversial. The context- and stage-specific effects of the different Wnt pathways produce complex and sometimes opposite outcomes that help to generate embryonic cell diversity. Although the results of recent studies of the Wnt/β-catenin pathway in ES cells appear to be surprising and controversial, they converge on the same conserved mechanism that leads to the inactivation of TCF3-mediated repression. PMID:21903672

  5. Induced pluripotent stem cells as a model for diabetes investigation

    PubMed Central

    Stepniewski, J.; Kachamakova-Trojanowska, N.; Ogrocki, D.; Szopa, M.; Matlok, M.; Beilharz, M.; Dyduch, G.; Malecki, M. T.; Jozkowicz, A.; Dulak, J.

    2015-01-01

    Mouse and human induced pluripotent stem cells (iPSCs) may represent a novel approach for modeling diabetes. Taking this into consideration, the aim of this study was to generate and evaluate differentiation potential of iPSCs from lepdb/db (db/db) mice, the model of diabetes type 2 as well as from patients with Maturity Onset Diabetes of the Young 3 (HNF1A MODY). Murine iPSC colonies from both wild type and db/db mice were positive for markers of pluripotency: Oct3/4A, Nanog, SSEA1, CDy1 and alkaline phosphatase and differentiated in vitro and in vivo into cells originating from three germ layers. However, our results suggest impaired differentiation of db/db cells into endothelial progenitor-like cells expressing CD34 and Tie2 markers and their reduced angiogenic potential. Human control and HNF1A MODY reprogrammed cells also expressed pluripotency markers: OCT3/4A, SSEA4, TRA-1–60, TRA-1-81, formed embryoid bodies (EBs) and differentiated into cells of three germ layers. Additionally, insulin expressing cells were obtained from those partially reprogrammed cells with direct as well as EB-mediated differentiation method. Our findings indicate that disease-specific iPSCs may help to better understand the mechanisms responsible for defective insulin production or vascular dysfunction upon differentiation toward cell types affected by diabetes. PMID:25716801

  6. Cripto is essential to capture mouse epiblast stem cell and human embryonic stem cell pluripotency.

    PubMed

    Fiorenzano, Alessandro; Pascale, Emilia; D'Aniello, Cristina; Acampora, Dario; Bassalert, Cecilia; Russo, Francesco; Andolfi, Gennaro; Biffoni, Mauro; Francescangeli, Federica; Zeuner, Ann; Angelini, Claudia; Chazaud, Claire; Patriarca, Eduardo J; Fico, Annalisa; Minchiotti, Gabriella

    2016-01-01

    Known molecular determinants of developmental plasticity are mainly transcription factors, while the extrinsic regulation of this process has been largely unexplored. Here we identify Cripto as one of the earliest epiblast markers and a key extracellular determinant of the naive and primed pluripotent states. We demonstrate that Cripto sustains mouse embryonic stem cell (ESC) self-renewal by modulating Wnt/β-catenin, whereas it maintains mouse epiblast stem cell (EpiSC) and human ESC pluripotency through Nodal/Smad2. Moreover, we provide unprecedented evidence that Cripto controls the metabolic reprogramming in ESCs to EpiSC transition. Remarkably, Cripto deficiency attenuates ESC lineage restriction in vitro and in vivo, and permits ESC transdifferentiation into trophectoderm lineage, suggesting that Cripto has earlier functions than previously recognized. All together, our studies provide novel insights into the current model of mammalian pluripotency and contribute to the understanding of the extrinsic regulation of the first cell lineage decision in the embryo. PMID:27586544

  7. Snapshots of Pluripotency.

    PubMed

    Tesar, Paul J

    2016-02-01

    Pluripotency is a unique developmental state that lays the foundation upon which the entire embryo is built. Pluripotent cells possess the unique capacity to generate, in an exquisitely defined sequence, all the distinct cell types comprising the fetal and adult organism. The discovery of pluripotent stem cells and now the ability to generate them from differentiated cells has had a profound impact on a vast array of scientific disciplines. In addition to their clinical potential as a source of therapeutic cell types, pluripotent stem cells provide scalable access to otherwise experimentally inaccessible development- and disease-associated biology. Here I provide my perspective on the continuum of pluripotency in the early mammalian embryo. I also discuss how novel genomic technologies are now enabling the capture of molecular "snapshots" of the several distinct pluripotent states that stem cells undergo during this pivotal developmental period. PMID:26833092

  8. Snapshots of Pluripotency

    PubMed Central

    Tesar, Paul J.

    2016-01-01

    Summary Pluripotency is a unique developmental state that lays the foundation upon which the entire embryo is built. Pluripotent cells possess the unique capacity to generate, in an exquisitely defined sequence, all the distinct cell types comprising the fetal and adult organism. The discovery of pluripotent stem cells and now the ability to generate them from differentiated cells has had a profound impact on a vast array of scientific disciplines. In addition to their clinical potential as a source of therapeutic cell types, pluripotent stem cells provide scalable access to otherwise experimentally inaccessible development- and disease-associated biology. Here I provide my perspective on the continuum of pluripotency in the early mammalian embryo. I also discuss how novel genomic technologies are now enabling the capture of molecular “snapshots” of the several distinct pluripotent states that stem cells undergo during this pivotal developmental period. PMID:26833092

  9. ‘Above all, do no harm’: safeguarding pluripotent stem cell therapy against iatrogenic tumorigenesis

    PubMed Central

    2014-01-01

    Human pluripotent stem cells are the foundations of regenerative medicine. However, the worst possible complication of using pluripotent stem cells in therapy could be iatrogenic cancerogenesis. Nevertheless, despite the rapid progress in the development of new techniques for induction of pluripotency and for directed differentiation, risks of cancerogenic transformation of therapeutically implanted pluripotent stem cells still persist. 'Above all, do no harm', as quoted from the Hippocratic Oath, is our ultimate creed. Therefore, the primary goal in designing any therapeutic regimes involving stem cells should be the elimination of any possibilities of their neoplasmic transformation. I review here the basic strategies that have been designed to attain this goal: sorting out undifferentiated, pluripotent stem cells with antibodies targeting surface-displayed biomarkers; sorting in differentiating cells, which express recombinant proteins as reporters; killing undifferentiated stem cells with toxic antibodies or antibody-guided toxins; eliminating undifferentiated stem cells with cytotoxic drugs; making potentially tumorigenic stem cells sensitive to pro-drugs by transformation with suicide-inducing genes; eradication of differentiation-refractive stem cells by self-triggered transgenic expression of human recombinant DNases. Every pluripotent undifferentiated stem cell poses a risk of neoplasmic transformation. Therefore, the aforementioned or other novel strategies that would safeguard against iatrogenic transformation of these stem cells should be considered for incorporation into every stem cell therapy trial. PMID:25158017

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

    PubMed Central

    Biswas, Dhruba; Jiang, Peng

    2016-01-01

    The ability to generate transplantable neural cells in a large quantity in the laboratory is a critical step in the field of developing stem cell regenerative medicine for neural repair. During the last few years, groundbreaking studies have shown that cell fate of adult somatic cells can be reprogrammed through lineage specific expression of transcription factors (TFs)-and defined culture conditions. This key concept has been used to identify a number of potent small molecules that could enhance the efficiency of reprogramming with TFs. Recently, a growing number of studies have shown that small molecules targeting specific epigenetic and signaling pathways can replace all of the reprogramming TFs. Here, we provide a detailed review of the studies reporting the generation of chemically induced pluripotent stem cells (ciPSCs), neural stem cells (ciNSCs), and neurons (ciN). We also discuss the main mechanisms of actions and the pathways that the small molecules regulate during chemical reprogramming. PMID:26861316

  11. Cardiac Regeneration Using Pluripotent Stem Cells – Progression to Large Animal Models

    PubMed Central

    Chong, James J.H.; Murry, Charles E.

    2014-01-01

    Pluripotent stem cells (PSCs) have indisputable cardiomyogenic potential and therefore have been intensively investigated as a potential cardiac regenerative therapy. Current directed differentiation protocols are able to produce high yields of cardiomyocytes from PSCs and studies in small animal models of cardiovascular disease have proven sustained engraftment and functional efficacy. Therefore, the time is ripe for cardiac regenerative therapies using PSC derivatives to be tested in large animal models that more closely resemble the hearts of humans. In this review, we discuss the results of our recent study using human embryonic stem cell derived cardiomyocytes (hESC-CM) in a non-human primate model of ischemic cardiac injury. Large scale remuscularization, electromechanical coupling and short-term arrhythmias demonstrated by our hESC-CM grafts are discussed in the context of other studies using Adult Stem Cells for cardiac regeneration. PMID:25087896

  12. The case for induced pluripotent stem cell-derived cardiomyocytes in pharmacological screening

    PubMed Central

    Khan, Jaffar M; Lyon, Alexander R; Harding, Sian E

    2013-01-01

    The current drug screening models are deficient, particularly in detecting cardiac side effects. Human stem cell-derived cardiomyocytes could aid both early cardiotoxicity detection and novel drug discovery. Work over the last decade has generated human embryonic stem cells as potentially accurate sources of human cardiomyocytes, but ethical constraints and poor efficacy in establishing cell lines limit their use. Induced pluripotent stem cells do not require the use of human embryos and have the added advantage of producing patient-specific cardiomyocytes, allowing both generic and disease- and patient-specific pharmacological screening, as well as drug development through disease modelling. A critical question is whether sufficient standards have been achieved in the reliable and reproducible generation of ‘adult-like’ cardiomyocytes from human fibroblast tissue to progress from validation to safe use in practice and drug discovery. This review will highlight the need for a new experimental system, assess the validity of human induced pluripotent stem cell-derived cardiomyocytes and explore what the future may hold for their use in pharmacology. LINKED ARTICLES This article is part of a themed section on Regenerative Medicine and Pharmacology: A Look to the Future. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-2 PMID:22845396

  13. Human Pluripotent Stem Cells for Modelling Human Liver Diseases and Cell Therapy

    PubMed Central

    Dianat, Noushin; Steichen, Clara; Vallier, Ludovic; Weber, Anne; Dubart-Kupperschmitt, Anne

    2013-01-01

    The liver is affected by many types of diseases, including metabolic disorders and acute liver failure. Orthotopic liver transplantation (OLT) is currently the only effective treatment for life-threatening liver diseases but transplantation of allogeneic hepatocytes has now become an alternative as it is less invasive than OLT and can be performed repeatedly. However, this approach is hampered by the shortage of organ donors, and the problems related to the isolation of high quality adult hepatocytes, their cryopreservation and their absence of proliferation in culture. Liver is also a key organ to assess the pharmacokinetics and toxicology of xenobiotics and for drug discovery, but appropriate cell culture systems are lacking. All these problems have highlighted the need to explore other sources of cells such as stem cells that could be isolated, expanded to yield sufficiently large populations and then induced to differentiate into functional hepatocytes. The presence of a niche of “facultative” progenitor and stem cells in the normal liver has recently been confirmed but they display no telomerase activity. The recent discovery that human induced pluripotent stem cells can be generated from somatic cells has renewed hopes for regenerative medicine and in vitro disease modelling, as these cells are easily accessible. We review here the present progresses, limits and challenges for the generation of functional hepatocytes from human pluripotent stem cells in view of their potential use in regenerative medicine and drug discovery. PMID:23444872

  14. Apoptotic Susceptibility to DNA Damage of Pluripotent Stem Cells Facilitates Pharmacologic Purging of Teratoma Risk

    PubMed Central

    Smith, Alyson J.; Nelson, Natalie G.; Oommen, Saji; Hartjes, Katherine A.; Folmes, Clifford D.; Terzic, Andre

    2012-01-01

    Pluripotent stem cells have been the focus of bioengineering efforts designed to generate regenerative products, yet harnessing therapeutic capacity while minimizing risk of dysregulated growth remains a challenge. The risk of residual undifferentiated stem cells within a differentiated progenitor population requires a targeted approach to eliminate contaminating cells prior to delivery. In this study we aimed to validate a toxicity strategy that could selectively purge pluripotent stem cells in response to DNA damage and avoid risk of uncontrolled cell growth upon transplantation. Compared with somatic cell types, embryonic stem cells and induced pluripotent stem cells displayed hypersensitivity to apoptotic induction by genotoxic agents. Notably, hypersensitivity in pluripotent stem cells was stage-specific and consistently lost upon in vitro differentiation, with the mean half-maximal inhibitory concentration increasing nearly 2 orders of magnitude with tissue specification. Quantitative polymerase chain reaction and Western blotting demonstrated that the innate response was mediated through upregulation of the BH3-only protein Puma in both natural and induced pluripotent stem cells. Pretreatment with genotoxic etoposide purged hypersensitive pluripotent stem cells to yield a progenitor population refractory to teratoma formation upon transplantation. Collectively, this study exploits a hypersensitive apoptotic response to DNA damage within pluripotent stem cells to decrease risk of dysregulated growth and augment the safety profile of transplant-ready, bioengineered progenitor cells. PMID:23197662

  15. Human induced pluripotent stem cells: A new source for brown and white adipocytes

    PubMed Central

    Hafner, Anne-Laure; Dani, Christian

    2014-01-01

    Mesenchymal stem cells (MSCs) derived from human induced pluripotent stem cells (hiPSCs) provide a novel source for generating adipocytes, thus opening new avenues for fundamental research and clinical medicine. We present the adipogenic potential of hiPSCs and the various methods to derive hiPSC-MSCs. We discuss the main characteristic of hiPSC-MSCs, which is their low adipogenic capacity as compared to adult-MSCs. Finally, we propose several hypotheses to explanation this feature, underlying a potential critical role of the micro-environment. We favour the hypothesis that the range of factors or culture conditions required to induce adipocyte differentiation of MSCs derived from adult tissues and from embryonic-like cells could differ. PMID:25258668

  16. Lessons from the embryonic neural stem cell niche for neural lineage differentiation of pluripotent stem cells.

    PubMed

    Solozobova, Valeriya; Wyvekens, Nicolas; Pruszak, Jan

    2012-09-01

    Pluripotent stem cells offer an abundant and malleable source for the generation of differentiated cells for transplantation as well as for in vitro screens. Patterning and differentiation protocols have been developed to generate neural progeny from human embryonic or induced pluripotent stem cells. However, continued refinement is required to enhance efficiency and to prevent the generation of unwanted cell types. We summarize and interpret insights gained from studies of embryonic neuroepithelium. A multitude of factors including soluble molecules, interactions with the extracellular matrix and neighboring cells cooperate to control neural stem cell self-renewal versus differentiation. Applying these findings and concepts to human stem cell systems in vitro may yield more appropriately patterned cell types for biomedical applications. PMID:22628111

  17. Pluripotent stem cells in disease modelling and drug discovery.

    PubMed

    Avior, Yishai; Sagi, Ido; Benvenisty, Nissim

    2016-03-01

    Experimental modelling of human disorders enables the definition of the cellular and molecular mechanisms underlying diseases and the development of therapies for treating them. The availability of human pluripotent stem cells (PSCs), which are capable of self-renewal and have the potential to differentiate into virtually any cell type, can now help to overcome the limitations of animal models for certain disorders. The ability to model human diseases using cultured PSCs has revolutionized the ways in which we study monogenic, complex and epigenetic disorders, as well as early- and late-onset diseases. Several strategies are used to generate such disease models using either embryonic stem cells (ES cells) or patient-specific induced PSCs (iPSCs), creating new possibilities for the establishment of models and their use in drug screening. PMID:26818440

  18. Autophagic response to cell culture stress in pluripotent stem cells.

    PubMed

    Gregory, Siân; Swamy, Sushma; Hewitt, Zoe; Wood, Andrew; Weightman, Richard; Moore, Harry

    2016-05-01

    Autophagy is an important conserved cellular process, both constitutively as a recycling pathway for long lived proteins and as an upregulated stress response. Recent findings suggest a fundamental role for autophagic processes in the maintenance of pluripotent stem cell function. In human embryonic stem cells (hESCS), autophagy was investigated by transfection of LC3-GFP to visualize autophagosomes and with an antibody to LC3B protein. The presence of the primary cilium (PC) in hESCs as the site of recruitment of autophagy-related proteins was also assessed. HESCs (mShef11) in vitro displayed basal autophagy which was upregulated in response to deprivation of culture medium replacement. Significantly higher levels of autophagy were exhibited on spontaneous differentiation of hESCs in vitro. The PC was confirmed to be present in hESCs and therefore may serve to coordinate autophagy function. PMID:26385182

  19. Generation of neuropeptidergic hypothalamic neurons from human pluripotent stem cells

    PubMed Central

    Merkle, Florian T.; Maroof, Asif; Wataya, Takafumi; Sasai, Yoshiki; Studer, Lorenz; Eggan, Kevin; Schier, Alexander F.

    2015-01-01

    Hypothalamic neurons orchestrate many essential physiological and behavioral processes via secreted neuropeptides, and are relevant to human diseases such as obesity, narcolepsy and infertility. We report the differentiation of human pluripotent stem cells into many of the major types of neuropeptidergic hypothalamic neurons, including those producing pro-opiolemelanocortin, agouti-related peptide, hypocretin/orexin, melanin-concentrating hormone, oxytocin, arginine vasopressin, corticotropin-releasing hormone (CRH) or thyrotropin-releasing hormone. Hypothalamic neurons can be generated using a ‘self-patterning’ strategy that yields a broad array of cell types, or via a more reproducible directed differentiation approach. Stem cell-derived human hypothalamic neurons share characteristic morphological properties and gene expression patterns with their counterparts in vivo, and are able to integrate into the mouse brain. These neurons could form the basis of cellular models, chemical screens or cellular therapies to study and treat common human diseases. PMID:25670790

  20. Methods of induced pluripotent stem cells for clinical application.

    PubMed

    Seki, Tomohisa; Fukuda, Keiichi

    2015-01-26

    Reprograming somatic cells using exogenetic gene expression represents a groundbreaking step in regenerative medicine. Induced pluripotent stem cells (iPSCs) are expected to yield novel therapies with the potential to solve many issues involving incurable diseases. In particular, applying iPSCs clinically holds the promise of addressing the problems of immune rejection and ethics that have hampered the clinical applications of embryonic stem cells. However, as iPSC research has progressed, new problems have emerged that need to be solved before the routine clinical application of iPSCs can become established. In this review, we discuss the current technologies and future problems of human iPSC generation methods for clinical use. PMID:25621111

  1. Effect of Induced Pluripotent Stem Cell Technology in Blood Banking.

    PubMed

    Focosi, Daniele; Pistello, Mauro

    2016-03-01

    Population aging has imposed cost-effective alternatives to blood donations. Artificial blood is still at the preliminary stages of development, and the need for viable cells seems unsurmountable. Because large numbers of viable cells must be promptly available for clinical use, stem cell technologies, expansion, and banking represent ideal tools to ensure a regular supply. Provided key donors can be identified, induced pluripotent stem cell (iPSC) technology could pave the way to a new era in transfusion medicine, just as it is already doing in many other fields of medicine. The present review summarizes the current state of research on iPSC technology in the field of blood banking, highlighting hurdles, and promises. PMID:26819256

  2. Lower Oncogenic Potential of Human Mesenchymal Stem Cells Derived from Cord Blood Compared to Induced Pluripotent Stem Cells

    PubMed Central

    Foroutan, T.; Najmi, M.; Kazemi, N.; Hasanlou, M.; Pedram, A.

    2015-01-01

    Background: In regenerative medicine, use of each of the mesenchymal stem cells derived from bone marrow, cord blood, and adipose tissue, has several cons and pros. Mesenchymal stem cells derived from cord blood have been considered the best source for precursor transplantation. Direct reprogramming of a somatic cell into induced pluripotent stem cells by over-expression of 6 transcription factors Oct4, Sox2, Klf4, lin28, Nanog, and c-Myc has great potential for regenerative medicine, eliminating the ethical issues of embryonic stem cells and the rejection problems of using non-autologous cells. Objective: To compare reprogramming and pluripotent markers OCT4, Sox-2, c-Myc, Klf4, Nanog, and lin28 in mesenchymal stem cells derived from cord blood and induced pluripotent stem cells. Methods: We analyzed the expression level of OCT4, Sox-2, c-Myc, Klf4, Nanog and lin28 genes in human mesenchymal stem cells derived from cord blood and induced pluripotent stem cells by cell culture and RT-PCR. Results: The expression level of pluripotent genes OCT4 and Sox-2, Nanog and lin28 in mesenchymal stem cells derived from cord blood were significantly higher than those in induced pluripotent stem cells. In contrast to OCT-4A and Sox-2, Nanog and lin28, the expression level of oncogenic factors c-Myc and Klf4 were significantly higher in induced pluripotent stem cells than in mesenchymal stem cells derived from cord blood. Conclusion: It could be concluded that mesenchymal stem cells derived from human cord blood have lower oncogenic potential compared to induced pluripotent stem cells. PMID:26306155

  3. Efficient Generation of Nonhuman Primate Induced Pluripotent Stem Cells

    PubMed Central

    Zhong, Bonan; Trobridge, Grant D.; Zhang, Xiaobing; Watts, Korashon L.; Ramakrishnan, Aravind; Wohlfahrt, Martin; Adair, Jennifer E.

    2011-01-01

    Induced pluripotent stem (iPS) cells have great potential for regenerative medicine and gene therapy. Thus far, iPS cells have typically been generated using integrating viral vectors expressing various reprogramming transcription factors; nonintegrating methods have been less effective and efficient. Because there is a significant risk of malignant transformation and cancer involved with the use of iPS cells, careful evaluation of transplanted iPS cells will be necessary in small and large animal studies before clinical application. Here, we have generated and characterized nonhuman primate iPS cells with the goal of evaluating iPS cell transplantation in a clinically relevant large animal model. We developed stable Phoenix-RD114-based packaging cell lines that produce OCT4, SOX2, c-MYC, and KLF4 (OSCK) expressing gammaretroviral vectors. Using these vectors in combination with small molecules, we were able to efficiently and reproducibly generate nonhuman primate iPS cells from pigtailed macaques (Macaca nemestrina). The established nonhuman primate iPS cells exhibited pluripotency and extensive self-renewal capacity. The facile and reproducible generation of nonhuman primate iPS cells using defined producer cells as a source of individual reprogramming factors should provide an important resource to optimize and evaluate iPS cell technology for studies involving stem cell biology and regenerative medicine. PMID:21058905

  4. Non integrative strategy decreases chromosome instability and improves endogenous pluripotency genes reactivation in porcine induced pluripotent-like stem cells

    PubMed Central

    Congras, Annabelle; Barasc, Harmonie; Canale-Tabet, Kamila; Plisson-Petit, Florence; Delcros, Chantal; Feraud, Olivier; Oudrhiri, Noufissa; Hadadi, Eva; Griscelli, Franck; Bennaceur-Griscelli, Annelise; Turhan, Ali; Afanassieff, Marielle; Ferchaud, Stéphane; Pinton, Alain; Yerle-Bouissou, Martine; Acloque, Hervé

    2016-01-01

    The pig is an emerging animal model, complementary to rodents for basic research and for biomedical and agronomical purposes. However despite the progress made on mouse and rat models to produce genuine pluripotent cells, it remains impossible to produce porcine pluripotent cell lines with germline transmission. Reprogramming of pig somatic cells using conventional integrative strategies remains also unsatisfactory. In the present study, we compared the outcome of both integrative and non-integrative reprogramming strategies on pluripotency and chromosome stability during pig somatic cell reprogramming. The porcine cell lines produced with integrative strategies express several pluripotency genes but they do not silence the integrated exogenes and present a high genomic instability upon passaging. In contrast, pig induced pluripotent-like stem cells produced with non-integrative reprogramming system (NI-iPSLCs) exhibit a normal karyotype after more than 12 months in culture and reactivate endogenous pluripotency markers. Despite the persistent expression of exogenous OCT4 and MYC, these cells can differentiate into derivatives expressing markers of the three embryonic germ layers and we propose that these NI-iPSLCs can be used as a model to bring new insights into the molecular factors controlling and maintaining pluripotency in the pig and other non-rodent mammalians. PMID:27245508

  5. Non integrative strategy decreases chromosome instability and improves endogenous pluripotency genes reactivation in porcine induced pluripotent-like stem cells.

    PubMed

    Congras, Annabelle; Barasc, Harmonie; Canale-Tabet, Kamila; Plisson-Petit, Florence; Delcros, Chantal; Feraud, Olivier; Oudrhiri, Noufissa; Hadadi, Eva; Griscelli, Franck; Bennaceur-Griscelli, Annelise; Turhan, Ali; Afanassieff, Marielle; Ferchaud, Stéphane; Pinton, Alain; Yerle-Bouissou, Martine; Acloque, Hervé

    2016-01-01

    The pig is an emerging animal model, complementary to rodents for basic research and for biomedical and agronomical purposes. However despite the progress made on mouse and rat models to produce genuine pluripotent cells, it remains impossible to produce porcine pluripotent cell lines with germline transmission. Reprogramming of pig somatic cells using conventional integrative strategies remains also unsatisfactory. In the present study, we compared the outcome of both integrative and non-integrative reprogramming strategies on pluripotency and chromosome stability during pig somatic cell reprogramming. The porcine cell lines produced with integrative strategies express several pluripotency genes but they do not silence the integrated exogenes and present a high genomic instability upon passaging. In contrast, pig induced pluripotent-like stem cells produced with non-integrative reprogramming system (NI-iPSLCs) exhibit a normal karyotype after more than 12 months in culture and reactivate endogenous pluripotency markers. Despite the persistent expression of exogenous OCT4 and MYC, these cells can differentiate into derivatives expressing markers of the three embryonic germ layers and we propose that these NI-iPSLCs can be used as a model to bring new insights into the molecular factors controlling and maintaining pluripotency in the pig and other non-rodent mammalians. PMID:27245508

  6. Human pluripotent stem cells: Towards therapeutic development for the treatment of lifestyle diseases

    PubMed Central

    Nishio, Miwako; Nakahara, Masako; Yuo, Akira; Saeki, Kumiko

    2016-01-01

    There are two types of human pluripotent stem cells: Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), both of which launched themselves on clinical trials after having taken measures to overcome problems: Blocking rejections by immunosuppressants regarding ESCs and minimizing the risk of tumorigenicity by depleting exogenous gene components regarding iPSCs. It is generally assumed that clinical applications of human pluripotent stem cells should be limited to those cases where there are no alternative measures for treatments because of the risk in transplanting those cells to living bodies. Regarding lifestyle diseases, we have already several therapeutic options, and thus, development of human pluripotent stem cell-based therapeutics tends to be avoided. Nevertheless, human pluripotent stem cells can contribute to the development of new therapeutics in this field. As we will show, there is a case where only a short-term presence of human pluripotent stem-derived cells can exert long-term therapeutic effects even after they are rejected. In those cases, immunologically rejections of ESC- or allogenic iPSC-derived cells may produce beneficial outcomes by nullifying the risk of tumorigenesis without deterioration of therapeutic effects. Another utility of human pluripotent stem cells is the provision of an innovative tool for drug discovery that are otherwise unavailable. For example, clinical specimens of human classical brown adipocytes (BAs), which has been attracting a great deal of attention as a new target of drug discovery for the treatment of metabolic disorders, are unobtainable from living individuals due to scarcity, fragility and ethical problems. However, BA can easily be produced from human pluripotent stem cells. In this review, we will contemplate potential contribution of human pluripotent stem cells to therapeutic development for lifestyle diseases. PMID:26981171

  7. Human pluripotent stem cells: Towards therapeutic development for the treatment of lifestyle diseases.

    PubMed

    Nishio, Miwako; Nakahara, Masako; Yuo, Akira; Saeki, Kumiko

    2016-02-26

    There are two types of human pluripotent stem cells: Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), both of which launched themselves on clinical trials after having taken measures to overcome problems: Blocking rejections by immunosuppressants regarding ESCs and minimizing the risk of tumorigenicity by depleting exogenous gene components regarding iPSCs. It is generally assumed that clinical applications of human pluripotent stem cells should be limited to those cases where there are no alternative measures for treatments because of the risk in transplanting those cells to living bodies. Regarding lifestyle diseases, we have already several therapeutic options, and thus, development of human pluripotent stem cell-based therapeutics tends to be avoided. Nevertheless, human pluripotent stem cells can contribute to the development of new therapeutics in this field. As we will show, there is a case where only a short-term presence of human pluripotent stem-derived cells can exert long-term therapeutic effects even after they are rejected. In those cases, immunologically rejections of ESC- or allogenic iPSC-derived cells may produce beneficial outcomes by nullifying the risk of tumorigenesis without deterioration of therapeutic effects. Another utility of human pluripotent stem cells is the provision of an innovative tool for drug discovery that are otherwise unavailable. For example, clinical specimens of human classical brown adipocytes (BAs), which has been attracting a great deal of attention as a new target of drug discovery for the treatment of metabolic disorders, are unobtainable from living individuals due to scarcity, fragility and ethical problems. However, BA can easily be produced from human pluripotent stem cells. In this review, we will contemplate potential contribution of human pluripotent stem cells to therapeutic development for lifestyle diseases. PMID:26981171

  8. Genome surveillance in pluripotent stem cells: Low apoptosis threshold and efficient antioxidant defense

    PubMed Central

    Dannenmann, Benjamin; Lehle, Simon; Essmann, Frank; Schulze-Osthoff, Klaus

    2016-01-01

    ABSTRACT Pluripotent stem cells must be endowed with efficient genome surveillance. Here we describe the multiple mechanisms that ensure their genome integrity, including high susceptibility to apoptosis and efficient prevention of DNA lesions. In induced pluripotent stem cells, apoptosis hypersensitivity is mediated by increased expression of proapoptotic BCL-2 protein, whereas DNA damage is prevented by the upregulation of several antioxidant enzymes. Antioxidants might be therefore employed for safer stem cell therapies. PMID:27308586

  9. Reprogram to pluripotency: a new logic and a chemical cocktail

    PubMed Central

    Song, Hongjun; Ming, Guo-li

    2015-01-01

    Somatic cells from animals and humans can be reprogrammed into pluripotent stem cells by pluripotency factors. Hongkui Deng and colleagues discovered that pluripotency can also be induced with exogenous lineage specifiers via balancing competing differentiation forces. In a related study they achieved, for the first time, restoration of pluripotency in adult somatic cells using a chemical cocktail alone. PMID:26998393

  10. Induced Pluripotent Stem Cells: Challenges and Opportunities for Cancer Immunotherapy

    PubMed Central

    Sachamitr, Patty; Hackett, Simon; Fairchild, Paul Jonathan

    2014-01-01

    Despite recent advances in cancer treatment over the past 30 years, therapeutic options remain limited and do not always offer a cure for malignancy. Given that tumor-associated antigens (TAA) are, by definition, self-proteins, the need to productively engage autoreactive T cells remains at the heart of strategies for cancer immunotherapy. These have traditionally focused on the administration of autologous monocyte-derived dendritic cells (moDC) pulsed with TAA, or the ex vivo expansion and adoptive transfer of tumor-infiltrating lymphocytes (TIL) as a source of TAA-specific cytotoxic T cells (CTL). Although such approaches have shown some efficacy, success has been limited by the poor capacity of moDC to cross present exogenous TAA to the CD8+ T-cell repertoire and the potential for exhaustion of CTL expanded ex vivo. Recent advances in induced pluripotency offer opportunities to generate patient-specific stem cell lines with the potential to differentiate in vitro into cell types whose properties may help address these issues. Here, we review recent success in the differentiation of NK cells from human induced pluripotent stem (iPS) cells as well as minor subsets of dendritic cells (DCs) with therapeutic potential, including CD141+XCR1+ DC, capable of cross presenting TAA to naïve CD8+ T cells. Furthermore, we review recent progress in the use of TIL as the starting material for the derivation of iPSC lines, thereby capturing their antigen specificity in a self-renewing stem cell line, from which potentially unlimited numbers of naïve TAA-specific T cells may be differentiated, free of the risks of exhaustion. PMID:24860566

  11. Proteomics and glycoproteomics of pluripotent stem-cell surface proteins.

    PubMed

    Sun, Bingyun

    2015-03-01

    Pluripotent stem cells are a unique cell type with promising potential in regenerative and personalized medicine. Yet the difficulty to understand and coax their seemingly stochastic differentiation and spontaneous self-renewal have largely limited their clinical applications. A call has been made by numerous researchers for a better characterization of surface proteins on these cells, in search of biomarkers that can dictate developmental stages and lineage specifications, and can help formulate mechanistic insight of stem-cell fate choices. In the past two decades, proteomics has gained significant recognition in profiling surface proteins at high throughput. This review will summarize the impact of these studies on stem-cell biology, and discuss the used proteomic techniques. A systematic comparison of all the techniques and their results is also attempted here to help reveal pros, cons, and the complementarity of the existing methods. This awareness should assist in selecting suitable strategies for stem-cell related research, and shed light on technical improvements that can be explored in the future. PMID:25211708

  12. Stem cell pluripotency and transcription factor Oct4.

    PubMed

    Pan, Guang Jin; Chang, Zeng Yi; Schöler, Hans R; Pei, Duanqing

    2002-12-01

    Mammalian cell totipotency is a subject that has fascinated scientists for generations. A long lasting question whether some of the somatic cells retains totipotency was answered by the cloning of Dolly at the end of the 20th century. The dawn of the 21st has brought forward great expectations in harnessing the power of totipotentcy in medicine. Through stem cell biology, it is possible to generate any parts of the human body by stem cell engineering. Considerable resources will be devoted to harness the untapped potentials of stem cells in the foreseeable future which may transform medicine as we know today. At the molecular level, totipotency has been linked to a singular transcription factor and its expression appears to define whether a cell should be totipotent. Named Oct4, it can activate or repress the expression of various genes. Curiously, very little is known about Oct4 beyond its ability to regulate gene expression. The mechanism by which Oct4 specifies totipotency remains entirely unresolved. In this review, we summarize the structure and function of Oct4 and address issues related to Oct4 function in maintaining totipotency or pluripotency of embryonic stem cells. PMID:12528890

  13. Achilles' heel of pluripotent stem cells: genetic, genomic and epigenetic variations during prolonged culture.

    PubMed

    Rebuzzini, Paola; Zuccotti, Maurizio; Redi, Carlo Alberto; Garagna, Silvia

    2016-07-01

    Pluripotent stem cells differentiate into almost any specialized adult cell type of an organism. PSCs can be derived either from the inner cell mass of a blastocyst-giving rise to embryonic stem cells-or after reprogramming of somatic terminally differentiated cells to obtain ES-like cells, named induced pluripotent stem cells. The potential use of these cells in the clinic, for investigating in vitro early embryonic development or for screening the effects of new drugs or xenobiotics, depends on capability to maintain their genome integrity during prolonged culture and differentiation. Both human and mouse PSCs are prone to genomic and (epi)genetic instability during in vitro culture, a feature that seriously limits their real potential use. Culture-induced variations of specific chromosomes or genes, are almost all unpredictable and, as a whole, differ among independent cell lines. They may arise at different culture passages, suggesting the absence of a safe passage number maintaining genome integrity and rendering the control of genomic stability mandatory since the very early culture passages. The present review highlights the urgency for further studies on the mechanisms involved in determining (epi)genetic and chromosome instability, exploiting the knowledge acquired earlier on other cell types. PMID:26961132

  14. Activin A directs striatal projection neuron differentiation of human pluripotent stem cells

    PubMed Central

    Arber, Charles; Precious, Sophie V.; Cambray, Serafí; Risner-Janiczek, Jessica R.; Kelly, Claire; Noakes, Zoe; Fjodorova, Marija; Heuer, Andreas; Ungless, Mark A.; Rodríguez, Tristan A.; Rosser, Anne E.; Dunnett, Stephen B.; Li, Meng

    2015-01-01

    The efficient generation of striatal neurons from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) is fundamental for realising their promise in disease modelling, pharmaceutical drug screening and cell therapy for Huntington's disease. GABAergic medium-sized spiny neurons (MSNs) are the principal projection neurons of the striatum and specifically degenerate in the early phase of Huntington's disease. Here we report that activin A induces lateral ganglionic eminence (LGE) characteristics in nascent neural progenitors derived from hESCs and hiPSCs in a sonic hedgehog-independent manner. Correct specification of striatal phenotype was further demonstrated by the induction of the striatal transcription factors CTIP2, GSX2 and FOXP2. Crucially, these human LGE progenitors readily differentiate into postmitotic neurons expressing the striatal projection neuron signature marker DARPP32, both in culture and following transplantation in the adult striatum in a rat model of Huntington's disease. Activin-induced neurons also exhibit appropriate striatal-like electrophysiology in vitro. Together, our findings demonstrate a novel route for efficient differentiation of GABAergic striatal MSNs from human pluripotent stem cells. PMID:25804741

  15. Neural Conversion and Patterning of Human Pluripotent Stem Cells: A Developmental Perspective

    PubMed Central

    Zirra, Alexandra; Wiethoff, Sarah; Patani, Rickie

    2016-01-01

    Since the reprogramming of adult human terminally differentiated somatic cells into induced pluripotent stem cells (hiPSCs) became a reality in 2007, only eight years have passed. Yet over this relatively short period, myriad experiments have revolutionized previous stem cell dogmata. The tremendous promise of hiPSC technology for regenerative medicine has fuelled rising expectations from both the public and scientific communities alike. In order to effectively harness hiPSCs to uncover fundamental mechanisms of disease, it is imperative to first understand the developmental neurobiology underpinning their lineage restriction choices in order to predictably manipulate cell fate to desired derivatives. Significant progress in developmental biology provides an invaluable resource for rationalising directed differentiation of hiPSCs to cellular derivatives of the nervous system. In this paper we begin by reviewing core developmental concepts underlying neural induction in order to provide context for how such insights have guided reductionist in vitro models of neural conversion from hiPSCs. We then discuss early factors relevant in neural patterning, again drawing upon crucial knowledge gained from developmental neurobiological studies. We conclude by discussing open questions relating to these concepts and how their resolution might serve to strengthen the promise of pluripotent stem cells in regenerative medicine. PMID:27069483

  16. Gingival Fibroblasts as Autologous Feeders for Induced Pluripotent Stem Cells.

    PubMed

    Yu, G; Okawa, H; Okita, K; Kamano, Y; Wang, F; Saeki, M; Yatani, H; Egusa, H

    2016-01-01

    Human gingival fibroblasts (hGFs) present an attractive source of induced pluripotent stem cells (iPSCs), which are expected to be a powerful tool for regenerative dentistry. However, problems to be addressed prior to clinical application include the use of animal-derived feeder cells for cultures. The aim of this study was to establish an autologous hGF-derived iPSC (hGF-iPSC) culture system by evaluating the feeder ability of hGFs. In both serum-containing and serum-free media, hGFs showed higher proliferation than human dermal fibroblasts (hDFs). Three hGF strains were isolated under serum-free conditions, although 2 showed impaired proliferation. When hGF-iPSCs were transferred onto mitomycin C-inactivated hGFs, hDFs, or mouse-derived SNL feeders, hGF and SNL feeders were clearly hGF-iPSC supportive for more than 50 passages, whereas hDF feeders were only able to maintain undifferentiated hGF-iPSC growth for a few passages. After 20 passages on hGF feeders, embryonic stem cell marker expression and CpG methylation at the NANOG and OCT3/4 promoters were similar for hGF-iPSCs cultured on hGF and SNL feeder cells. Long-term cultures of hGF-iPSCs on hGF feeders sustained their normal karyotype and pluripotency. On hGF feeders, hGF-iPSC colonies were surrounded by many colony-derived fibroblast-like cells, and the size of intact colonies at 7 d after passage was significantly larger than that on SNL feeders. Allogeneic hGF strains also maintained hGF-iPSCs for 10 passages. Compared with hDFs, hGFs showed a higher production of laminin-332, laminin α5 chain, and insulin-like growth factor-II, which have been reported to sustain the long-term self-renewal of pluripotent stem cells. These results suggest that hGFs possess an excellent feeder capability and thus can be used as alternatives to conventional mouse-derived SNL and hDF feeders. In addition, our findings suggest that hGF feeders are promising candidates for animal component-free ex vivo expansion of

  17. RNA Polymerase III Transcriptomes in Human Embryonic Stem Cells and Induced Pluripotent Stem Cells, and Relationships with Pluripotency Transcription Factors

    PubMed Central

    Alla, Ravi K.; Cairns, Bradley R.

    2014-01-01

    Recent genomic approaches have revealed that the repertoire of RNA Pol III-transcribed genes varies in different human cell types, and that this variation is likely determined by a combination of the chromatin landscape, cell-specific DNA-binding transcription factors, and collaboration with RNA Pol II. Although much is known about this regulation in differentiated human cells, there is presently little understanding of this aspect of the Pol III system in human ES cells. Here, we determine the occupancy profiles of Pol III components in human H1 ES cells, and also induced pluripotent cells, and compare to known profiles of chromatin, transcription factors, and RNA expression. We find a relatively large fraction of the Pol III repertoire occupied in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). In ES cells we find clear correlations between Pol III occupancy and active chromatin. Interestingly, we find a highly significant fraction of Pol III-occupied genes with adjacent binding events by pluripotency factors in ES cells, especially NANOG. Notably, in human ES cells we find H3K27me3 adjacent to but not overlapping many active Pol III loci. We observe in all such cases, a peak of H3K4me3 and/or RNA Pol II, between the H3K27me3 and Pol III binding peaks, suggesting that H3K4me3 and Pol II activity may “insulate” Pol III from neighboring repressive H3K27me3. Further, we find iPSCs have a larger Pol III repertoire than their precursors. Finally, the active Pol III genome in iPSCs is not completely reprogrammed to a hESC like state and partially retains the transcriptional repertoire of the precursor. Together, our correlative results are consistent with Pol III binding and activity in human ES cells being enabled by active/permissive chromatin that is shaped in part by the pluripotency network of transcription factors and RNA Pol II activity. PMID:24465633

  18. Comparison of American mink embryonic stem and induced pluripotent stem cell transcriptomes

    PubMed Central

    2015-01-01

    Background Recently fibroblasts of many mammalian species have been reprogrammed to pluripotent state using overexpression of several transcription factors. This technology allows production of induced pluripotent stem (iPS) cells with properties similar to embryonic stem (ES) cells. The completeness of reprogramming process is well studied in such species as mouse and human but there is not enough data on other species. We produced American mink (Neovison vison) ES and iPS cells and compared these cells using transcriptome analysis. Results We report the generation of 10 mink ES and 22 iPS cell lines. The majority of the analyzed cell lines had normal diploid chromosome number. The only ES cell line with XX chromosome set had both X-chromosomes in active state that is characteristic of pluripotent cells. The pluripotency of ES and iPS cell lines was confirmed by formation of teratomas with cell types representing all three germ layers. Transcriptome analysis of mink embryonic fibroblasts (EF), two ES and two iPS cell lines allowed us to identify 11831 assembled contigs which were annotated. These led to a number of 6891 unique genes. Of these 3201 were differentially expressed between mink EF and ES cells. We analyzed expression levels of these genes in iPS cell lines. This allowed us to show that 80% of genes were correctly reprogrammed in iPS cells, whereas approximately 6% had an intermediate expression pattern, about 7% were not reprogrammed and about 5% had a "novel" expression pattern. We observed expression of pluripotency marker genes such as Oct4, Sox2 and Rex1 in ES and iPS cell lines with notable exception of Nanog. Conclusions We had produced and characterized American mink ES and iPS cells. These cells were pluripotent by a number of criteria and iPS cells exhibited effective reprogramming. Interestingly, we had showed lack of Nanog expression and consider it as a species-specific feature. PMID:26694224

  19. Derivation of Induced Pluripotent Stem Cells for Human Disease Modeling

    PubMed Central

    Narsinh, Kamileh; Narsinh, Kazim H.; Wu, Joseph C.

    2011-01-01

    The successful derivation of human induced pluripotent stem cells (hiPSCs) by de-differentiation of somatic cells offers significant potential to overcome obstacles in the field of cardiovascular disease. hiPSC derivatives offer incredible potential for new disease models and regenerative medicine therapies. However, many questions remain regarding the optimal starting materials and methods to enable safe, efficient derivation of hiPSCs suitable for clinical applications. Initial reprogramming experiments were carried out using lentiviral or retroviral gene delivery methods. More recently, various non-viral methods that avoid permanent and random transgene insertion have emerged as alternatives. These include transient DNA transfection approaches using transposons or minicircle plasmids, protein transduction approaches, and RNA transfection approaches. In addition, several small molecules have been found to significantly augment iPSC derivation efficiency, allowing the use of a fewer number of genes during pluripotency induction. Here, we review these various methods for the derivation of hiPSCs, focusing on their ultimate clinical applicability, with an emphasis on their potential for use as cardiovascular therapies and disease modeling platforms. PMID:21527744

  20. Preclinical Studies for Induced Pluripotent Stem Cell-based Therapeutics*

    PubMed Central

    Harding, John; Mirochnitchenko, Oleg

    2014-01-01

    Induced pluripotent stem cells (iPSCs) and their differentiated derivatives can potentially be applied to cell-based therapy for human diseases. The properties of iPSCs are being studied intensively both to understand the basic biology of pluripotency and cellular differentiation and to solve problems associated with therapeutic applications. Examples of specific preclinical applications summarized briefly in this minireview include the use of iPSCs to treat diseases of the liver, nervous system, eye, and heart and metabolic conditions such as diabetes. Early stage studies illustrate the potential of iPSC-derived cells and have identified several challenges that must be addressed before moving to clinical trials. These include rigorous quality control and efficient production of required cell populations, improvement of cell survival and engraftment, and development of technologies to monitor transplanted cell behavior for extended periods of time. Problems related to immune rejection, genetic instability, and tumorigenicity must be solved. Testing the efficacy of iPSC-based therapies requires further improvement of animal models precisely recapitulating human disease conditions. PMID:24362021

  1. Induced pluripotent stem cells: from Nobel Prizes to clinical applications.

    PubMed

    Rashid, S Tamir; Alexander, Graeme J M

    2013-03-01

    Advances in basic hepatology have been constrained for many years by the inability to culture primary hepatocytes in vitro, until just over five years ago when the scientific playing field was changed beyond recognition with the demonstration that human skin fibroblasts could be reprogrammed to resemble embryonic cells. The reprogrammed cells, known as induced pluripotent stem cells (iPSCs), were then shown to have the capacity to re-differentiate into almost any human cell type, including hepatocytes. The unlimited number and isogenic nature of the cells that can be generated from tiny fragments of tissue have massive implications for the study of human liver diseases in vitro. Of more immediate clinical importance were recent data demonstrating precision gene therapy on patient specific iPSCs, which opens up the real and exciting possibility of autologous hepatocyte transplantation as a substitute for allogeneic whole liver transplantation, which has been an effective approach to end-stage liver disease, but one that has now been outstripped by demand. In this review, we describe the historical development, current technology and potential clinical applications of induced pluripotency, concluding with a perspective on possible future directions in this dynamic field. PMID:23131523

  2. Microfluidic technology enhances the potential of human pluripotent stem cells.

    PubMed

    Gagliano, Onelia; Elvassore, Nicola; Luni, Camilla

    2016-05-01

    Since the discovery of human somatic cell reprogramming, human induced pluripotent stem cells (hiPSC) have been increasingly recognized as the landmark for development of organs-on-chip. hiPSCs show a remarkable plasticity that is related to their ability to promptly respond to the surrounding environment. In vitro, the soluble culture microenvironment, with its critical balance between exogenous and cell-secreted factors, plays a great role in inducing hiPSC response, for both preserving pluripotency and controlling differentiation stages. Exploring the complexity of hiPSC microenvironment requires new experimental tools, as a tight control is limited within conventional culture dishes. Microfluidic technology is particularly attractive in hiPSC research because of its ability to mimic specific environmental cues by accurate control of soluble factors with high spatiotemporal resolution and in a high-throughput fashion. In this review, we highlight recent progress in hiPSC research enabled by microfluidic technology as well as new emerging scenarios. PMID:26772885

  3. Generation of induced pluripotent stem cells from the prairie vole.

    PubMed

    Manoli, Devanand S; Subramanyam, Deepa; Carey, Catriona; Sudin, Erik; Van Westerhuyzen, Julie A; Bales, Karen L; Blelloch, Robert; Shah, Nirao M

    2012-01-01

    The vast majority of animals mate more or less promiscuously. A few mammals, including humans, utilize more restrained mating strategies that entail a longer term affiliation with a single mating partner. Such pair bonding mating strategies have been resistant to genetic analysis because of a lack of suitable model organisms. Prairie voles are small mouse-like rodents that form enduring pair bonds in the wild as well as in the laboratory, and consequently they have been used widely to study social bonding behavior. The lack of targeted genetic approaches in this species however has restricted the study of the molecular and neural circuit basis of pair bonds. As a first step in rendering the prairie vole amenable to reverse genetics, we have generated induced pluripotent stem cell (IPSC) lines from prairie vole fibroblasts using retroviral transduction of reprogramming factors. These IPSC lines display the cellular and molecular hallmarks of IPSC cells from other organisms, including mice and humans. Moreover, the prairie vole IPSC lines have pluripotent differentiation potential since they can give rise to all three germ layers in tissue culture and in vivo. These IPSC lines can now be used to develop conditions that facilitate homologous recombination and eventually the generation of prairie voles bearing targeted genetic modifications to study the molecular and neural basis of pair bond formation. PMID:22675440

  4. Young at Heart: Pioneering Approaches to Model Nonischaemic Cardiomyopathy with Induced Pluripotent Stem Cells

    PubMed Central

    Gowran, Aoife; Rasponi, Marco; Perrucci, Gianluca L.; Righetti, Stefano; Zanobini, Marco; Pompilio, Giulio

    2016-01-01

    A mere 9 years have passed since the revolutionary report describing the derivation of induced pluripotent stem cells from human fibroblasts and the first in-patient translational use of cells obtained from these stem cells has already been achieved. From the perspectives of clinicians and researchers alike, the promise of induced pluripotent stem cells is alluring if somewhat beguiling. It is now evident that this technology is nascent and many areas for refinement have been identified and need to be considered before induced pluripotent stem cells can be routinely used to stratify, treat and cure patients, and to faithfully model diseases for drug screening purposes. This review specifically addresses the pioneering approaches to improve induced pluripotent stem cell based models of nonischaemic cardiomyopathy. PMID:27110250

  5. Young at Heart: Pioneering Approaches to Model Nonischaemic Cardiomyopathy with Induced Pluripotent Stem Cells.

    PubMed

    Gowran, Aoife; Rasponi, Marco; Visone, Roberta; Nigro, Patrizia; Perrucci, Gianluca L; Righetti, Stefano; Zanobini, Marco; Pompilio, Giulio

    2016-01-01

    A mere 9 years have passed since the revolutionary report describing the derivation of induced pluripotent stem cells from human fibroblasts and the first in-patient translational use of cells obtained from these stem cells has already been achieved. From the perspectives of clinicians and researchers alike, the promise of induced pluripotent stem cells is alluring if somewhat beguiling. It is now evident that this technology is nascent and many areas for refinement have been identified and need to be considered before induced pluripotent stem cells can be routinely used to stratify, treat and cure patients, and to faithfully model diseases for drug screening purposes. This review specifically addresses the pioneering approaches to improve induced pluripotent stem cell based models of nonischaemic cardiomyopathy. PMID:27110250

  6. Nanotopography Promotes Pancreatic Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.

    PubMed

    Kim, Jong Hyun; Kim, Hyung Woo; Cha, Kyoung Je; Han, Jiyou; Jang, Yu Jin; Kim, Dong Sung; Kim, Jong-Hoon

    2016-03-22

    Although previous studies suggest that nanotopographical features influence properties and behaviors of stem cells, only a few studies have attempted to derive clinically useful somatic cells from human pluripotent stem cells using nanopatterned surfaces. In the present study, we report that polystyrene nanopore-patterned surfaces significantly promote the pancreatic differentiation of human embryonic and induced pluripotent stem cells. We compared different diameters of nanopores and showed that 200 nm nanopore-patterned surfaces highly upregulated the expression of PDX1, a critical transcription factor for pancreatic development, leading to an approximately 3-fold increase in the percentage of differentiating PDX1(+) pancreatic progenitors compared with control flat surfaces. Furthermore, in the presence of biochemical factors, 200 nm nanopore-patterned surfaces profoundly enhanced the derivation of pancreatic endocrine cells producing insulin, glucagon, or somatostatin. We also demonstrate that nanopore-patterned surface-induced upregulation of PDX1 is associated with downregulation of TAZ, suggesting the potential role of TAZ in nanopore-patterned surface-mediated mechanotransduction. Our study suggests that appropriate cytokine treatments combined with nanotopographical stimulation could be a powerful tool for deriving a high purity of desired cells from human pluripotent stem cells. PMID:26900863

  7. Induced pluripotent stem cells from a spinal muscular atrophy patient

    PubMed Central

    Ebert, Allison D.; Yu, Junying; Rose, Ferrill F.; Mattis, Virginia B.; Lorson, Christian L.; Thomson, James A.; Svendsen, Clive N.

    2009-01-01

    Spinal muscular atrophy (SMA) is one of the most common inherited forms of neurological disease leading to infant mortality. Patients exhibit selective loss of lower motor neurons resulting in muscle weakness, paralysis, and often death. Although patient fibroblasts have been used extensively to study SMA, motor neurons have a unique anatomy and physiology which may underlie their vulnerability to the disease process. Here we report the generation of induced pluripotent stem (iPS) cells from skin fibroblast samples taken from a child with SMA. These cells expanded robustly in culture, maintained the disease genotype, and generated motor neurons that showed selective deficits compared to those derived from the child's unaffected mother. This is the first study to show human iPS cells can be used to model the specific pathology seen in a genetically inherited disease. As such, it represents a promising resource to study disease mechanisms, screen novel drug compounds, and develop new therapies. PMID:19098894

  8. Cryopreservation of human pluripotent stem cells in defined medium.

    PubMed

    Liu, Weiwei; Chen, Guokai

    2014-01-01

    This unit describes a cryopreservation procedure using an enzyme-free dissociation method to harvest cells and preserve cells in albumin-free chemically defined E8 medium for human pluripotent stem cells (hPSCs). The dissociation by EDTA/PBS produces small cell aggregates that allow high survival efficiency in passaging and cryopreservation. Cryopreservation in E8 medium eliminates serum and other animal products, and is suitable for dealing with the increasing demand for high-quality hPSCs in translational research. In combination with the special feature of EDTA/PBS dissociation, the protocols in this unit allow for efficient cryopreservation in a more time-saving manner. PMID:25366897

  9. Efficient Generation of Hypothalamic Neurons from Human Pluripotent Stem Cells.

    PubMed

    Wang, Liheng; Egli, Dieter; Leibel, Rudolph L

    2016-01-01

    The hypothalamus comprises neuronal clusters that are essential for body weight regulation and other physiological functions. Insights into the complex cellular physiology of this region of the brain are critical to understanding the pathogenesis of obesity, but human hypothalamic cells are largely inaccessible for direct study. Here we describe a technique for generation of arcuate-like hypothalamic neurons from human pluripotent stem (hPS) cells. Early activation of SHH signaling and inhibition of BMP and TGFβ signaling, followed by timed inhibition of NOTCH, can efficiently differentiate hPS cells into NKX2.1+ hypothalamic progenitors. Subsequent incubation with BDNF induces the differentiation and maturation of pro-opiomelanocortin and neuropeptide Y neurons, which are major cell types in the arcuate hypothalamus. These neurons have molecular and cellular characteristics consistent with arcuate neurons. © 2016 by John Wiley & Sons, Inc. PMID:27367166

  10. Generation of serotonin neurons from human pluripotent stem cells

    PubMed Central

    Lu, Jianfeng; Zhong, Xuefei; Liu, Huisheng; Hao, Ling; Huang, Cindy Tzu-Ling; Sherafat, Mohammad Amin; Jones, Jeffrey; Ayala, Melvin; Li, Lingjun; Zhang, Su-Chun

    2016-01-01

    Serotonin neurons located in the raphe nucleus of the hindbrain have crucial roles in regulating brain functions and have been implicated in various psychiatric disorders. Yet functional human serotonin neurons are not available for in vitro studies. Through manipulation of the WNT pathway, we demonstrate efficient differentiation of human pluripotent stem cells (hPSCs) to cells resembling central serotonin neurons, primarily those located in the rhombomeric segments 2–3 of the rostral raphe, which participate in high-order brain functions. The serotonin neurons express a series of molecules essential for serotonergic development, including tryptophan hydroxylase 2, exhibit typical electrophysiological properties and release serotonin in an activity-dependent manner. When treated with the FDA-approved drugs tramadol and escitalopram oxalate, they release or uptake serotonin in a dose- and time-dependent manner, suggesting the utility of these cells for the evaluation of drug candidates. PMID:26655496

  11. Auxetic nuclei in embryonic stem cells exiting pluripotency

    NASA Astrophysics Data System (ADS)

    Pagliara, Stefano; Franze, Kristian; McClain, Crystal R.; Wylde, George W.; Fisher, Cynthia L.; Franklin, Robin J. M.; Kabla, Alexandre J.; Keyser, Ulrich F.; Chalut, Kevin J.

    2014-06-01

    Embryonic stem cells (ESCs) self-renew in a state of naïve pluripotency in which they are competent to generate all somatic cells. It has been hypothesized that, before irreversibly committing, ESCs pass through at least one metastable transition state. This transition would represent a gateway for differentiation and reprogramming of somatic cells. Here, we show that during the transition, the nuclei of ESCs are auxetic: they exhibit a cross-sectional expansion when stretched and a cross-sectional contraction when compressed, and their stiffness increases under compression. We also show that the auxetic phenotype of transition ESC nuclei is driven at least in part by global chromatin decondensation. Through the regulation of molecular turnover in the differentiating nucleus by external forces, auxeticity could be a key element in mechanotransduction. Our findings highlight the importance of nuclear structure in the regulation of differentiation and reprogramming.

  12. Using human pluripotent stem cells to study Friedreich ataxia cardiomyopathy.

    PubMed

    Crombie, Duncan E; Pera, Martin F; Delatycki, Martin B; Pébay, Alice

    2016-06-01

    Friedreich ataxia (FRDA) is the most common of the inherited ataxias. It is an autosomal recessive disease characterised by degeneration of peripheral sensory neurons, regions of the central nervous system and cardiomyopathy. FRDA is usually due to homozygosity for trinucleotide GAA repeat expansions found within first intron of the FRATAXIN (FXN) gene, which results in reduced levels of the mitochondrial protein FXN. Reduced FXN protein results in mitochondrial dysfunction and iron accumulation leading to increased oxidative stress and cell death in the nervous system and heart. Yet the precise functions of FXN and the underlying mechanisms leading to disease pathology remain elusive. This is particularly true of the cardiac aspect of FRDA, which remains largely uncharacterized at the cellular level. Here, we summarise current knowledge on experimental models in which to study FRDA cardiomyopathy, with a particular focus on the use of human pluripotent stem cells as a disease model. PMID:27019046

  13. A Method for Genome Editing in Human Pluripotent Stem Cells.

    PubMed

    Smith, Cory; Ye, Zhaohui; Cheng, Linzhao

    2016-01-01

    Human pluripotent stem cells (PSCs) hold great potential for regenerative medicine and currently are being used as a research tool for basic discovery and disease modeling. To evaluate the role of a single genetic variant, a system of genome editing is needed to precisely mutate any desired DNA sequence in isolation and measure its effect on phenotype when compared to the isogenic parental PSC from which it was derived. This protocol describes the general targeting schemes used by researchers to edit PSCs to knock out, knock-in, or precisely alter a single nucleotide, covering conditions for electroporation, clonal isolation, and screening of edited PSCs for the targeted mutation. These recent advances simplify the procedure for genome editing, allowing individual researchers to induce nearly any desired mutation to further study its function or to reverse a disease-causing variant for future applications in regenerative medicine. PMID:27037073

  14. Human pluripotent stem cell-derived mesenchymal stem cells prevent allergic airway inflammation in mice.

    PubMed

    Sun, Yue-Qi; Deng, Meng-Xia; He, Jia; Zeng, Qing-Xiang; Wen, Weiping; Wong, David S H; Tse, Hung-Fat; Xu, Geng; Lian, Qizhou; Shi, Jianbo; Fu, Qing-Ling

    2012-12-01

    We previously found that mesenchymal stem cells (MSCs) derived from human-induced pluripotent stem cells (iPSCs) exerted immunomodulatory effects on Th2-mediated allergic rhinitis in vitro. However, their contribution to the asthma and allergic rhinitis in animal models remains unclear. In this study, we developed a mouse model of ovalbumin (OVA)-induced allergic inflammation in both the upper and lower airways and evaluated the effects of the systemic administration of human iPSC-MSCs and bone marrow-derived MSCs (BM-MSCs) on allergic inflammation. Our results showed that treatments with both the iPSC-MSCs and BM-MSCs before the challenge phase protected the animals from the majority of allergy-specific pathological changes. This protection included an inhibition of inflammatory cell infiltration and mucus production in the lung, a reduction in eosinophil infiltration in the nose, and a decrease in inflammatory cell infiltration in both the bronchoalveolar and nasal lavage fluids. In addition, treatment with iPSC-MSCs or BM-MSCs before the challenge phase resulted in reduced serum levels of Th2 immunoglobulins (e.g., IgE) and decreased levels of Th2 cytokines including interleukin (IL)-4, IL-5, or IL-13 in the bronchoalveolar and/or nasal lavage fluids. Similar therapeutic effects were observed when the animals were pretreated with human iPSC-MSCs before the sensitization phase. These data suggest that iPSC-MSCs may be used as an alternative strategy to adult MSCs in the treatment of asthma and allergic rhinitis. PMID:22987325

  15. HEXIM1 Induces Differentiation of Human Pluripotent Stem Cells

    PubMed Central

    Ding, Vanessa; Lew, Qiao Jing; Chu, Kai Ling; Natarajan, Subaashini; Rajasegaran, Vikneswari; Gurumurthy, Meera; Choo, Andre B. H.; Chao, Sheng-Hao

    2013-01-01

    Hexamethylene bisacetamide inducible protein 1 (HEXIM1) is best known as the inhibitor of positive transcription elongation factor b (P-TEFb), which is composed of cyclin-dependent kinase 9 (CDK9)/cyclin T1. P-TEFb is an essential regulator for the transcriptional elongation by RNA polymerase II. A genome-wide study using human embryonic stem cells shows that most mRNA synthesis is regulated at the stage of transcription elongation, suggesting a possible role for P-TEFb/HEXIM1 in the gene regulation of stem cells. In this report, we detected a marked increase in HEXIM1 protein levels in the differentiated human pluripotent stem cells (hPSCs) induced by LY294002 treatment. Since no changes in CDK9 and cyclin T1 were observed in the LY294002-treated cells, increased levels of HEXIM1 might lead to inhibition of P-TEFb activity. However, treatment with a potent P-TEFb inhibiting compound, flavopiridol, failed to induce hPSC differentiation, ruling out the possible requirement for P-TEFb kinase activity in hPSC differentiation. Conversely, differentiation was observed when hPSCs were incubated with hexamethylene bisacetamide, a HEXIM1 inducing reagent. The involvement of HEXIM1 in the regulation of hPSCs was further supported when overexpression of HEXIM1 concomitantly induced hPSC differentiation. Collectively, our study demonstrates a novel role of HEXIM1 in regulating hPSC fate through a P-TEFb-independent pathway. PMID:23977357

  16. Derivation of Ethnically Diverse Human Induced Pluripotent Stem Cell Lines.

    PubMed

    Chang, Eun Ah; Tomov, Martin L; Suhr, Steven T; Luo, Jiesi; Olmsted, Zachary T; Paluh, Janet L; Cibelli, Jose

    2015-01-01

    The human genome with all its ethnic variations contributes to differences in human development, aging, disease, repair, and response to medical treatments and is an exciting area of research and clinical study. The availability of well-characterized ethnically diverse stem cell lines is limited and has not kept pace with other advances in stem cell research. Here we derived xenofree ethnically diverse-human induced pluripotent stem cell (ED-iPSC) lines from fibroblasts obtained from individuals of African American, Hispanic-Latino, Asian, and Caucasian ethnic origin and have characterized the lines under a uniform platform for comparative analysis. Derived ED-iPSC lines are low passage number and evaluated in vivo by teratoma formation and in vitro by high throughput microarray analysis of EB formation and early differentiation for tri-lineage commitment to endoderm, ectoderm and mesoderm. These new xenofree ED-iPSC lines represent a well-characterized valuable resource with potential for use in future research in drug discovery or clinical investigations. PMID:26482195

  17. Retinal Organoids from Pluripotent Stem Cells Efficiently Recapitulate Retinogenesis

    PubMed Central

    Völkner, Manuela; Zschätzsch, Marlen; Rostovskaya, Maria; Overall, Rupert W.; Busskamp, Volker; Anastassiadis, Konstantinos; Karl, Mike O.

    2016-01-01

    Summary The plasticity of pluripotent stem cells provides new possibilities for studying development, degeneration, and regeneration. Protocols for the differentiation of retinal organoids from embryonic stem cells have been developed, which either recapitulate complete eyecup morphogenesis or maximize photoreceptor genesis. Here, we have developed a protocol for the efficient generation of large, 3D-stratified retinal organoids that does not require evagination of optic-vesicle-like structures, which so far limited the organoid yield. Analysis of gene expression in individual organoids, cell birthdating, and interorganoid variation indicate efficient, reproducible, and temporally regulated retinogenesis. Comparative analysis of a transgenic reporter for PAX6, a master regulator of retinogenesis, shows expression in similar cell types in mouse in vivo, and in mouse and human retinal organoids. Early or late Notch signaling inhibition forces cell differentiation, generating organoids enriched with cone or rod photoreceptors, respectively, demonstrating the power of our improved organoid system for future research in stem cell biology and regenerative medicine. PMID:27050948

  18. Smooth Muscle Precursor Cells Derived from Human Pluripotent Stem Cells for Treatment of Stress Urinary Incontinence.

    PubMed

    Wang, Zhe; Wen, Yan; Li, Yan Hui; Wei, Yi; Green, Morgaine; Wani, Prachi; Zhang, Pengbo; Pera, Renee Reijo; Chen, Bertha

    2016-03-15

    There is great interest in using stem cells (SC) to regenerate a deficient urethral sphincter in patients with urinary incontinence. The smooth muscle component of the sphincter is a significant contributor to sphincter function. However, current translational efforts for sphincter muscle restoration focus only on skeletal muscle regeneration because they rely on adult mesenchymal SC as cell source. These adult SC do not yield sufficient smooth muscle cells (SMCs) for transplantation. We may be able to overcome this limitation by using pluripotent stem cell (PSC) to derive SMCs. Hence, we sought to investigate whether smooth muscle precursor cells (pSMCs) derived from human PSCs can restore urethral function in an animal model generated by surgical urethrolysis and ovariectomy. Rats were divided into four groups: control (no intervention), sham saline (surgery + saline injection), bladder SMC (surgery + human bladder SMC injection), and treatment (surgery + pSMC injection, which includes human embryonic stem cell (hESC) H9-derived pSMC, episomal reprogrammed induced pluripotent stem cells (iPSCs)-derived pSMC, or viral reprogrammed iPSC-derived pSMC). pSMCs (2 × 10(6) cells/rat) were injected periurethrally 3 weeks postsurgery. Leak point pressure (LPP) and baseline external urethral sphincter electromyography were measured 5 weeks postinjection. Both iPSC-derived pSMC treatment groups showed significantly higher LPP compared to the sham saline group, consistent with restoration of urethral sphincter function. While the difference between the H9-derived pSMC treatment and sham saline group was not significant, it did show a trend toward restoration of the LPP to the level of intact controls. Our data indicate that pSMCs derived from human PSCs (hESC and iPSC) can restore sphincter function. PMID:26785911

  19. The convergence of cochlear implantation with induced pluripotent stem cell therapy.

    PubMed

    Gunewardene, Niliksha; Dottori, Mirella; Nayagam, Bryony A

    2012-09-01

    According to 2010 estimates from The National Institute on Deafness and other Communication Disorders, approximately 17% (36 million) American adults have reported some degree of hearing loss. Currently, the only clinical treatment available for those with severe-to-profound hearing loss is a cochlear implant, which is designed to electrically stimulate the auditory nerve in the absence of hair cells. Whilst the cochlear implant has been revolutionary in terms of providing hearing to the severe-to-profoundly deaf, there are variations in cochlear implant performance which may be related to the degree of degeneration of auditory neurons following hearing loss. Hence, numerous experimental studies have focused on enhancing the efficacy of cochlear implants by using neurotrophins to preserve the auditory neurons, and more recently, attempting to replace these dying cells with new neurons derived from stem cells. As a result, several groups are now investigating the potential for both embryonic and adult stem cells to replace the degenerating sensory elements in the deaf cochlea. Recent advances in our knowledge of stem cells and the development of induced pluripotency by Takahashi and Yamanaka in 2006, have opened a new realm of science focused on the use of induced pluripotent stem (iPS) cells for therapeutic purposes. This review will provide a broad overview of the potential benefits and challenges of using iPS cells in combination with a cochlear implant for the treatment of hearing loss, including differentiation of iPS cells into an auditory neural lineage and clinically relevant transplantation approaches. PMID:21956409

  20. Smooth Muscle Precursor Cells Derived from Human Pluripotent Stem Cells for Treatment of Stress Urinary Incontinence

    PubMed Central

    Wang, Zhe; Li, Yan Hui; Wei, Yi; Green, Morgaine; Wani, Prachi; Zhang, Pengbo; Pera, Renee Reijo; Chen, Bertha

    2016-01-01

    There is great interest in using stem cells (SC) to regenerate a deficient urethral sphincter in patients with urinary incontinence. The smooth muscle component of the sphincter is a significant contributor to sphincter function. However, current translational efforts for sphincter muscle restoration focus only on skeletal muscle regeneration because they rely on adult mesenchymal SC as cell source. These adult SC do not yield sufficient smooth muscle cells (SMCs) for transplantation. We may be able to overcome this limitation by using pluripotent stem cell (PSC) to derive SMCs. Hence, we sought to investigate whether smooth muscle precursor cells (pSMCs) derived from human PSCs can restore urethral function in an animal model generated by surgical urethrolysis and ovariectomy. Rats were divided into four groups: control (no intervention), sham saline (surgery + saline injection), bladder SMC (surgery + human bladder SMC injection), and treatment (surgery + pSMC injection, which includes human embryonic stem cell (hESC) H9-derived pSMC, episomal reprogrammed induced pluripotent stem cells (iPSCs)-derived pSMC, or viral reprogrammed iPSC-derived pSMC). pSMCs (2 × 106 cells/rat) were injected periurethrally 3 weeks postsurgery. Leak point pressure (LPP) and baseline external urethral sphincter electromyography were measured 5 weeks postinjection. Both iPSC-derived pSMC treatment groups showed significantly higher LPP compared to the sham saline group, consistent with restoration of urethral sphincter function. While the difference between the H9-derived pSMC treatment and sham saline group was not significant, it did show a trend toward restoration of the LPP to the level of intact controls. Our data indicate that pSMCs derived from human PSCs (hESC and iPSC) can restore sphincter function. PMID:26785911

  1. Generation of Mouse Induced Pluripotent Stem Cells by Protein Transduction

    PubMed Central

    Nemes, Csilla; Varga, Eszter; Polgar, Zsuzsanna; Klincumhom, Nuttha; Pirity, Melinda K.

    2014-01-01

    Somatic cell reprogramming has generated enormous interest after the first report by Yamanaka and his coworkers in 2006 on the generation of induced pluripotent stem cells (iPSCs) from mouse fibroblasts. Here we report the generation of stable iPSCs from mouse fibroblasts by recombinant protein transduction (Klf4, Oct4, Sox2, and c-Myc), a procedure designed to circumvent the risks caused by integration of exogenous sequences in the target cell genome associated with gene delivery systems. The recombinant proteins were fused in the frame to the glutathione-S-transferase tag for affinity purification and to the transactivator transcription-nuclear localization signal polypeptide to facilitate membrane penetration and nuclear localization. We performed the reprogramming procedure on embryonic fibroblasts from inbred (C57BL6) and outbred (ICR) mouse strains. The cells were treated with purified proteins four times, at 48-h intervals, and cultured on mitomycin C treated mouse embryonic fibroblast (MEF) cells in complete embryonic stem cell (ESC) medium until colonies formed. The iPSCs generated from the outbred fibroblasts exhibited similar morphology and growth properties to ESCs and were sustained in an undifferentiated state for more than 20 passages. The cells were checked for pluripotency-related markers (Oct4, Sox2, Klf4, cMyc, Nanog) by immunocytochemistry and by reverse transcription–polymerase chain reaction. The protein iPSCs (piPSCs) formed embryoid bodies and subsequently differentiated towards all three germ layer lineages. Importantly, the piPSCs could incorporate into the blastocyst and led to variable degrees of chimerism in newborn mice. These data show that recombinant purified cell-penetrating proteins are capable of reprogramming MEFs to iPSCs. We also demonstrated that the cells of the generated cell line satisfied all the requirements of bona fide mouse ESCs: form round colonies with defined boundaries; have a tendency to attach together with

  2. Graphene for improved femtosecond laser based pluripotent stem cell transfection.

    PubMed

    Mthunzi, Patience; He, Kuang; Ngcobo, Sandile; Khanyile, Thulile; Warner, Jamie H

    2014-05-01

    Pluripotent stem cells are hugely attractive in the tissue engineering research field as they can self-renew and be selectively differentiated into various cell types. For stem cell and tissue engineering research it is important to develop new, biocompatible scaffold materials and graphene has emerged as a promising material in this area as it does not compromise cell proliferation and accelerates specific cell differentiation. Previous studies have shown a non-invasive optical technique for mouse embryonic stem (mES) cell differentiation and transfection using femtosecond (fs) laser pulses. To investigate cellular responses to the influence of graphene and laser irradiation, here we present for the first time a study of mES cell fs laser transfection on graphene coated substrates. First we studied the impact of graphene on Chinese Hamster Ovary (CHO-K1) cell viability and cell cytotoxicity in the absence of laser exposure. These were tested via evaluating the mitochondrial activity through adenosine triphosphates (ATP) luminescence and breakages on the cell plasma membrane assessed using cytosolic lactate dehydrogenase (LDH) screening. Secondly, the effects of fs laser irradiation on cell viability and cytotoxicity at 1064 and 532 nm for cells plated and grown on graphene and pure glass were assessed. Finally, optical transfection of CHO-K1 and mES cells was performed on graphene coated versus plain glass substrates. Our results show graphene stimulated cell viability whilst triggering a mild release of intracellular LDH. We also observed that compared to pure glass substrates; laser irradiation at 1064 nm on graphene plates was less cytotoxic. Finally, in mES cells efficient optical transfection at 1064 (82%) and 532 (25%) nm was obtained due to the presence of a graphene support as compared to pristine glass. Here we hypothesize an up-regulation of cell adhesion promoting peptides or laminin-related receptors of the extracellular matrix (ECM) in cell samples

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

    PubMed

    Sanal, Madhusudana Girija

    2014-01-01

    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 the question of

  4. Identification of novel proteins differentially expressed in pluripotent embryonic stem cells and differentiated cells.

    PubMed

    Enomoto, Kei; Watanabe-Susaki, Kanako; Kowno, Megumi; Takada, Hitomi; Intoh, Atsushi; Yamanaka, Yuko; Hirano, Hisashi; Sugino, Hiromu; Asashima, Makoto; Kurisaki, Akira

    2015-01-01

    Mammalian pluripotent stem cells possess properties of self-renewal and pluripotency. These abilities are maintained by the strict regulation of pluripotent stem cell-specific transcription factor network and unique properties of chromatin in the stem cells. Although these major signaling pathways robustly control the characteristics of stem cells, other regulatory factors, such as metabolic pathways, are also known to modulate stem cell proliferation and differentiation. In this study, we fractionated protein samples from mouse embryonic stem (ES) cells cultured with or without the leukemia inhibitory factor (LIF). Protein expression was quantified by 2-dimensional differential gel electrophoresis (2D-DIGE). In total, 44 proteins were identified as being differentially expressed in the pluripotent stem cells and the differentiated cells. Surprisingly, half of the identified proteins were the proteins localized in mitochondria, which supply cellular energy and regulate cell cycle, development, and cell death. Some of these identified proteins are involved in the metabolic function and the regulation of pluripotency. Further analysis of the identified proteins could provide new information for the manipulation of pluripotency in ES cells. PMID:26399336

  5. In Vitro Gamete Differentiation from Pluripotent Stem Cells as a Promising Therapy for Infertility.

    PubMed

    Mouka, Aurélie; Tachdjian, Gérard; Dupont, Joëlle; Drévillon, Loïc; Tosca, Lucie

    2016-04-01

    Generation of gametes derived in vitro from pluripotent stem cells holds promising prospects for future reproductive applications. Indeed, it provides information on molecular and cellular mechanisms underlying germ cell (GC) development and could offer a new potential treatment for infertility. Great progress has been made in derivation of gametes from embryonic stem cells, despite ethical issues. Induced pluripotent stem cells (iPSCs) technology allows the reprogramming of a differentiated somatic cell, possibly emanating from the patient, into a pluripotent state. With the emergence of iPSCs, several studies created primordial GC stage to mature gamete-like cells in vitro in mice and humans. Recent findings in GC derivation suggest that in mice, functional gametes can be generated in vitro. This strengthens the idea that it might be possible in the future to generate functional human sperm and oocytes from pluripotent stem cells in culture. PMID:26873432

  6. Advancements in Induced Pluripotent Stem Cell Technology for Cardiac Regenerative Medicine

    PubMed Central

    Suh, Carol Y.; Wang, Zelun; Bártulos, Oscar; Qyang, Yibing

    2014-01-01

    Cardiovascular diseases remain the leading causes of morbidity and mortality in the developed world. Cellular based cardiac regenerative therapy serves as a potential approach to treating cardiovascular diseases. Although various cellular types have been tested, induced pluripotent stem cells are regarded as a promising cell source for therapy. In this review, we will highlight some of the advances in generating induced pluripotent stem cells and differentiation to cardiac cells. We will also discuss the progress in modeling cardiovascular diseases using induced pluripotent stem cell derived cardiac cells. As we continue to make progress in induced pluripotent stem cell and cardiac differentiation technology, we will become closer to application of cardiac regenerative medicine. PMID:24651517

  7. Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells Using Sendai Virus.

    PubMed

    Soares, Filipa A C; Pedersen, Roger A; Vallier, Ludovic

    2016-01-01

    This protocol describes the efficient isolation of peripheral blood mononuclear cells from circulating blood via density gradient centrifugation and subsequent generation of integration-free human induced pluripotent stem cells. Peripheral blood mononuclear cells are cultured for 9 days to allow expansion of the erythroblast population. The erythroblasts are then used to derive human induced pluripotent stem cells using Sendai viral vectors, each expressing one of the four reprogramming factors Oct4, Sox2, Klf4, and c-Myc. PMID:25687300

  8. Loss of pluripotency in human embryonic stem cells directly correlates with an increase in nuclear zinc.

    SciTech Connect

    Finney, L.; Vogt, S.; Wolford, J. L.; Chishti, Y.; Jin, Q.; Ward, J.; Chen, L.

    2010-01-01

    The pluripotency of human embryonic stem cells (hESCs) is important to investigations of early development and to cell replacement therapy, but the mechanism behind pluripotency is incompletely understood. Zinc has been shown to play a key role in differentiation of non-pluripotent cell types, but here its role in hESCs is directly examined. By mapping the distribution of metals in hESCs at high resolution by x-ray fluorescence microprobe (XFM) and by analyzing subcellular metal content, we have found evidence that loss of pluripotency is directly correlated with an increase in nuclear zinc. Zinc elevation not only redefines our understanding of the mechanisms that support pluripotency, but also may act as a biomarker and an intervention point for stem cell differentiation.

  9. Embryonic Stem Cells/Induced Pluripotent Stem Cells

    PubMed Central

    Feng, Xuezhu; Zhang, Jiuchun; Smuga-Otto, Kimberly; Tian, Shulan; Yu, Junying; Stewart, Ron; Thomson, James A.

    2012-01-01

    Unlike mouse embryonic stem cells (ESCs), which are closely related to the inner cell mass, human ESCs appear to be more closely related to the later primitive ectoderm. For example, human ESCs and primitive ectoderm share a common epithelial morphology, growth factor requirements, and the potential to differentiate to all three embryonic germ layers. However, it has previously been shown that human ESCs can also differentiate to cells expressing markers of trophoblast, an extraembryonic lineage formed before the formation of primitive ectoderm. Here, we show that phorbol ester 12-O-tetradecanoylphorbol 13-acetate causes human ESCs to undergo an epithelial mesenchymal transition and to differentiate into cells expressing markers of parietal endoderm, another extraembryonic lineage. We further confirmed that this differentiation is through the activation of protein kinase C (PKC) pathway and demonstrated that a particular PKC subtype, PKC-δ, is most responsible for this transition. PMID:22213079

  10. DNA methylation dynamics in human induced pluripotent stem cells.

    PubMed

    Nishino, Koichiro; Umezawa, Akihiro

    2016-07-01

    Indeed human induced pluripotent stem cells (hiPSCs) are considered to be powerful tools in regenerative medicine. To enable the use of hiPSCs in the field of regenerative medicine, it is necessary to understand the mechanisms of reprogramming during the transformation of somatic cells into hiPSCs. Genome-wide epigenetic modification constitutes a critical event in the generation of iPSCs. In other words, to analyze epigenetic changes in iPSCs means to elucidate reprogramming processes. We have established a large number of hiPSCs derived from various human tissues and have obtained their DNA methylation profiles. Comparison analyses indicated that the epigenetic patterns of various hiPSCs, irrespective of their source tissue, were very similar to one another and were similar to those of human embryonic stem cells (hESCs). However, the profiles of hiPSCs and hESCs exhibited epigenetic differences, which were caused by random aberrant hypermethylation at early passages. Interestingly, continuous passaging of the hiPSCs diminished the differences between DNA methylation profiles of hiPSCs and hESCs. The number of aberrant DNA methylation regions may thus represent a useful epigenetic index for evaluating hiPSCs in the context of therapeutic applications. PMID:27083573

  11. Application of Induced Pluripotent Stem Cells in Liver Diseases

    PubMed Central

    Yu, Yue; Wang, Xuehao; Nyberg, Scott L.

    2014-01-01

    Tens of millions of patients are affected by liver disease worldwide. Many of these patients can benefit from therapy involving hepatocyte transplantation. Liver transplantation is presently the only proven treatment for many medically refractory liver diseases including end-stage liver failure and inherited metabolic liver disease. However, the shortage in transplantable livers prevents over 40% of listed patients per year from receiving a liver transplant; many of these patients die before receiving an organ offer or become too sick to transplant. Therefore, new therapies are needed to supplement whole-organ liver transplantation and reduce mortality on waiting lists worldwide. Furthermore, the remarkable regenerative capacity of hepatocytes in vivo is exemplified by the increasing number of innovative cell-based therapies and animal models of human liver disorders. Induced pluripotent stem cells (iPSCs) have similar properties to those of embryonic stem cells (ESCs) but bypass the ethical concerns of embryo destruction. Therefore, generation of hepatocyte-like cells (HLCs) using iPSC technology may be beneficial for the treatment of severe liver diseases, screening of drug toxicities, basic research of several hepatocytic disorders, and liver transplantation. Here we briefly summarize the growing number of potential applications of iPSCs for treatment of liver disease. PMID:26858888

  12. Induced pluripotent stem cells for modeling neurological disorders.

    PubMed

    Russo, Fabiele B; Cugola, Fernanda R; Fernandes, Isabella R; Pignatari, Graciela C; Beltrão-Braga, Patricia C B

    2015-12-24

    Several diseases have been successfully modeled since the development of induced pluripotent stem cell (iPSC) technology in 2006. Since then, methods for increased reprogramming efficiency and cell culture maintenance have been optimized and many protocols for differentiating stem cell lines have been successfully developed, allowing the generation of several cellular subtypes in vitro. Gene editing technologies have also greatly advanced lately, enhancing disease-specific phenotypes by creating isogenic cell lines, allowing mutations to be corrected in affected samples or inserted in control lines. Neurological disorders have benefited the most from iPSC-disease modeling for its capability for generating disease-relevant cell types in vitro from the central nervous system, such as neurons and glial cells, otherwise only available from post-mortem samples. Patient-specific iPSC-derived neural cells can recapitulate the phenotypes of these diseases and therefore, considerably enrich our understanding of pathogenesis, disease mechanism and facilitate the development of drug screening platforms for novel therapeutic targets. Here, we review the accomplishments and the current progress in human neurological disorders by using iPSC modeling for Alzheimer's disease, Parkinson's disease, Huntington's disease, spinal muscular atrophy, amyotrophic lateral sclerosis, duchenne muscular dystrophy, schizophrenia and autism spectrum disorders, which include Timothy syndrome, Fragile X syndrome, Angelman syndrome, Prader-Willi syndrome, Phelan-McDermid, Rett syndrome as well as Nonsyndromic Autism. PMID:26722648

  13. Mimicking Retinal Development and Disease With Human Pluripotent Stem Cells.

    PubMed

    Sinha, Divya; Phillips, Jenny; Joseph Phillips, M; Gamm, David M

    2016-04-01

    As applications of human pluripotent stem cells (hPSCs) continue to be refined and pursued, it is important to keep in mind that the strengths and weaknesses of this technology lie with its developmental origins. The remarkable capacity of differentiating hPSCs to recapitulate cell and tissue genesis has provided a model system to study stages of human development that were not previously amenable to investigation and experimentation. Furthermore, demonstration of developmentally appropriate, stepwise differentiation of hPSCs to specific cell types offers support for their authenticity and their suitability for use in disease modeling and cell replacement therapies. However, limitations to farming cells and tissues in an artificial culture environment, as well as the length of time required for most cells to mature, are some of the many issues to consider before using hPSCs to study or treat a particular disease. Given the overarching need to understand and modulate the dynamics of lineage-specific differentiation in stem cell cultures, this review will first examine the capacity of hPSCs to serve as models of retinal development. Thereafter, we will discuss efforts to model retinal disorders with hPSCs and present challenges that face investigators who aspire to use such systems to study disease pathophysiology and/or screen for therapeutics. We also refer readers to recent publications that provide additional insight and details on these rapidly evolving topics. PMID:27116663

  14. Induced pluripotent stem cells for modeling neurological disorders

    PubMed Central

    Russo, Fabiele B; Cugola, Fernanda R; Fernandes, Isabella R; Pignatari, Graciela C; Beltrão-Braga, Patricia C B

    2015-01-01

    Several diseases have been successfully modeled since the development of induced pluripotent stem cell (iPSC) technology in 2006. Since then, methods for increased reprogramming efficiency and cell culture maintenance have been optimized and many protocols for differentiating stem cell lines have been successfully developed, allowing the generation of several cellular subtypes in vitro. Gene editing technologies have also greatly advanced lately, enhancing disease-specific phenotypes by creating isogenic cell lines, allowing mutations to be corrected in affected samples or inserted in control lines. Neurological disorders have benefited the most from iPSC-disease modeling for its capability for generating disease-relevant cell types in vitro from the central nervous system, such as neurons and glial cells, otherwise only available from post-mortem samples. Patient-specific iPSC-derived neural cells can recapitulate the phenotypes of these diseases and therefore, considerably enrich our understanding of pathogenesis, disease mechanism and facilitate the development of drug screening platforms for novel therapeutic targets. Here, we review the accomplishments and the current progress in human neurological disorders by using iPSC modeling for Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, spinal muscular atrophy, amyotrophic lateral sclerosis, duchenne muscular dystrophy, schizophrenia and autism spectrum disorders, which include Timothy syndrome, Fragile X syndrome, Angelman syndrome, Prader-Willi syndrome, Phelan-McDermid, Rett syndrome as well as Nonsyndromic Autism. PMID:26722648

  15. Germline Competent Pluripotent Mouse Stem Cells Generated by Plasmid Vectors.

    PubMed

    Chen, Chien-Hong; Su, Yu-Hsiu; Lee, Kun-Hsiung; Chuang, Chin-Kai

    2016-07-01

    We developed nonintegrated methods to reprogram mouse embryonic fibroblast (MEF) cells into induced pluripotent stem cells (iPSCs) using pig pOct4, pSox2, and pc-Myc as well as human hKLF4, hAID, and hTDG that were carried by plasmid vectors. The 4F method employed pOct4, pSox2, pc-Myc, and hKLF4 to derive iPSC clones with naive embryonic stem cell (ESC)-like morphology. These 4F clones expressed endogenous mouse Nanog protein and could generate chimeras. In addition to the four conventional reprogramming factors used in the 4F method, hAID and hTDG were utilized in a 6F method to increase the conversion efficiency of reprogramming by approximately five-fold. One of the 6F plasmid derived iPSC (piPSC) clones was shown to be germline transmission competent. PMID:26980563

  16. Induced pluripotent stem cells: origins, applications, and future perspectives.

    PubMed

    Zhao, Jing; Jiang, Wen-jie; Sun, Chen; Hou, Cong-zhe; Yang, Xiao-Mei; Gao, Jian-gang

    2013-12-01

    Embryonic stem (ES) cells are widely used for different purposes, including gene targeting, cell therapy, tissue repair, organ regeneration, and so on. However, studies and applications of ES cells are hindered by ethical issues regarding cell sources. To circumvent ethical disputes, great efforts have been taken to generate ES cell-like cells, which are not derived from the inner cell mass of blastocyst-stage embryos. In 2006, Yamanaka et al. first reprogrammed mouse embryonic fibroblasts into ES cell-like cells called induced pluripotent stem (iPS) cells. About one year later, Yamanaka et al. and Thomson et al. independently reprogrammed human somatic cells into iPS cells. Since the first generation of iPS cells, they have now been derived from quite a few different kinds of cell types. In particular, the use of peripheral blood facilitates research on iPS cells because of safety, easy availability, and plenty of cell sources. Now iPS cells have been used for cell therapy, disease modeling, and drug discovery. In this review, we describe the generations, applications, potential issues, and future perspectives of iPS cells. PMID:24302707

  17. Generating trunk neural crest from human pluripotent stem cells

    PubMed Central

    Huang, Miller; Miller, Matthew L.; McHenry, Lauren K.; Zheng, Tina; Zhen, Qiqi; Ilkhanizadeh, Shirin; Conklin, Bruce R.; Bronner, Marianne E.; Weiss, William A.

    2016-01-01

    Neural crest cells (NCC) are stem cells that generate different lineages, including neuroendocrine, melanocytic, cartilage, and bone. The differentiation potential of NCC varies according to the level from which cells emerge along the neural tube. For example, only anterior “cranial” NCC form craniofacial bone, whereas solely posterior “trunk” NCC contribute to sympathoadrenal cells. Importantly, the isolation of human fetal NCC carries ethical and scientific challenges, as NCC induction typically occur before pregnancy is detectable. As a result, current knowledge of NCC biology derives primarily from non-human organisms. Important differences between human and non-human NCC, such as expression of HNK1 in human but not mouse NCC, suggest a need to study human NCC directly. Here, we demonstrate that current protocols to differentiate human pluripotent stem cells (PSC) to NCC are biased toward cranial NCC. Addition of retinoic acid drove trunk-related markers and HOX genes characteristic of a posterior identity. Subsequent treatment with bone morphogenetic proteins (BMPs) enhanced differentiation to sympathoadrenal cells. Our approach provides methodology for detailed studies of human NCC, and clarifies roles for retinoids and BMPs in the differentiation of human PSC to trunk NCC and to sympathoadrenal lineages. PMID:26812940

  18. Finding the rhythm of sudden cardiac death: new opportunities using induced pluripotent stem cell-derived cardiomyocytes.

    PubMed

    Sallam, Karim; Li, Yingxin; Sager, Philip T; Houser, Steven R; Wu, Joseph C

    2015-06-01

    Sudden cardiac death is a common cause of death in patients with structural heart disease, genetic mutations, or acquired disorders affecting cardiac ion channels. A wide range of platforms exist to model and study disorders associated with sudden cardiac death. Human clinical studies are cumbersome and are thwarted by the extent of investigation that can be performed on human subjects. Animal models are limited by their degree of homology to human cardiac electrophysiology, including ion channel expression. Most commonly used cellular models are cellular transfection models, which are able to mimic the expression of a single-ion channel offering incomplete insight into changes of the action potential profile. Induced pluripotent stem cell-derived cardiomyocytes resemble, but are not identical, adult human cardiomyocytes and provide a new platform for studying arrhythmic disorders leading to sudden cardiac death. A variety of platforms exist to phenotype cellular models, including conventional and automated patch clamp, multielectrode array, and computational modeling. Induced pluripotent stem cell-derived cardiomyocytes have been used to study long QT syndrome, catecholaminergic polymorphic ventricular tachycardia, hypertrophic cardiomyopathy, and other hereditary cardiac disorders. Although induced pluripotent stem cell-derived cardiomyocytes are distinct from adult cardiomyocytes, they provide a robust platform to advance the science and clinical care of sudden cardiac death. PMID:26044252

  19. Therapeutics from Adult Stem Cells and the Hype Curve.

    PubMed

    Maguire, Greg

    2016-05-12

    The Gartner curve for regenerative and stem cell therapeutics is currently climbing out of the "trough of disillusionment" and into the "slope of enlightenment". Understanding that the early years of stem cell therapy relied on the model of embryonic stem cells (ESCs), and then moved into a period of the overhype of induced pluripotent stem cells (iPSCs), instead of using the model of 40 years of success, i.e. adult stem cells used in bone marrow transplants, the field of stem cell therapy has languished for years, trying to move beyond the early and poorly understood success of bone marrow transplants. Recent studies in the lab and clinic show that adult stem cells of various types, and the molecules that they release, avoid the issues associated with ESCs and iPSCs and lead to better therapeutic outcomes and into the slope of enlightenment. PMID:27190588

  20. Mass Spectrometry–based Proteomic Analysis of the Matrix Microenvironment in Pluripotent Stem Cell Culture*

    PubMed Central

    Hughes, Chris; Radan, Lida; Chang, Wing Y.; Stanford, William L.; Betts, Dean H.; Postovit, Lynne-Marie; Lajoie, Gilles A.

    2012-01-01

    The cellular microenvironment comprises soluble factors, support cells, and components of the extracellular matrix (ECM) that combine to regulate cellular behavior. Pluripotent stem cells utilize interactions between support cells and soluble factors in the microenvironment to assist in the maintenance of self-renewal and the process of differentiation. However, the ECM also plays a significant role in shaping the behavior of human pluripotent stem cells, including embryonic stem cells (hESCs) and induced pluripotent stem cells. Moreover, it has recently been observed that deposited factors in a hESC-conditioned matrix have the potential to contribute to the reprogramming of metastatic melanoma cells. Therefore, the ECM component of the pluripotent stem cell microenvironment necessitates further analysis. In this study we first compared the self-renewal and differentiation properties of hESCs grown on Matrigel™ pre-conditioned by hESCs to those on unconditioned Matrigel™. We determined that culture on conditioned Matrigel™ prevents differentiation when supportive growth factors are removed from the culture medium. To investigate and identify factors potentially responsible for this beneficial effect, we performed a defined SILAC MS-based proteomics screen of hESC-conditioned Matrigel™. From this proteomics screen, we identified over 80 extracellular proteins in matrix conditioned by hESCs and induced pluripotent stem cells. These included matrix-associated factors that participate in key stem cell pluripotency regulatory pathways, such as Nodal/Activin and canonical Wnt signaling. This work represents the first investigation of stem-cell-derived matrices from human pluripotent stem cells using a defined SILAC MS-based proteomics approach. PMID:23023296

  1. Mesenchymal stem cells and induced pluripotent stem cells as therapies for multiple sclerosis.

    PubMed

    Xiao, Juan; Yang, Rongbing; Biswas, Sangita; Qin, Xin; Zhang, Min; Deng, Wenbin

    2015-01-01

    Multiple sclerosis (MS) is a chronic, autoimmune, inflammatory demyelinating disorder of the central nervous system that leads to permanent neurological deficits. Current MS treatment regimens are insufficient to treat the irreversible neurological disabilities. Tremendous progress in the experimental and clinical applications of cell-based therapies has recognized stem cells as potential candidates for regenerative therapy for many neurodegenerative disorders including MS. Mesenchymal stem cells (MSC) and induced pluripotent stem cell (iPSCs) derived precursor cells can modulate the autoimmune response in the central nervous system (CNS) and promote endogenous remyelination and repair process in animal models. This review highlights studies involving the immunomodulatory and regenerative effects of mesenchymal stem cells and iPSCs derived cells in animal models, and their translation into immunomodulatory and neuroregenerative treatment strategies for MS. PMID:25918935

  2. Naive Pluripotent Stem Cells Derived Directly from Isolated Cells of the Human Inner Cell Mass.

    PubMed

    Guo, Ge; von Meyenn, Ferdinand; Santos, Fatima; Chen, Yaoyao; Reik, Wolf; Bertone, Paul; Smith, Austin; Nichols, Jennifer

    2016-04-12

    Conventional generation of stem cells from human blastocysts produces a developmentally advanced, or primed, stage of pluripotency. In vitro resetting to a more naive phenotype has been reported. However, whether the reset culture conditions of selective kinase inhibition can enable capture of naive epiblast cells directly from the embryo has not been determined. Here, we show that in these specific conditions individual inner cell mass cells grow into colonies that may then be expanded over multiple passages while retaining a diploid karyotype and naive properties. The cells express hallmark naive pluripotency factors and additionally display features of mitochondrial respiration, global gene expression, and genome-wide hypomethylation distinct from primed cells. They transition through primed pluripotency into somatic lineage differentiation. Collectively these attributes suggest classification as human naive embryonic stem cells. Human counterparts of canonical mouse embryonic stem cells would argue for conservation in the phased progression of pluripotency in mammals. PMID:26947977

  3. Naive Pluripotent Stem Cells Derived Directly from Isolated Cells of the Human Inner Cell Mass

    PubMed Central

    Guo, Ge; von Meyenn, Ferdinand; Santos, Fatima; Chen, Yaoyao; Reik, Wolf; Bertone, Paul; Smith, Austin; Nichols, Jennifer

    2016-01-01

    Summary Conventional generation of stem cells from human blastocysts produces a developmentally advanced, or primed, stage of pluripotency. In vitro resetting to a more naive phenotype has been reported. However, whether the reset culture conditions of selective kinase inhibition can enable capture of naive epiblast cells directly from the embryo has not been determined. Here, we show that in these specific conditions individual inner cell mass cells grow into colonies that may then be expanded over multiple passages while retaining a diploid karyotype and naive properties. The cells express hallmark naive pluripotency factors and additionally display features of mitochondrial respiration, global gene expression, and genome-wide hypomethylation distinct from primed cells. They transition through primed pluripotency into somatic lineage differentiation. Collectively these attributes suggest classification as human naive embryonic stem cells. Human counterparts of canonical mouse embryonic stem cells would argue for conservation in the phased progression of pluripotency in mammals. PMID:26947977

  4. Pluripotency and differentiation of cells from human testicular sperm extraction: An investigation of cell stemness.

    PubMed

    Sadeghian-Nodoushan, Fatemeh; Aflatoonian, Reza; Borzouie, Zahra; Akyash, Fatemeh; Fesahat, Farzaneh; Soleimani, Mehrdad; Aghajanpour, Samaneh; Moore, Harry D; Aflatoonian, Behrouz

    2016-04-01

    Human male germ-line stem cells (hmGSCs) and human testis-derived embryonic stem cell-like (htESC-like) cells are claimed to be in vitro pluripotent counterparts of spermatogonial stem cells (SSCs), but the origin and pluripotency of human testis-derived cell cultures are still under debate. The aim of this study was to generate putative pluripotent stem cells in vitro from human testicular sperm-extracted (TESE) samples of infertile men, and to assess their pluripotency and capacity to differentiate. TESE samples were minced, enzymatically disaggregated and dispersed into single-cell or cluster suspensions, and then cultured. Initially, cell clusters resembled those described for hmGSCs and htESC-like cells, and were positive for markers such as OCT4/POU5F1, NANOG, and TRA-2-54. Prolonged propagation of cell clusters expressing pluripotency markers did not thrive; instead, the cells that emerged possessed characteristics of mesenchymal stromal cells (MSCs) such as STRO-1, CD105/EGLN1, CD13/ANPEP, SOX9, vimentin, and fibronectin. KIT, SOX2, and CD44 were not expressed by these MSCs. The multipotential differentiation capacity of these cells was confirmed using Oil Red-O and Alizarin Red staining after induction with specific culture conditions. It is therefore concluded that pluripotent stem cells could not be derived using the conditions previously reported to be successful for TESE samples. PMID:27077675

  5. Present state and future perspectives of using pluripotent stem cells in toxicology research

    PubMed Central

    Löser, Peter

    2011-01-01

    The use of novel drugs and chemicals requires reliable data on their potential toxic effects on humans. Current test systems are mainly based on animals or in vitro–cultured animal-derived cells and do not or not sufficiently mirror the situation in humans. Therefore, in vitro models based on human pluripotent stem cells (hPSCs) have become an attractive alternative. The article summarizes the characteristics of pluripotent stem cells, including embryonic carcinoma and embryonic germ cells, and discusses the potential of pluripotent stem cells for safety pharmacology and toxicology. Special attention is directed to the potential application of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) for the assessment of developmental toxicology as well as cardio- and hepatotoxicology. With respect to embryotoxicology, recent achievements of the embryonic stem cell test (EST) are described and current limitations as well as prospects of embryotoxicity studies using pluripotent stem cells are discussed. Furthermore, recent efforts to establish hPSC-based cell models for testing cardio- and hepatotoxicity are presented. In this context, methods for differentiation and selection of cardiac and hepatic cells from hPSCs are summarized, requirements and implications with respect to the use of these cells in safety pharmacology and toxicology are presented, and future challenges and perspectives of using hPSCs are discussed. PMID:21225242

  6. Small-Molecule-Driven Hepatocyte Differentiation of Human Pluripotent Stem Cells

    PubMed Central

    Siller, Richard; Greenhough, Sebastian; Naumovska, Elena; Sullivan, Gareth J.

    2015-01-01

    Summary The differentiation of pluripotent stem cells to hepatocytes is well established, yet current methods suffer from several drawbacks. These include a lack of definition and reproducibility, which in part stems from continued reliance on recombinant growth factors. This has remained a stumbling block for the translation of the technology into industry and the clinic for reasons associated with cost and quality. We have devised a growth-factor-free protocol that relies on small molecules to differentiate human pluripotent stem cells toward a hepatic phenotype. The procedure can efficiently direct both human embryonic stem cells and induced pluripotent stem cells to hepatocyte-like cells. The final population of cells demonstrates marker expression at the transcriptional and protein levels, as well as key hepatic functions such as serum protein production, glycogen storage, and cytochrome P450 activity. PMID:25937370

  7. Haplotype-based banking of human pluripotent stem cells for transplantation: potential and limitations.

    PubMed

    Zimmermann, Anna; Preynat-Seauve, Olivier; Tiercy, Jean-Marie; Krause, Karl-Heinz; Villard, Jean

    2012-09-01

    High expectations surround the area of stem cells therapeutics. However, the cells' source-adult or embryonic-and the cells' origin-patient-derived autologous or healthy donor genetically unrelated-remain subjects of debate. Autologous origins have the advantage of a theoretical absence of immune rejection by the recipient. However, this approach has several limitations with regard to the disease of the recipient and to potential problems with the generation, expansion, and manipulation of autologous induced pluripotent stem cells (iPS cells) preparation. An alternative to using autologous cells is the establishment of a bank of well-characterized adult cells that would be used to generate iPS cells and their derivatives. In the context of transplantation, such cells would come from genetically unrelated donors and the immune system of the recipient would reject the graft without immunosuppressive therapy. To minimize the risk of rejection, human leukocyte antigen (HLA) compatibility is certainly the best option, and the establishment of an HLA-organized bank would mean having a limited number of stem cells that would be sufficient for a large number of recipients. The concept of haplobanking with HLA homozygous cell lines would also limit the number of HLA mismatches, but such an approach will not necessarily be less immunogenic in terms of selection criteria, because of the limited number of HLA-compatible loci and the level of HLA typing resolution. PMID:22559254

  8. New muscle for old hearts: engineering tissue from pluripotent stem cells.

    PubMed

    Martin, Ulrich

    2015-05-01

    Stem cell-based therapies are considered to be promising and innovative therapeutic strategies for heart repair. Patient-derived induced pluripotent stem cells (iPSCs) are now available, which combine the advantages of autologous adult stem cells with the unlimited potential of embryonic stem cells for proliferation and differentiation. Intense research has driven dramatic progress in various areas of iPSC technology relevant for clinically applicable iPSC-based cellular therapies. At this point, it is already possible to generate transgene-free autologous iPSCs from small blood samples or hair, to scale up the expansion and differentiation of iPSCs to clinically required dimensions, and to obtain highly enriched cardiomyocyte preparations. On the other hand, critical hurdles such as the targeted specification of distinct cardiomyocyte subpopulations, survival and proper functional integration of cellular transplants after myocardial infarction, and in vitro engineering of prevascularized muscle patches have yet to be overcome. Nevertheless, concepts of cellular cardiomyoplasty seem to have come of age and the first clinical applications of iPSC-based heart repair can be expected within the coming years. PMID:25915101

  9. Intricacies of Pluripotency.

    PubMed

    Bhartiya, Deepa

    2015-01-01

    Pluripotent stem cells have the potential to differentiate into 200 odd cell types present in adult body. Pluripotent stem cells available for regenerative medicine include embryonic stem (ES) cells, induced pluripotent stem (iPS) cells and very small ES-like stem (VSELs) cells. Nuclear OCT-4 is one of the crucial factors that dictate pluripotent state. Compared to ES/iPS cells grown in Petri dish, VSELs exist in adult body organs and results are emerging to suggest that they may have better potential to regenerate adult organs. This is because of their distinct epigenetic status as they are closer to the primordial germ cells from the epiblast-stage embryo compared to inner cell mass from which ES cells are obtained in vitro. We need to make special efforts to study them as they are very small in size and tend to get lost during processing. VSELs exist in adult organs, get mobilized in response to stress, undergo asymmetric cell divisions to give rise to tissue specific progenitors which further differentiate into various cell types and are possibly better candidates for regenerative medicine because they have no associated risk of tumor formation or immunological rejection. They are possibly also the 'embryonic remnants' in adult organs responsible for initiating cancer. Thus, rather than not accepting VSELs because they neither form teratoma nor divide in vitro like ES cells, it is time that scientific community should think of revising the definition of the term 'pluripotency'. PMID:26195889

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

    PubMed Central

    Ruiz, Sergio; Lopez-Contreras, Andres J.; Gabut, Mathieu; Marion, Rosa M.; Gutierrez-Martinez, Paula; Bua, Sabela; Ramirez, Oscar; Olalde, Iñigo; Rodrigo-Perez, Sara; Li, Han; Marques-Bonet, Tomas; Serrano, Manuel; Blasco, Maria A.; Batada, Nizar N.; Fernandez-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. Increasing the levels of the checkpoint kinase 1 (CHK1) reduces reprogramming-induced replication stress and increases the efficiency of iPSC generation. Similarly, nucleoside supplementation during reprogramming reduces the load of DNA damage and genomic rearrangements on iPSC. Our data reveal that lowering replication stress during reprogramming, genetically or chemically, provides a simple strategy to reduce genomic instability on mouse and human iPSC. PMID:26292731

  11. Induced pluripotent stem cell-derived cardiomyocytes: boutique science or valuable arrhythmia model?

    PubMed

    Knollmann, Björn C

    2013-03-15

    This article reviews the strengths and limitations of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) as models of cardiac arrhythmias. Specifically, the article attempts to answer the following questions: Which clinical arrhythmias can be modeled by iPSC-CM? How well can iPSC-CM model adult ventricular myocytes? What are the strengths and limitations of published iPSC-CM arrhythmia models? What new mechanistic insight has been gained? What is the evidence that would support using iPSC-CM to personalize antiarrhythmic drug therapy? The review also discusses the pros and cons of using the iPSC-CM technology for modeling specific genetic arrhythmia disorders, such as long QT syndrome, Brugada Syndrome, or Catecholaminergic Polymorphic Ventricular Tachycardia. PMID:23569106

  12. Engineering bone tissue substitutes from human induced pluripotent stem cells

    PubMed Central

    de Peppo, Giuseppe Maria; Marcos-Campos, Iván; Kahler, David John; Alsalman, Dana; Shang, Linshan; Vunjak-Novakovic, Gordana; Marolt, Darja

    2013-01-01

    Congenital defects, trauma, and disease can compromise the integrity and functionality of the skeletal system to the extent requiring implantation of bone grafts. Engineering of viable bone substitutes that can be personalized to meet specific clinical needs represents a promising therapeutic alternative. The aim of our study was to evaluate the utility of human-induced pluripotent stem cells (hiPSCs) for bone tissue engineering. We first induced three hiPSC lines with different tissue and reprogramming backgrounds into the mesenchymal lineages and used a combination of differentiation assays, surface antigen profiling, and global gene expression analysis to identify the lines exhibiting strong osteogenic differentiation potential. We then engineered functional bone substitutes by culturing hiPSC-derived mesenchymal progenitors on osteoconductive scaffolds in perfusion bioreactors and confirmed their phenotype stability in a subcutaneous implantation model for 12 wk. Molecular analysis confirmed that the maturation of bone substitutes in perfusion bioreactors results in global repression of cell proliferation and an increased expression of lineage-specific genes. These results pave the way for growing patient-specific bone substitutes for reconstructive treatments of the skeletal system and for constructing qualified experimental models of development and disease. PMID:23653480

  13. Fast and Efficient Multitransgenic Modification of Human Pluripotent Stem Cells

    PubMed Central

    Schwanke, Kristin; Merkert, Sylvia; Kempf, Henning; Hartung, Susann; Jara-Avaca, Monica; Templin, Christian; Göhring, Gudrun; Haverich, Axel

    2014-01-01

    Abstract Human pluripotent stem cells (hPSCs) represent a prime cell source for pharmacological research and regenerative therapies because of their extensive expansion potential and their ability to differentiate into essentially all somatic lineages in vitro. Improved methods to stably introduce multiple transgenes into hPSCs will promote, for example, their preclinical testing by facilitating lineage differentiation and purification in vitro and the subsequent in vivo monitoring of respective progenies after their transplantation into relevant animal models. To date, the establishment of stable transgenic hPSC lines is still laborious and time-consuming. Current limitations include the low transfection efficiency of hPSCs via nonviral methods, the inefficient recovery of genetically engineered clones, and the silencing of transgene expression. Here we describe a fast, electroporation-based method for the generation of multitransgenic hPSC lines by overcoming the need for any preadaptation of conventional hPSC cultures to feeder-free conditions before genetic manipulation. We further show that the selection for a single antibiotic resistance marker encoded on one plasmid allowed for the stable genomic (co-)integration of up to two additional, independent expression plasmids. The method thereby enables the straightforward, nonviral generation of valuable multitransgenic hPSC lines in a single step. Practical applicability of the method is demonstrated for antibiotic-based lineage enrichment in vitro and for sodium iodide symporter transgene-based in situ cell imaging after intramyocardial cell infusion into explanted pig hearts. PMID:24483184

  14. A New Class of Pluripotent Stem Cell Cytotoxic Small Molecules

    PubMed Central

    Goh, Gwendoline Tze Wei; Seng, Eng Khuan; Guo, Xu Ming; Tan, Cherine Mei Fong; Chan, Woon-Khiong; Lee, Joel Mun Kin

    2014-01-01

    A major concern in Pluripotent Stem Cell (PSC)-derived cell replacement therapy is the risk of teratoma formation from contaminating undifferentiated cells. Removal of undifferentiated cells from differentiated cultures is an essential step before PSC-based cell therapies can be safely deployed in a clinical setting. We report a group of novel small molecules that are cytotoxic to PSCs. Our data indicates that these molecules are specific and potent in their activity allowing rapid eradication of undifferentiated cells. Experiments utilizing mixed PSC and primary human neuronal and cardiomyocyte cultures demonstrate that up to a 6-fold enrichment for specialized cells can be obtained without adversely affecting cell viability and function. Several structural variants were synthesized to identify key functional groups and to improve specificity and efficacy. Comparative microarray analysis and ensuing RNA knockdown studies revealed involvement of the PERK/ATF4/DDIT3 ER stress pathway. Surprisingly, cell death following ER stress induction was associated with a concomitant decrease in endogenous ROS levels in PSCs. Undifferentiated cells treated with these molecules preceding transplantation fail to form teratomas in SCID mice. Furthermore, these molecules remain non-toxic and non-teratogenic to zebrafish embryos suggesting that they may be safely used in vivo. PMID:24647085

  15. Induced Pluripotent Stem Cell Therapies for Cervical Spinal Cord Injury.

    PubMed

    Doulames, Vanessa M; Plant, Giles W

    2016-01-01

    Cervical-level injuries account for the majority of presented spinal cord injuries (SCIs) to date. Despite the increase in survival rates due to emergency medicine improvements, overall quality of life remains poor, with patients facing variable deficits in respiratory and motor function. Therapies aiming to ameliorate symptoms and restore function, even partially, are urgently needed. Current therapeutic avenues in SCI seek to increase regenerative capacities through trophic and immunomodulatory factors, provide scaffolding to bridge the lesion site and promote regeneration of native axons, and to replace SCI-lost neurons and glia via intraspinal transplantation. Induced pluripotent stem cells (iPSCs) are a clinically viable means to accomplish this; they have no major ethical barriers, sources can be patient-matched and collected using non-invasive methods. In addition, the patient's own cells can be used to establish a starter population capable of producing multiple cell types. To date, there is only a limited pool of research examining iPSC-derived transplants in SCI-even less research that is specific to cervical injury. The purpose of the review herein is to explore both preclinical and clinical recent advances in iPSC therapies with a detailed focus on cervical spinal cord injury. PMID:27070598

  16. Directed Myogenic Differentiation of Human Induced Pluripotent Stem Cells.

    PubMed

    Shoji, Emi; Woltjen, Knut; Sakurai, Hidetoshi

    2016-01-01

    Patient-derived induced pluripotent stem cells (iPSCs) have opened the door to recreating pathological conditions in vitro using differentiation into diseased cells corresponding to each target tissue. Yet for muscular diseases, a method for reproducible and efficient myogenic differentiation from human iPSCs is required for in vitro modeling. Here, we introduce a myogenic differentiation protocol mediated by inducible transcription factor expression that reproducibly and efficiently drives human iPSCs into myocytes. Delivering a tetracycline-inducible, myogenic differentiation 1 (MYOD1) piggyBac (PB) vector to human iPSCs enables the derivation of iPSCs that undergo uniform myogenic differentiation in a short period of time. This differentiation protocol yields a homogenous skeletal muscle cell population, reproducibly reaching efficiencies as high as 70-90 %. MYOD1-induced myocytes demonstrate characteristics of mature myocytes such as cell fusion and cell twitching in response to electric stimulation within 14 days of differentiation. This differentiation protocol can be applied widely in various types of patient-derived human iPSCs and has great prospects in disease modeling particularly with inherited diseases that require studies of early pathogenesis and drug screening. PMID:25971915

  17. Building a microphysiological skin model from induced pluripotent stem cells

    PubMed Central

    2013-01-01

    The discovery of induced pluripotent stem cells (iPSCs) in 2006 was a major breakthrough for regenerative medicine. The establishment of patient-specific iPSCs has created the opportunity to model diseases in culture systems, with the potential to rapidly advance the drug discovery field. Current methods of drug discovery are inefficient, with a high proportion of drug candidates failing during clinical trials due to low efficacy and/or high toxicity. Many drugs fail toxicity testing during clinical trials, since the cells on which they have been tested do not adequately model three-dimensional tissues or their interaction with other organs in the body. There is a need to develop microphysiological systems that reliably represent both an intact tissue and also the interaction of a particular tissue with other systems throughout the body. As the port of entry for many drugs is via topical delivery, the skin is the first line of exposure, and also one of the first organs to demonstrate a reaction after systemic drug delivery. In this review, we discuss our strategy to develop a microphysiological system using iPSCs that recapitulates human skin for analyzing the interactions of drugs with the skin. PMID:24564920

  18. The potential of induced pluripotent stem cell derived hepatocytes.

    PubMed

    Hannoun, Zara; Steichen, Clara; Dianat, Noushin; Weber, Anne; Dubart-Kupperschmitt, Anne

    2016-07-01

    Orthotopic liver transplantation remains the only curative treatment for liver disease. However, the number of patients who die while on the waiting list (15%) has increased in recent years as a result of severe organ shortages; furthermore the incidence of liver disease is increasing worldwide. Clinical trials involving hepatocyte transplantation have provided encouraging results. However, transplanted cell function appears to often decline after several months, necessitating liver transplantation. The precise aetiology of the loss of cell function is not clear, but poor engraftment and immune-mediated loss appear to be important factors. Also, primary human hepatocytes (PHH) are not readily available, de-differentiate, and die rapidly in culture. Hepatocytes are available from other sources, such as tumour-derived human hepatocyte cell lines and immortalised human hepatocyte cell lines or porcine hepatocytes. However, all these cells suffer from various limitations such as reduced or differences in functions or risk of zoonotic infections. Due to their significant potential, one possible inexhaustible source of hepatocytes is through the directed differentiation of human induced pluripotent stem cells (hiPSCs). This review will discuss the potential applications and existing limitations of hiPSC-derived hepatocytes in regenerative medicine, drug screening, in vitro disease modelling and bioartificial livers. PMID:26916529

  19. Generation of kidney organoids from human pluripotent stem cells.

    PubMed

    Takasato, Minoru; Er, Pei X; Chiu, Han S; Little, Melissa H

    2016-09-01

    The human kidney develops from four progenitor populations-nephron progenitors, ureteric epithelial progenitors, renal interstitial progenitors and endothelial progenitors-resulting in the formation of maximally 2 million nephrons. Until recently, the reported methods differentiated human pluripotent stem cells (hPSCs) into either nephron progenitor or ureteric epithelial progenitor cells, consequently forming only nephrons or collecting ducts, respectively. Here we detail a protocol that simultaneously induces all four progenitors to generate kidney organoids within which segmented nephrons are connected to collecting ducts and surrounded by renal interstitial cells and an endothelial network. As evidence of functional maturity, proximal tubules within organoids display megalin-mediated and cubilin-mediated endocytosis, and they respond to a nephrotoxicant to undergo apoptosis. This protocol consists of 7 d of monolayer culture for intermediate mesoderm induction, followed by 18 d of 3D culture to facilitate self-organizing renogenic events leading to organoid formation. Personnel experienced in culturing hPSCs are required to conduct this protocol. PMID:27560173

  20. The business of exploiting induced pluripotent stem cells.

    PubMed

    Prescott, Catherine

    2011-08-12

    Induced pluripotent stem cells (iPS cells) can be exploited for both research and clinical applications. The first part of this review seeks to provide an understanding of the financial drivers and key elements of a successful business strategy that underpin a company focused on developing iPS-related products and services targeted at the research market. The latter part of the review highlights some of the reasons as to why the reprogramming of somatic cells is currently being used to develop cell-based models to screen for small molecules with drug-like properties rather than to develop cell-based regenerative medicines per se. The latter may be used to repair or replace a patient's damaged cells and thereby have the potential to 'cure' a disease and, in doing so, prevent or delay the onset of associated medical conditions. However, the cost of an expensive regenerative medicine and time to accrue any benefit linked to a decrease in co-morbidity expenditure may not outweigh the benefit for a healthcare community that has finite resources. The implications of this are discussed together with evidence that the UK National Institute for Health and Clinical Excellence (NICE) and the National Health Service (NHS) have established a precedent for a cost-sharing strategy with the pharmaceutical industry. PMID:21727138

  1. Induced Pluripotent Stem Cell Therapies for Cervical Spinal Cord Injury

    PubMed Central

    Doulames, Vanessa M.; Plant, Giles W.

    2016-01-01

    Cervical-level injuries account for the majority of presented spinal cord injuries (SCIs) to date. Despite the increase in survival rates due to emergency medicine improvements, overall quality of life remains poor, with patients facing variable deficits in respiratory and motor function. Therapies aiming to ameliorate symptoms and restore function, even partially, are urgently needed. Current therapeutic avenues in SCI seek to increase regenerative capacities through trophic and immunomodulatory factors, provide scaffolding to bridge the lesion site and promote regeneration of native axons, and to replace SCI-lost neurons and glia via intraspinal transplantation. Induced pluripotent stem cells (iPSCs) are a clinically viable means to accomplish this; they have no major ethical barriers, sources can be patient-matched and collected using non-invasive methods. In addition, the patient’s own cells can be used to establish a starter population capable of producing multiple cell types. To date, there is only a limited pool of research examining iPSC-derived transplants in SCI—even less research that is specific to cervical injury. The purpose of the review herein is to explore both preclinical and clinical recent advances in iPSC therapies with a detailed focus on cervical spinal cord injury. PMID:27070598

  2. The business of exploiting induced pluripotent stem cells

    PubMed Central

    Prescott, Catherine

    2011-01-01

    Induced pluripotent stem cells (iPS cells) can be exploited for both research and clinical applications. The first part of this review seeks to provide an understanding of the financial drivers and key elements of a successful business strategy that underpin a company focused on developing iPS-related products and services targeted at the research market. The latter part of the review highlights some of the reasons as to why the reprogramming of somatic cells is currently being used to develop cell-based models to screen for small molecules with drug-like properties rather than to develop cell-based regenerative medicines per se. The latter may be used to repair or replace a patient's damaged cells and thereby have the potential to ‘cure’ a disease and, in doing so, prevent or delay the onset of associated medical conditions. However, the cost of an expensive regenerative medicine and time to accrue any benefit linked to a decrease in co-morbidity expenditure may not outweigh the benefit for a healthcare community that has finite resources. The implications of this are discussed together with evidence that the UK National Institute for Health and Clinical Excellence (NICE) and the National Health Service (NHS) have established a precedent for a cost-sharing strategy with the pharmaceutical industry. PMID:21727138

  3. A new class of pluripotent stem cell cytotoxic small molecules.

    PubMed

    Richards, Mark; Phoon, Chee Wee; Goh, Gwendoline Tze Wei; Seng, Eng Khuan; Guo, Xu Ming; Tan, Cherine Mei Fong; Chan, Woon-Khiong; Lee, Joel Mun Kin

    2014-01-01

    A major concern in Pluripotent Stem Cell (PSC)-derived cell replacement therapy is the risk of teratoma formation from contaminating undifferentiated cells. Removal of undifferentiated cells from differentiated cultures is an essential step before PSC-based cell therapies can be safely deployed in a clinical setting. We report a group of novel small molecules that are cytotoxic to PSCs. Our data indicates that these molecules are specific and potent in their activity allowing rapid eradication of undifferentiated cells. Experiments utilizing mixed PSC and primary human neuronal and cardiomyocyte cultures demonstrate that up to a 6-fold enrichment for specialized cells can be obtained without adversely affecting cell viability and function. Several structural variants were synthesized to identify key functional groups and to improve specificity and efficacy. Comparative microarray analysis and ensuing RNA knockdown studies revealed involvement of the PERK/ATF4/DDIT3 ER stress pathway. Surprisingly, cell death following ER stress induction was associated with a concomitant decrease in endogenous ROS levels in PSCs. Undifferentiated cells treated with these molecules preceding transplantation fail to form teratomas in SCID mice. Furthermore, these molecules remain non-toxic and non-teratogenic to zebrafish embryos suggesting that they may be safely used in vivo. PMID:24647085

  4. Pluripotent stem cells: An in vitro model for nanotoxicity assessments.

    PubMed

    Handral, Harish K; Tong, Huei Jinn; Islam, Intekhab; Sriram, Gopu; Rosa, Vinicus; Cao, Tong

    2016-10-01

    The advent of technology has led to an established range of engineered nanoparticles that are used in diverse applications, such as cell-cell interactions, cell-material interactions, medical therapies and the target modulation of cellular processes. The exponential increase in the utilization of nanomaterials and the growing number of associated criticisms has highlighted the potential risks of nanomaterials to human health and the ecosystem. The existing in vivo and in vitro platforms show limitations, with fluctuations being observed in the results of toxicity assessments. Pluripotent stem cells (PSCs) are viable source of cells that are capable of developing into specialized cells of the human body. PSCs can be efficiently used to screen new biomaterials/drugs and are potential candidates for studying impairments of biophysical morphology at both the cellular and tissue levels during interactions with nanomaterials and for diagnosing toxicity. Three-dimensional in vitro models obtained using PSC-derived cells would provide a realistic, patient-specific platform for toxicity assessments and in drug screening applications. The current review focuses on PSCs as an alternative in vitro platform for assessing the hazardous effects of nanomaterials on health systems and highlights the importance of PSC-derived in vitro platforms. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27241574

  5. Modeling Familial Cancer with Induced Pluripotent Stem Cells

    PubMed Central

    Lee, Dung-Fang; Su, Jie; Kim, Huen Suk; Chang, Betty; Papatsenko, Dmitri; Zhao, Ruiying; Yuan, Ye; Gingold, Julian; Xia, Weiya; Darr, Henia; Mirzayans, Razmik; Hung, Mien-Chie; Schaniel, Christoph; Lemischka, Ihor R.

    2015-01-01

    SUMMARY In vitro modeling of human disease has recently become feasible with induced pluripotent stem cell (iPSC) technology. Here, we established patient-derived iPSCs from a Li-Fraumeni Syndrome (LFS) family and investigated the role of mutant p53 in the development of osteosarcoma (OS). LFS iPSC-derived osteoblasts (OBs) recapitulated OS features including defective osteoblastic differentiation as well as tumorigenic ability. Systematic analyses revealed that the expression of genes enriched in LFS-derived OBs strongly correlated with decreased time to tumor recurrence and poor patient survival. Furthermore, LFS OBs exhibited impaired upregulation of the imprinted gene H19 during osteogenesis. Restoration of H19 expression in LFS OBs facilitated osteoblastic differentiation and repressed tumorigenic potential. By integrating human imprinted gene network (IGN) into functional genomic analyses, we found that H19 mediates suppression of LFS-associated OS through the IGN component DECORIN (DCN). In summary, these findings demonstrate the feasibility of studying inherited human cancer syndromes with iPSCs. PMID:25860607

  6. Modeling Rett Syndrome Using Human Induced Pluripotent Stem Cells.

    PubMed

    Andoh-Noda, Tomoko; Inouye, Michiko O; Miyake, Kunio; Kubota, Takeo; Okano, Hideyuki; Akamatsu, Wado

    2016-01-01

    Rett syndrome (RTT) is one of a group of neurodevelopmental disorders typically characterized by deficits in the X-linked gene MECP2 (methyl-CpG binding protein 2). The MECP2 gene encodes a multifunctional protein involved in transcriptional repression, transcriptional activation, chromatin remodeling, and RNA splicing. Genetic deletion of Mecp2 in mice revealed neuronal disabilities including RTT-like phenotypes and provided an excellent platform for understanding the pathogenesis of RTT. So far, there are no effective pharmacological treatments for RTT because the role of MECP2 in RTT is incompletely understood. Recently, human induced pluripotent stem cell (hiPSC) technologies have improved our knowledge of neurological and neurodevelopmental diseases including RTT because neurons derived from RTT-hiPSCs can be used for disease modeling to understand RTT phenotypes and to perform high throughput pharmaceutical drug screening. In this review, we provide an overview of RTT, including MeCP2 function and mouse models of RTT. In addition, we introduce recent advances in disease modeling of RTT using hiPSC-derived neural cells. PMID:27071793

  7. A Method to Identify and Isolate Pluripotent Human Stem Cells and Mouse Epiblast Stem Cells Using Lipid Body-Associated Retinyl Ester Fluorescence

    PubMed Central

    Muthusamy, Thangaselvam; Mukherjee, Odity; Menon, Radhika; Megha, P.B.; Panicker, Mitradas M.

    2014-01-01

    Summary We describe the use of a characteristic blue fluorescence to identify and isolate pluripotent human embryonic stem cells and human-induced pluripotent stem cells. The blue fluorescence emission (450–500 nm) is readily observed by fluorescence microscopy and correlates with the expression of pluripotency markers (OCT4, SOX2, and NANOG). It allows easy identification and isolation of undifferentiated human pluripotent stem cells, high-throughput fluorescence sorting and subsequent propagation. The fluorescence appears early during somatic reprogramming. We show that the blue fluorescence arises from the sequestration of retinyl esters in cytoplasmic lipid bodies. The retinoid-sequestering lipid bodies are specific to human and mouse pluripotent stem cells of the primed or epiblast-like state and absent in naive mouse embryonic stem cells. Retinol, present in widely used stem cell culture media, is sequestered as retinyl ester specifically by primed pluripotent cells and also can induce the formation of these lipid bodies. PMID:25068130

  8. Generation of induced pluripotent stem cells from domestic goats.

    PubMed

    Sandmaier, Shelley E S; Nandal, Anjali; Powell, Anne; Garrett, Wesley; Blomberg, Leann; Donovan, David M; Talbot, Neil; Telugu, Bhanu P

    2015-09-01

    The creation of genetically modified goats provides a powerful approach for improving animal health, enhancing production traits, animal pharming, and for ensuring food safety all of which are high-priority goals for animal agriculture. The availability of goat embryonic stem cells (ESCs) that are characteristically immortal in culture would be of enormous benefit for developing genetically modified animals. As an alternative to long-sought goat ESCs, we generated induced pluripotent stem cells (iPSC) by forced expression of bovine POU5F1, SOX2, MYC, KLF4, LIN-28, and NANOG reprogramming factors in combination with a MIR302/367 cluster, delivered by lentiviral vectors. In order to minimize integrations, the reprogramming factor coding sequences were assembled with porcine teschovirus-1 2A (P2A) self-cleaving peptides that allowed for tri-cistronic expression from each vector. The lentiviral-transduced cells were cultured on irradiated mouse feeder cells in a semi-defined, serum-free medium containing fibroblast growth factor (FGF) and/or leukemia inhibitory factor (LIF). The resulting goat iPSC exhibit cell and colony morphology typical of human and mouse ESCs-that is, well-defined borders, a high nuclear-to-cytoplasmic ratio, a short cell-cycle interval, alkaline phosphatase expression, and the ability to generate teratomas in vivo. Additionally, these goat iPSC demonstrated the ability to differentiate into directed lineages in vitro. These results constitute the first steps in establishing integration and footprint-free iPSC from ruminants. Mol. Reprod. Dev. 82: 709-721, 2015. © 2015 Wiley Periodicals, Inc. PMID:26118622

  9. Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells

    PubMed Central

    Rostovskaya, Maria; Bredenkamp, Nicholas; Smith, Austin

    2015-01-01

    Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or cell replacement therapy. Type I diabetes is considered a major target for stem cell applications due to the shortage of primary human beta cells. Several protocols have been reported for generating pancreatic progenitors by in vitro differentiation of human pluripotent stem cells. Here we first assessed one of these protocols on a panel of pluripotent stem cell lines for capacity to engender glucose sensitive insulin-producing cells after engraftment in immunocompromised mice. We observed variable outcomes with only one cell line showing a low level of glucose response. We, therefore, undertook a systematic comparison of different methods for inducing definitive endoderm and subsequently pancreatic differentiation. Of several protocols tested, we identified a combined approach that robustly generated pancreatic progenitors in vitro from both embryo-derived and induced pluripotent stem cells. These findings suggest that, although there are intrinsic differences in lineage specification propensity between pluripotent stem cell lines, optimal differentiation procedures may consistently direct a substantial fraction of cells into pancreatic specification. PMID:26416676

  10. Prospects and Challenges of Induced Pluripotent Stem Cells in Equine Health.

    PubMed

    Donadeu, F Xavier; Esteves, Cristina L

    2015-01-01

    Pluripotent stem cells (PSCs) hold, through the capacity to differentiate into virtually all body cell types, unprecedented promise for human and animal medicine. PSCs are naturally found in the early embryo, and in rodents and humans they can be robustly harvested and grown in culture in the form of embryonic stem cells (ESCs); however, the availability of ESCs from horses is limited. ES-like cells named induced pluripotent stem cells (iPSCs) can be derived in vitro by transcription factor-mediated reprogramming of adult cells. As such, iPSCs can be generated in a patient-specific manner providing unmatched potential for tissue transplantation and in vitro disease modeling. In humans, clinical trials using iPSC-derived cells are already taking place and the use of in vitro iPSC models has identified novel mechanisms of disease and therapeutic targets. Although to a more limited extent, iPSCs have also been generated from horses, a species in which, after humans, these cells are likely to hold the greatest potential in regenerative medicine. Before a clinical use can be envisioned, however, significant challenges will need to be addressed in relation to the robust derivation, long-term culture, differentiation, and clinical safety of equine iPSCs. Toward this objective, recent studies have reported significant improvement in culture conditions and the successful derivation for the first time of functional cell types from equine iPSCs. Given the wide range of exciting applications they could have, it is hoped future research will make the biomedical promise of iPSCs a reality not only for humans but also horses. PMID:26664986

  11. Survival and differentiation of adenovirus-generated induced pluripotent stem cells transplanted into the rat striatum.

    PubMed

    Fink, Kyle D; Rossignol, Julien; Lu, Ming; Lévêque, Xavier; Hulse, Travis D; Crane, Andrew T; Nerriere-Daguin, Veronique; Wyse, Robert D; Starski, Phillip A; Schloop, Matthew T; Dues, Dylan J; Witte, Steve J; Song, Cheng; Vallier, Ludovic; Nguyen, Tuan H; Naveilhan, Philippe; Anegon, Ignacio; Lescaudron, Laurent; Dunbar, Gary L

    2014-01-01

    Induced pluripotent stem cells (iPSCs) offer certain advantages over embryonic stem cells in cell replacement therapy for a variety of neurological disorders. However, reliable procedures, whereby transplanted iPSCs can survive and differentiate into functional neurons, without forming tumors, have yet to be devised. Currently, retroviral or lentiviral reprogramming methods are often used to reprogram somatic cells. Although the use of these viruses has proven to be effective, formation of tumors often results following in vivo transplantation, possibly due to the integration of the reprogramming genes. The goal of the current study was to develop a new approach, using an adenovirus for reprogramming cells, characterize the iPSCs in vitro, and test their safety, survivability, and ability to differentiate into region-appropriate neurons following transplantation into the rat brain. To this end, iPSCs were derived from bone marrow-derived mesenchymal stem cells and tail-tip fibroblasts using a single cassette lentivirus or a combination of adenoviruses. The reprogramming efficiency and levels of pluripotency were compared using immunocytochemistry, flow cytometry, and real-time polymerase chain reaction. Our data indicate that adenovirus-generated iPSCs from tail-tip fibroblasts are as efficient as the method we used for lentiviral reprogramming. All generated iPSCs were also capable of differentiating into neuronal-like cells in vitro. To test the in vivo survivability and the ability to differentiate into region-specific neurons in the absence of tumor formation, 400,000 of the iPSCs derived from tail-tip fibroblasts that were transfected with the adenovirus pair were transplanted into the striatum of adult, immune-competent rats. We observed that these iPSCs produced region-specific neuronal phenotypes, in the absence of tumor formation, at 90 days posttransplantation. These results suggest that adenovirus-generated iPSCs may provide a safe and viable means for

  12. Bone morphogenetic protein 4 and retinoic acid trigger bovine VASA homolog expression in differentiating bovine induced pluripotent stem cells.

    PubMed

    Malaver-Ortega, Luis F; Sumer, Huseyin; Jain, Kanika; Verma, Paul J

    2016-02-01

    Primordial germ cells (PGCs) are the earliest identifiable and completely committed progenitors of female and male gametes. They are obvious targets for genome editing because they assure the transmission of desirable or introduced traits to future generations. PGCs are established at the earliest stages of embryo development and are difficult to propagate in vitro--two characteristics that pose a problem for their practical application. One alternative method to enrich for PGCs in vitro is to differentiate them from pluripotent stem cells derived from adult tissues. Here, we establish a reporter system for germ cell identification in bovine pluripotent stem cells based on green fluorescent protein expression driven by the minimal essential promoter of the bovine Vasa homolog (BVH) gene, whose regulatory elements were identified by orthologous modelling of regulatory units. We then evaluated the potential of bovine induced pluripotent stem cell (biPSC) lines carrying the reporter construct to differentiate toward the germ cell lineage. Our results showed that biPSCs undergo differentiation as embryoid bodies, and a fraction of the differentiating cells expressed BVH. The rate of differentiation towards BVH-positive cells increased up to tenfold in the presence of bone morphogenetic protein 4 or retinoic acid. Finally, we determined that the expression of key PGC genes, such as BVH or SOX2, can be modified by pre-differentiation cell culture conditions, although this increase is not necessarily mirrored by an increase in the rate of differentiation. PMID:26660942

  13. The promise of induced pluripotent stem cells in research and therapy

    PubMed Central

    Robinton, Daisy A.; Daley, George Q.

    2013-01-01

    The field of stem-cell biology has been catapulted forward by the startling development of reprogramming technology. The ability to restore pluripotency to somatic cells through the ectopic co-expression of reprogramming factors has created powerful new opportunities for modelling human diseases and offers hope for personalized regenerative cell therapies. While the field is racing ahead, some researchers are pausing to evaluate whether induced pluripotent stem cells are indeed the true equivalents of embryonic stem cells and whether subtle differences between these cells might affect their research applications and therapeutic potential. PMID:22258608

  14. Small-Molecule Induction of Canine Embryonic Stem Cells Toward Naïve Pluripotency.

    PubMed

    Tobias, Ian C; Brooks, Courtney R; Teichroeb, Jonathan H; Villagómez, Daniel A; Hess, David A; Séguin, Cheryle A; Betts, Dean H

    2016-08-15

    Naïve and primed pluripotent stem cells (PSCs) reflect discrete pluripotent states that approximate the inner cell mass or the progressively lineage-restricted perigastrulation epiblast, respectively. Cells that occupy primed pluripotency have distinct epigenetic landscapes, transcriptional circuitry, and trophic requirements compared with their naïve counterparts. The existence of multiple pluripotent states has not been explored in dogs, which show promise as outbred biomedical models with more than 300 inherited diseases that also afflict humans. However, our understanding of canine embryogenesis and embryo-derived stem cells is limited. Herein, we converted leukemia inhibitory factor (LIF)-dependent and fibroblast growth factor 2 (FGF2)-dependent canine embryonic stem cells (cESCs) resembling primed PSCs toward a naïve pluripotent state using LIF and inhibitors of glycogen synthase kinase 3β and mitogen-activated protein kinase kinase 1/2 [called 2i and LIF (2iL)]. cESCs propagated in 2iL exhibited significant induction of genes associated with the naïve pluripotent state (eg, REX1, TBX3) and downregulation of primed pluripotency markers (eg, OTX2, FGF5) (P < 0.05). Differential phosphorylation of signal transducer and activator of transcription 3 (STAT3) and cell fate decisions on exposure to bone morphogenetic protein 4 (BMP4) suggested that a novel pluripotent identity has been established with 2iL. Accordingly, cESCs cultured with 2iL formed colonies at a greater efficiency than LIF-FGF2 cESCs following single-cell dissociation. Total genomic DNA methylation and histone H3 lysine 27 trimethylation signals were reduced in 2iL-treated cESCs. Our data suggest that 2iL culture conditions promote the conversion of cESCs toward an epigenetically distinct pluripotent state resembling naïve PSCs. PMID:27392793

  15. Expression of stem cell pluripotency factors during regeneration in the earthworm Eisenia foetida.

    PubMed

    Zheng, Pengfei; Shao, Qiang; Diao, Xiaoping; Li, Zandong; Han, Qian

    2016-01-01

    Stem cell pluripotency factors can induce somatic cells to form induced pluripotent stem cells, which are involved in cell reprogramming and dedifferentiation. The tissue regeneration in the earthworm Eisenia foetida may involve cell dedifferentiation. There is limited information about associations between pluripotency factors and the regeneration. In this report, cDNA sequences of pluripotency factors, oct4, nanog, sox2, c-myc and lin28 genes from the earthworm E. foetida were cloned, and quantitative PCR analysis was performed for their mRNA expressions in the head, clitellum and tail. The maximum up-regulation of oct4, nanog, sox2, c-myc and lin28 occurred at 12h, 4 days, 12h, 2 days, and 24h after amputation for 110, 178, 21, 251 and 325-fold, respectively, in comparison with the controls. The results suggest that the tissues are regenerated via cellular dedifferentiation and reprogramming. PMID:26299657

  16. Inference of Transcriptional Network for Pluripotency in Mouse Embryonic Stem Cells

    NASA Astrophysics Data System (ADS)

    Aburatani, S.

    2015-01-01

    In embryonic stem cells, various transcription factors (TFs) maintain pluripotency. To gain insights into the regulatory system controlling pluripotency, I inferred the regulatory relationships between the TFs expressed in ES cells. In this study, I applied a method based on structural equation modeling (SEM), combined with factor analysis, to 649 expression profiles of 19 TF genes measured in mouse Embryonic Stem Cells (ESCs). The factor analysis identified 19 TF genes that were regulated by several unmeasured factors. Since the known cell reprogramming TF genes (Pou5f1, Sox2 and Nanog) are regulated by different factors, each estimated factor is considered to be an input for signal transduction to control pluripotency in mouse ESCs. In the inferred network model, TF proteins were also arranged as unmeasured factors that control other TFs. The interpretation of the inferred network model revealed the regulatory mechanism for controlling pluripotency in ES cells.

  17. Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated genes.

    PubMed

    Mali, Prashant; Chou, Bin-Kuan; Yen, Jonathan; Ye, Zhaohui; Zou, Jizhong; Dowey, Sarah; Brodsky, Robert A; Ohm, Joyce E; Yu, Wayne; Baylin, Stephen B; Yusa, Kosuke; Bradley, Allan; Meyers, David J; Mukherjee, Chandrani; Cole, Philip A; Cheng, Linzhao

    2010-04-01

    We report here that butyrate, a naturally occurring fatty acid commonly used as a nutritional supplement and differentiation agent, greatly enhances the efficiency of induced pluripotent stem (iPS) cell derivation from human adult or fetal fibroblasts. After transient butyrate treatment, the iPS cell derivation efficiency is enhanced by 15- to 51-fold using either retroviral or piggyBac transposon vectors expressing 4 to 5 reprogramming genes. Butyrate stimulation is more remarkable (>100- to 200-fold) on reprogramming in the absence of either KLF4 or MYC transgene. Butyrate treatment did not negatively affect properties of iPS cell lines established by either 3 or 4 retroviral vectors or a single piggyBac DNA transposon vector. These characterized iPS cell lines, including those derived from an adult patient with sickle cell disease by either the piggyBac or retroviral vectors, show normal karyotypes and pluripotency. To gain insights into the underlying mechanisms of butyrate stimulation, we conducted genome-wide gene expression and promoter DNA methylation microarrays and other epigenetic analyses on established iPS cells and cells from intermediate stages of the reprogramming process. By days 6 to 12 during reprogramming, butyrate treatment enhanced histone H3 acetylation, promoter DNA demethylation, and the expression of endogenous pluripotency-associated genes, including DPPA2, whose overexpression partially substitutes for butyrate stimulation. Thus, butyrate as a cell permeable small molecule provides a simple tool to further investigate molecular mechanisms of cellular reprogramming. Moreover, butyrate stimulation provides an efficient method for reprogramming various human adult somatic cells, including cells from patients that are more refractory to reprogramming. PMID:20201064

  18. A murine-ES like state facilitates transgenesis and homologous recombination in human pluripotent stem cells

    PubMed Central

    Buecker, Christa; Chen, Hsu-Hsin; Polo, Jose; Daheron, Laurence; Bu, Lei; Barakat, Tahsin Stefan; Okwieka, Patricia; Porter, Andrew; Gribnau, Joost; Hochedlinger, Konrad; Geijsen, Niels

    2010-01-01

    Murine embryonic stem cells have been shown to exist in two functionally distinct pluripotent states, embryonic stem cells (ES cell)- and epiblast stem cells (EpiSCs), which are defined by the culture growth factor conditions. Human ES cells appear to exist in an epiblast-like state, which in comparison to their murine counterparts, is relatively difficult to propagate and manipulate. As a result, gene targeting is difficult and to-date only a handful of human knock-in or knock-out cell lines exist. We explored whether an alternative stem cell state exists for human stem cells as well, and demonstrate that manipulation of the growth factor milieu allows the derivation of a novel human stem cell type that displays morphological, molecular and functional properties of murine ES cells and facilitates gene targeting. As such, the murine ES-like state provides a powerful tool for the generation of recombinant human pluripotent stem cell lines. PMID:20569691

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

    PubMed Central

    2014-01-01

    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 the question of

  20. Protocols for Cryopreservation of Intact Hair Follicle That Maintain Pluripotency of Nestin-Expressing Hair-Follicle-Associated Pluripotent (HAP) Stem Cells.

    PubMed

    Kajiura, Satoshi; Mii, Sumiyuki; Aki, Ryoichi; Hamada, Yuko; Arakawa, Nobuko; Kawahara, Katsumasa; Li, Lingna; Katsuoka, Kensei; Hoffman, Robert M; Amoh, Yasuyuki

    2016-01-01

    Hair follicles contain nestin-expressing pluripotent stem cells, the origin of which is above the bulge area, below the sebaceous gland. We have termed these cells hair-follicle-associated pluripotent (HAP) stem cells. Cryopreservation methods of the hair follicle that maintain the pluripotency of HAP stem cells are described in this chapter. Intact hair follicles from green fluorescent protein (GFP) transgenic mice were cryopreserved by slow-rate cooling in TC-Protector medium and storage in liquid nitrogen. After thawing, the upper part of the hair follicle was isolated and cultured in DMEM with fetal bovine serum (FBS). After 4 weeks culture, cells from the upper part of the hair follicles grew out. The growing cells were transferred to DMEM/F12 without FBS. After 1 week culture, the growing cells formed hair spheres, each containing approximately 1 × 10(2) HAP stem cells. The hair spheres contained cells which could differentiate to neurons, glial cells, and other cell types. The formation of hair spheres by the thawed and cultured upper part of the hair follicle produced almost as many pluripotent hair spheres as fresh follicles. The hair spheres derived from cryopreserved hair follicles were as pluripotent as hair spheres from fresh hair follicles. These results suggest that the cryopreservation of the whole hair follicle is an effective way to store HAP stem cells for personalized regenerative medicine, enabling any individual to maintain a bank of pluripotent stem cells for future clinical use. PMID:27431257

  1. Trophoblast lineage cells derived from human induced pluripotent stem cells

    SciTech Connect

    Chen, Ying; Wang, Kai; Chandramouli, Gadisetti V.R.; Knott, Jason G.; Leach, Richard

    2013-07-12

    Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.

  2. Role of bioinspired polymers in determination of pluripotent stem cell fate

    PubMed Central

    Abraham, Sheena; Eroshenko, Nikolai; Rao, Raj R

    2009-01-01

    Human pluripotent stem cells, including embryonic and induced pluripotent stem cells, hold enormous potential for the treatment of many diseases, owing to their ability to generate cell types useful for therapeutic applications. Currently, many stem cell culture propagation and differentiation systems incorporate animal-derived components for promoting self-renewal and differentiation. However, use of these components is labor intensive, carries the risk of xenogeneic contamination and yields compromised experimental results that are difficult to duplicate. From a biomaterials perspective, the generation of an animal- and cell-free biomimetic microenvironment that provides the appropriate physical and chemical cues for stem cell self-renewal or differentiation into specialized cell types would be ideal. This review presents the use of natural and synthetic polymers that support propagation and differentiation of stem cells, in an attempt to obtain a clear understanding of the factors responsible for the determination of stem cell fate. PMID:19580405

  3. Stem-Cell Work Yielding New Approach to Disease: Induced Pluripotent Stem-Cell Research Soars, Spurring Dreams of Clinical Applications.

    PubMed

    Mertz, Leslie

    2016-01-01

    Interest in stem cells escalated in 2006 when scientists figured out how to reprogram some specialized adult cells to assume a stem-cell-like state. Called induced pluripotent stem cells (iPSCs), these cells opened the door to a range of potential applications, including generating cells and tissues to replace those that are faulty or missing in patients with cancer, diabetes, cardiovascular disease, or other maladies (Figure 1). Visions of new treatments and even cures for debilitating and fatal illnesses proliferated, and some of that work is well under way (see "A Wealth of Research"). Now, ten years later, those visions are looking more like real possibilities as research moves from the lab to the clinic and expands toward a greater understanding of the basic science behind stem cells and its applications. PMID:27414628

  4. Traceability in stem cell research: from participant sample to induced pluripotent stem cell and back.

    PubMed

    Morrison, Michael; Moraia, Linda Briceño; Steele, Jane C

    2016-01-01

    This paper describes a traceability system developed for the Stem cells for Biological Assays of Novel drugs and prediCtive toxiCology consortium. The system combines records and labels that to biological material across geographical locations and scientific processes from sample donation to induced pluripotent stem cell line. The labeling system uses a unique identification number to link every aliquot of sample at every stage of the reprogramming pathway back to the original donor. Only staff at the clinical recruitment site can reconnect the unique identification number to the identifying details of a specific donor. This ensures the system meets ethical and legal requirements for protecting privacy while allowing full traceability of biological material. The system can be adapted to other projects and for use with different primary sample types. PMID:26679283

  5. Lineage Conversion of Murine Extraembryonic Trophoblast Stem Cells to Pluripotent Stem Cells▿†

    PubMed Central

    Kuckenberg, Peter; Peitz, Michael; Kubaczka, Caroline; Becker, Astrid; Egert, Angela; Wardelmann, Eva; Zimmer, Andreas; Brüstle, Oliver; Schorle, Hubert

    2011-01-01

    In mammals, the first cell fate decision is initialized by cell polarization at the 8- to 16-cell stage of the preimplantation embryo. At this stage, outside cells adopt a trophectoderm (TE) fate, whereas the inside cell population gives rise to the inner cell mass (ICM). Prior to implantation, transcriptional interaction networks and epigenetic modifications divide the extraembryonic and embryonic fate irrevocably. Here, we report that extraembryonic trophoblast stem cell (TSC) lines are converted to induced pluripotent stem cells (TSC-iPSCs) by overexpressing Oct4, Sox2, Klf4, and cMyc. Methylation studies and gene array analyses indicated that TSC-iPSCs had adopted a pluripotent potential. The rate of conversion was lower than those of somatic reprogramming experiments, probably due to the unique genetic network controlling extraembryonic lineage fixation. Both in vitro and in vivo, TSC-iPSCs differentiated into tissues representing all three embryonic germ layers, indicating that somatic cell fate could be induced. Finally, TSC-iPSCs chimerized the embryo proper and contributed to the germ line of mice, indicating that these cells had acquired full somatic differentiation potential. These results lead to a better understanding of the molecular processes that govern the first lineage decision in mammals. PMID:21300784

  6. Differentiation of Definitive Endoderm from Human Induced Pluripotent Stem Cells on hMSCs Feeder in a Defined Medium

    PubMed Central

    Jaafarpour, Zahra; Soleimani, Masoud; Hosseinkhani, Saman; Karimi, Mohammad Hossein; Yaghmaei, Parichehreh; Mobarra, Naser; Geramizadeh, Bita

    2016-01-01

    Background: The Definitive Endoderm (DE) differentiation using the undefined media and non-human feeders can cause contaminations in the generated cells for therapeutic applications. Therefore, generating safer and more appropriate DE cells is needed. This study compared five different methods to establish an appropriate method for inducing an efficient DE differentiation from Human Induced Pluripotent Stem Cells (hiPSCs) on an appropriate feeder in a more defined medium. Methods: Human Induced Pluripotent Stem Cells (hiPSCs) were cultured on inactivated feeders. Passaged hiPSCs, without feeder, were incubated for three days with Activin-A and different endodermal differentiation media including 1-FBS, 2-B27, 3-ITS and albumin fraction-V, 4-B27 and ITS and 5-like the third medium. The feeder cells in the first four methods were Mouse Embryonic Fibroblasts (MEFs) and in the fifth method were human adult bone marrow Mesenchymal Stem Cells (hMSCs). DE markers FOXA2, SOX17 and CXCR4 and also pluripotency marker OCT4 were evaluated using qRT-PCR, as well as FOXA2 by the immunocytochemistry. Results: QRT-PCR analysis showed that after three days, the expression levels of DE and pluripotency markers in the differentiated hiPSCs among all five groups did not have any significant differences. Similarly, the immunocytochemistry analysis demonstrated that the differentiated hiPSCs expressed FOXA2, with no significant differences. Conclusion: Despite this similarity in the results, the third differentiation medium has more defined and cost effective components. Furthermore, hMSC, a human feeder, is safer than MEF. Therefore, the fifth method is preferable among other DE differentiation methods and can serve as a fundamental method helping the development of regenerative medicine. PMID:26855729

  7. Standardized Generation and Differentiation of Neural Precursor Cells from Human Pluripotent Stem Cells

    PubMed Central

    Kozhich, O; Hamilton, RS; Mallon, BS

    2012-01-01

    Precise, robust and scalable directed differentiation of pluripotent stem cells is an important goal with respect to disease modeling or future therapies. Using the AggreWell™400 system we have standardized the differentiation of human embryonic and induced pluripotent stem cells to a neuronal fate using defined conditions. This allows reproducibility in replicate experiments and facilitates the direct comparison of cell lines. Since the starting point for EB formation is a single cell suspension, this protocol is suitable for standard and novel methods of pluripotent stem cell culture. Moreover, an intermediate population of neural precursor cells, which are routinely >95% NCAMpos and Tra-1–60neg by FACS analysis, may be expanded and frozen prior to differentiation allowing a convenient starting point for downstream experiments. PMID:22388559

  8. An ex vivo gene therapy approach to treat muscular dystrophy using inducible pluripotent stem cells.

    PubMed

    Filareto, Antonio; Parker, Sarah; Darabi, Radbod; Borges, Luciene; Iacovino, Michelina; Schaaf, Tory; Mayerhofer, Timothy; Chamberlain, Jeffrey S; Ervasti, James M; McIvor, R Scott; Kyba, Michael; Perlingeiro, Rita C R

    2013-01-01

    Duchenne muscular dystrophy is a progressive and incurable neuromuscular disease caused by genetic and biochemical defects of the dystrophin-glycoprotein complex. Here we show the regenerative potential of myogenic progenitors derived from corrected dystrophic induced pluripotent stem cells generated from fibroblasts of mice lacking both dystrophin and utrophin. We correct the phenotype of dystrophic induced pluripotent stem cells using a Sleeping Beauty transposon system carrying the micro-utrophin gene, differentiate these cells into skeletal muscle progenitors and transplant them back into dystrophic mice. Engrafted muscles displayed large numbers of micro-utrophin-positive myofibers, with biochemically restored dystrophin-glycoprotein complex and improved contractile strength. The transplanted cells seed the satellite cell compartment, responded properly to injury and exhibit neuromuscular synapses. We also detect muscle engraftment after systemic delivery of these corrected progenitors. These results represent an important advance towards the future treatment of muscular dystrophies using genetically corrected autologous induced pluripotent stem cells. PMID:23462992

  9. Protein post-translational modifications and regulation of pluripotency in human stem cells

    PubMed Central

    Wang, Yu-Chieh; Peterson, Suzanne E; Loring, Jeanne F

    2014-01-01

    Post-translational modifications (PTMs) are known to be essential mechanisms used by eukaryotic cells to diversify their protein functions and dynamically coordinate their signaling networks. Defects in PTMs have been linked to numerous developmental disorders and human diseases, highlighting the importance of PTMs in maintaining normal cellular states. Human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), are capable of self-renewal and differentiation into a variety of functional somatic cells; these cells hold a great promise for the advancement of biomedical research and clinical therapy. The mechanisms underlying cellular pluripotency in human cells have been extensively explored in the past decade. In addition to the vast amount of knowledge obtained from the genetic and transcriptional research in hPSCs, there is a rapidly growing interest in the stem cell biology field to examine pluripotency at the protein and PTM level. This review addresses recent progress toward understanding the role of PTMs (glycosylation, phosphorylation, acetylation and methylation) in the regulation of cellular pluripotency. PMID:24217768

  10. Induced pluripotent stem cell-derived mesenchymal stem cells: A leap toward personalized therapies.

    PubMed

    Whitt, Jason; Vallabhaneni, Krishna C; Penfornis, Patrice; Pochampally, Radhika

    2016-01-01

    Mesenchymal Stem/stromal cell (MSCs) transplantation procedures have been used since the 1960's to treat leukemia and other diseases, but due to the risks involved only patients with life threatening illnesses were typically subjected to the transplantation procedure until the last decade. Recent advancements in transplantation techniques have made it more feasible to use it for non-life-threatening diseases. However, the potential uses for stem cells are still limited by their rarity, and, in the case of allogeneic transplants, graft-vs.-host complications. An evolving alternative to conventional stem cell therapies is induced pluripotent stem-cell derived mesenchymal stem/stromal cells (iPSC- MSCs), which have a multi-lineage potential comparable to conventionally acquired MSCs with the added benefit of being less immunoreactive. However there are still many hurdles left to be overcome before they can be used regularly for personalized therapies. This review will focus on recent advancements that have been made regarding the role MSCs play in tumor development and the potential uses iPSC-MSCs may have in future cancer treatment. PMID:26423301

  11. Human-induced pluripotent stem cells from blood cells of healthy donors and patients with acquired blood disorders

    PubMed Central

    Ye, Zhaohui; Zhan, Huichun; Mali, Prashant; Dowey, Sarah; Williams, Donna M.; Jang, Yoon-Young; Dang, Chi V.; Spivak, Jerry L.; Moliterno, Alison R.

    2009-01-01

    Human induced pluripotent stem (iPS) cells derived from somatic cells hold promise to develop novel patient-specific cell therapies and research models for inherited and acquired diseases. We and others previously reprogrammed human adherent cells, such as postnatal fibroblasts to iPS cells, which resemble adherent embryonic stem cells. Here we report derivation of iPS cells from postnatal human blood cells and the potential of these pluripotent cells for disease modeling. Multiple human iPS cell lines were generated from previously frozen cord blood or adult CD34+ cells of healthy donors, and could be redirected to hematopoietic differentiation. Multiple iPS cell lines were also generated from peripheral blood CD34+ cells of 2 patients with myeloproliferative disorders (MPDs) who acquired the JAK2-V617F somatic mutation in their blood cells. The MPD-derived iPS cells containing the mutation appeared normal in phenotypes, karyotype, and pluripotency. After directed hematopoietic differentiation, the MPD-iPS cell-derived hematopoietic progenitor (CD34+CD45+) cells showed the increased erythropoiesis and gene expression of specific genes, recapitulating features of the primary CD34+ cells of the corresponding patient from whom the iPS cells were derived. These iPS cells provide a renewable cell source and a prospective hematopoiesis model for investigating MPD pathogenesis. PMID:19797525

  12. Reprogramming to a pluripotent state modifies mesenchymal stem cell resistance to oxidative stress

    PubMed Central

    Asensi, Karina D; Fortunato, Rodrigo S; dos Santos, Danúbia S; Pacheco, Thaísa S; de Rezende, Danielle F; Rodrigues, Deivid C; Mesquita, Fernanda C P; Kasai-Brunswick, Tais H; de Carvalho, Antonio C Campos; Carvalho, Denise P; Carvalho, Adriana B; Goldenberg, Regina C dos S

    2014-01-01

    Properties of induced pluripotent stem cells (iPSC) have been extensively studied since their first derivation in 2006. However, the modification in reactive oxygen species (ROS) production and detoxification caused by reprogramming still needs to be further elucidated. The objective of this study was to compare the response of iPSC generated from menstrual blood–derived mesenchymal stem cells (mb-iPSC), embryonic stem cells (H9) and adult menstrual blood–derived mesenchymal stem cells (mbMSC) to ROS exposure and investigate the effects of reprogramming on cellular oxidative stress (OS). mbMSC were extremely resistant to ROS exposure, however, mb-iPSC were 10-fold less resistant to H2O2, which was very similar to embryonic stem cell sensitivity. Extracellular production of ROS was also similar in mb-iPSC and H9 and almost threefold lower than in mbMSC. Furthermore, intracellular amounts of ROS were higher in mb-iPSC and H9 when compared with mbMSC. As the ability to metabolize ROS is related to antioxidant enzymes, we analysed enzyme activities in these cell types. Catalase and superoxide dismutase activities were reduced in mb-iPSC and H9 when compared with mbMSC. Finally, cell adhesion under OS conditions was impaired in mb-iPSC when compared with mbMSC, albeit similar to H9. Thus, reprogramming leads to profound modifications in extracellular ROS production accompanied by loss of the ability to handle OS. PMID:24528612

  13. Elimination of Tumorigenic Human Pluripotent Stem Cells by a Recombinant Lectin-Toxin Fusion Protein

    PubMed Central

    Tateno, Hiroaki; Onuma, Yasuko; Ito, Yuzuru; Minoshima, Fumi; Saito, Sayoko; Shimizu, Madoka; Aiki, Yasuhiko; Asashima, Makoto; Hirabayashi, Jun

    2015-01-01

    Summary The application of stem-cell-based therapies in regenerative medicine is hindered by the tumorigenic potential of residual human pluripotent stem cells. Previously, we identified a human pluripotent stem-cell-specific lectin probe, called rBC2LCN, by comprehensive glycome analysis using high-density lectin microarrays. Here we developed a recombinant lectin-toxin fusion protein of rBC2LCN with a catalytic domain of Pseudomonas aeruginosa exotoxin A, termed rBC2LCN-PE23, which could be expressed as a soluble form from the cytoplasm of Escherichia coli and purified to homogeneity by one-step affinity chromatography. rBC2LCN-PE23 bound to human pluripotent stem cells, followed by its internalization, allowing intracellular delivery of a cargo of cytotoxic protein. The addition of rBC2LCN-PE23 to the culture medium was sufficient to completely eliminate human pluripotent stem cells. Thus, rBC2LCN-PE23 has the potential to contribute to the safety of stem-cell-based therapies. PMID:25866158

  14. Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines.

    PubMed

    Bignone, Paola A; Krupa, Rachel A; West, Michael D; Larocca, David

    2016-01-01

    The ability of human pluripotent stem cells (hPS) to both self-renew and differentiate into virtually any cell type makes them a promising source of cells for cell-based regenerative therapies. However, stem cell identity, purity, and scalability remain formidable challenges that need to be overcome for translation of pluripotent stem cell research into clinical applications. Directed differentiation from hPS cells is inefficient and residual contamination with pluripotent cells that have the potential to form tumors remains problematic. The derivation of scalable (self-renewing) embryonic progenitor stem cell lines offers a solution because they are well defined and clonally pure. Clonally pure progenitor stem cell lines also provide a means for identifying cell surface targeting reagents that are useful for identification, tracking, and repeated derivation of the corresponding progenitor stem cell types from additional hPS cell sources. Such stem cell targeting reagents can then be applied to the manufacture of genetically diverse banks of human embryonic progenitor cell lines for drug screening, disease modeling, and cell therapy. Here we present methods to identify human embryonic progenitor stem cell targeting peptides by selection of phage display libraries on clonal embryonic progenitor cell lines and demonstrate their use for targeting quantum dots (Qdots) for stem cell labeling. PMID:25410289

  15. Polymeric nanofibrous substrates stimulate pluripotent stem cells to form three-dimensional multilayered patty-like spheroids in feeder-free culture and maintain their pluripotency.

    PubMed

    Alamein, Mohammad A; Wolvetang, Ernst J; Ovchinnikov, Dmitry A; Stephens, Sebastien; Sanders, Katherine; Warnke, Patrick H

    2015-09-01

    Expansion of pluripotent stem cells in defined media devoid of animal-derived feeder cells to generate multilayered three-dimensional (3D) bulk preparations or spheroids, rather than two-dimensional (2D) monolayers, is advantageous for many regenerative, biological or disease-modelling studies. Here we show that electrospun polymer matrices comprised of nanofibres that mimic the architecture of the natural fibrous extracellular matrix allow for feeder-free expansion of pluripotent human induced pluripotent stem cells (IPSCs) and human embryonic stem cells (HESCs) into multilayered 3D 'patty-like' spheroid structures in defined xeno-free culture medium. The observation that IPSCs and HESCs readily revert to 2D growth in the absence of the synthetic nanofibre membranes suggests that this 3D expansion behaviour is mediated by the physical microenvironment and artificial niche provided by the nanofibres only. Importantly, we could show that such 3D growth as patties maintained the pluripotency of cells as long as they were kept on nanofibres. The generation of complex multilayered 3D structures consisting of only pluripotent cells on biodegradable nanofibre matrices of the desired shape and size will enable both industrial-scale expansion and intricate organ-tissue engineering applications with human pluripotent stem cells, where simultaneous coupling of differentiation pathways of all germ layers from one stem cell source may be required for organ formation. PMID:25423911

  16. Towards an Optimized Culture Medium for the Generation of Mouse Induced Pluripotent Stem Cells*

    PubMed Central

    Chen, Jiekai; Liu, Jing; Han, Qingkai; Qin, Dajiang; Xu, Jianyong; Chen, You; Yang, Jiaqi; Song, Hong; Yang, Dongshan; Peng, Meixiu; He, Wenzhi; Li, Ronghui; Wang, Hao; Gan, Yi; Ding, Ke; Zeng, Lingwen; Lai, Liangxue; Esteban, Miguel A.; Pei, Duanqing

    2010-01-01

    Generation of induced pluripotent stem cells from somatic cells using defined factors has potential relevant applications in regenerative medicine and biology. However, this promising technology remains inefficient and time consuming. We have devised a serum free culture medium termed iSF1 that facilitates the generation of mouse induced pluripotent stem cells. This optimization of the culture medium is sensitive to the presence of Myc in the reprogramming factors. Moreover, we could reprogram meningeal cells using only two factors Oct4/Klf4. Therefore, iSF1 represents a basal medium that may be used for mechanistic studies and testing new reprogramming approaches. PMID:20595395

  17. Unveiling the critical role of REX1 in the regulation of human stem cell pluripotency.

    PubMed

    Son, Mi-Young; Choi, Hoonsung; Han, Yong-Mahn; Cho, Yee Sook

    2013-11-01

    Reduced expression 1 (REX1) is a widely used pluripotency marker, but little is known about its roles in pluripotency. Here, we show that REX1 is functionally important in the reacquisition and maintenance of pluripotency. REX1-depleted human pluripotent stem cells (hPSCs) lose their self-renewal capacity and full differentiation potential, especially their mesoderm lineage potential. Cyclin B1/B2 expression was found to parallel that of REX1. REX1 positively regulates the transcriptional activity of cyclin B1/B2 through binding to their promoters. REX1 induces the phosphorylation of DRP1 at Ser616 by cyclin B/CDK1, which leads to mitochondrial fission and appears to be important for meeting the high-energy demands of highly glycolytic hPSCs. During reprogramming to pluripotency by defined factors (OCT4, SOX2, KLF4, and c-MYC), the reprogramming kinetics and efficiency are markedly improved by adding REX1 or replacing KLF4 with REX1. These improvements are achieved by lowering reprogramming barriers (growth arrest and apoptosis), by enhancing mitochondrial fission, and by conversion to glycolytic metabolism, dependent on the cyclin B1/B2-DRP1 pathway. Our results show that a novel pluripotency regulator, REX1, is essential for pluripotency and reprogramming. PMID:23939908

  18. LIN28 Regulates Stem Cell Metabolism and Conversion to Primed Pluripotency.

    PubMed

    Zhang, Jin; Ratanasirintrawoot, Sutheera; Chandrasekaran, Sriram; Wu, Zhaoting; Ficarro, Scott B; Yu, Chunxiao; Ross, Christian A; Cacchiarelli, Davide; Xia, Qing; Seligson, Marc; Shinoda, Gen; Xie, Wen; Cahan, Patrick; Wang, Longfei; Ng, Shyh-Chang; Tintara, Supisara; Trapnell, Cole; Onder, Tamer; Loh, Yuin-Han; Mikkelsen, Tarjei; Sliz, Piotr; Teitell, Michael A; Asara, John M; Marto, Jarrod A; Li, Hu; Collins, James J; Daley, George Q

    2016-07-01

    The RNA-binding proteins LIN28A and LIN28B play critical roles in embryonic development, tumorigenesis, and pluripotency, but their exact functions are poorly understood. Here, we show that, like LIN28A, LIN28B can function effectively with NANOG, OCT4, and SOX2 in reprogramming to pluripotency and that reactivation of both endogenous LIN28A and LIN28B loci are required for maximal reprogramming efficiency. In human fibroblasts, LIN28B is activated early during reprogramming, while LIN28A is activated later during the transition to bona fide induced pluripotent stem cells (iPSCs). In murine cells, LIN28A and LIN28B facilitate conversion from naive to primed pluripotency. Proteomic and metabolomic analysis highlighted roles for LIN28 in maintaining the low mitochondrial function associated with primed pluripotency and in regulating one-carbon metabolism, nucleotide metabolism, and histone methylation. LIN28 binds to mRNAs of proteins important for oxidative phosphorylation and modulates protein abundance. Thus, LIN28A and LIN28B play cooperative roles in regulating reprogramming, naive/primed pluripotency, and stem cell metabolism. PMID:27320042

  19. Mucin-Inspired Thermoresponsive Synthetic Hydrogels Induce Stasis in Human Pluripotent Stem Cells and Human Embryos

    PubMed Central

    2016-01-01

    Human pluripotent stem cells (hPSCs; both embryonic and induced pluripotent) rapidly proliferate in adherent culture to maintain their undifferentiated state. However, for mammals exhibiting delayed gestation (diapause), mucin-coated embryos can remain dormant for days or months in utero, with their constituent PSCs remaining pluripotent under these conditions. Here we report cellular stasis for both hPSC colonies and preimplantation embryos immersed in a wholly synthetic thermoresponsive gel comprising poly(glycerol monomethacrylate)-poly(2-hydroxypropyl methacrylate) [PGMA55-PHPMA135] diblock copolymer worms. This hydroxyl-rich mucin-mimicking nonadherent 3D gel maintained PSC viability and pluripotency in the quiescent G0 state without passaging for at least 14 days. Similarly, gel-coated human embryos remain in a state of suspended animation (diapause) for up to 8 days. The discovery of a cryptic cell arrest mechanism for both hPSCs and embryos suggests an important connection between the cellular mechanisms that evoke embryonic diapause and pluripotency. Moreover, such synthetic worm gels offer considerable utility for the short-term (weeks) storage of either pluripotent stem cells or human embryos without cryopreservation. PMID:27163030

  20. Mucin-Inspired Thermoresponsive Synthetic Hydrogels Induce Stasis in Human Pluripotent Stem Cells and Human Embryos.

    PubMed

    Canton, Irene; Warren, Nicholas J; Chahal, Aman; Amps, Katherine; Wood, Andrew; Weightman, Richard; Wang, Eugenia; Moore, Harry; Armes, Steven P

    2016-02-24

    Human pluripotent stem cells (hPSCs; both embryonic and induced pluripotent) rapidly proliferate in adherent culture to maintain their undifferentiated state. However, for mammals exhibiting delayed gestation (diapause), mucin-coated embryos can remain dormant for days or months in utero, with their constituent PSCs remaining pluripotent under these conditions. Here we report cellular stasis for both hPSC colonies and preimplantation embryos immersed in a wholly synthetic thermoresponsive gel comprising poly(glycerol monomethacrylate)-poly(2-hydroxypropyl methacrylate) [PGMA55-PHPMA135] diblock copolymer worms. This hydroxyl-rich mucin-mimicking nonadherent 3D gel maintained PSC viability and pluripotency in the quiescent G0 state without passaging for at least 14 days. Similarly, gel-coated human embryos remain in a state of suspended animation (diapause) for up to 8 days. The discovery of a cryptic cell arrest mechanism for both hPSCs and embryos suggests an important connection between the cellular mechanisms that evoke embryonic diapause and pluripotency. Moreover, such synthetic worm gels offer considerable utility for the short-term (weeks) storage of either pluripotent stem cells or human embryos without cryopreservation. PMID:27163030

  1. Intricacies of Pluripotency

    PubMed Central

    Bhartiya, Deepa

    2015-01-01

    Pluripotent stem cells have the potential to differentiate into 200 odd cell types present in adult body. Pluripotent stem cells available for regenerative medicine include embryonic stem (ES) cells, induced pluripotent stem (iPS) cells and very small ES-like stem (VSELs) cells. Nuclear OCT-4 is one of the crucial factors that dictate pluripotent state. Compared to ES/iPS cells grown in Petri dish, VSELs exist in adult body organs and results are emerging to suggest that they may have better potential to regenerate adult organs. This is because of their distinct epigenetic status as they are closer to the primordial germ cells from the epiblast-stage embryo compared to inner cell mass from which ES cells are obtained in vitro. We need to make special efforts to study them as they are very small in size and tend to get lost during processing. VSELs exist in adult organs, get mobilized in response to stress, undergo asymmetric cell divisions to give rise to tissue specific progenitors which further differentiate into various cell types and are possibly better candidates for regenerative medicine because they have no associated risk of tumor formation or immunological rejection. They are possibly also the ‘embryonic remnants’ in adult organs responsible for initiating cancer. Thus, rather than not accepting VSELs because they neither form teratoma nor divide in vitro like ES cells, it is time that scientific community should think of revising the definition of the term ‘pluripotency’. PMID:26195889

  2. Modulating the biochemical and biophysical culture environment to enhance osteogenic differentiation and maturation of human pluripotent stem cell-derived mesenchymal progenitors

    PubMed Central

    2013-01-01

    Advances in the fields of stem cell biology, biomaterials, and tissue engineering over the last decades have brought the possibility of constructing tissue substitutes with a broad range of applications in regenerative medicine, disease modeling, and drug discovery. Different types of human stem cells have been used, each presenting a unique set of advantages and limitations with regard to the desired research goals. Whereas adult stem cells are at the frontier of research for tissue and organ regeneration, pluripotent stem cells represent a more challenging cell source for clinical translation. However, with their unlimited growth and wide differentiation potential, pluripotent stem cells represent an unprecedented resource for the construction of advanced human tissue models for biological studies and drug discovery. At the heart of these applications lies the challenge to reproducibly expand, differentiate, and organize stem cells into mature, stable tissue structures. In this review, we focus on the derivation of mesenchymal tissue progenitors from human pluripotent stem cells and the control of their osteogenic differentiation and maturation by modulation of the biophysical culture environment. Similarly to enhancing bone development, the described principles can be applied to the construction of other mesenchymal tissues for basic and applicative studies. PMID:24004835

  3. Pluripotent stem cells derived from mouse primordial germ cells by small molecule compounds.

    PubMed

    Kimura, Tohru; Kaga, Yoshiaki; Sekita, Yoichi; Fujikawa, Keita; Nakatani, Tsunetoshi; Odamoto, Mika; Funaki, Soichiro; Ikawa, Masahito; Abe, Kuniya; Nakano, Toru

    2015-01-01

    Primordial germ cells (PGCs) can give rise to pluripotent stem cells known as embryonic germ cells (EGCs) when cultured with basic fibroblast growth factor (bFGF), stem cell factor (SCF), and leukemia inhibitory factor. Somatic cells can give rise to induced pluripotent stem cells (iPSCs) by introduction of the reprogramming transcription factors Oct4, Sox2, and Klf4. The effects of Sox2 and Klf4 on somatic cell reprogramming can be reproduced using the small molecule compounds, transforming growth factor-β receptor (TGFβR) inhibitor and Kempaullone, respectively. Here we examined the effects of TGFβR inhibitor and Kempaullone on EGC derivation from PGCs. Treatment of PGCs with TGFβR inhibitor and/or Kempaullone generated pluripotent stem cells under standard embryonic stem cell (ESC) culture conditions without bFGF and SCF, which we termed induced EGCs (iEGCs). The derivation efficiency of iEGCs was dependent on the differentiation stage and sex. DNA methylation levels of imprinted genes in iEGCs were reduced, with the exception of the H19 gene. The promoters of genes involved in germline development were generally hypomethylated in PGCs, but three germline genes showed comparable DNA methylation levels among iEGs, ESCs, and iPSCs. These results show that PGCs can be reprogrammed into pluripotent state using small molecule compounds, and that DNA methylation of these germline genes is not maintained in iEGCs. PMID:25186651

  4. Suspension Culture of Human Pluripotent Stem Cells in Controlled, Stirred Bioreactors

    PubMed Central

    Olmer, Ruth; Lange, Andreas; Selzer, Sebastian; Kasper, Cornelia; Haverich, Axel

    2012-01-01

    Therapeutic and industrial applications of pluripotent stem cells and their derivatives require large cell quantities generated in defined conditions. To this end, we have translated single cell-inoculated suspension cultures of human pluripotent stem cells (hPSCs; including human induced pluripotent stem cells [hiPS] and human embryonic stem cells [hESC]) to stirred tank bioreactors. These systems that are widely used in biopharmaceutical industry allow straightforward scale up and detailed online monitoring of key process parameters. To ensure minimum medium consumption, but in parallel functional integration of all probes mandatory for process monitoring, that is, for pO2 and pH, experiments were performed in 100 mL culture volume in a “mini reactor platform” consisting of four independently controlled vessels. By establishing defined parameters for tightly controlled cell inoculation and aggregate formation up to 2×108 hiPSCs/100 mL were generated in a single process run in 7 days. Expression of pluripotency markers and ability of cells to differentiate into derivates of all three germ layers in vitro was maintained, underlining practical utility of this new process. The presented data provide key steps toward scalable mass expansion of human iPS and ES cells thereby enabling translation of stem cell research to (pre)clinical application in relevant large animal models and valuable in vitro assays for drug development and validation as well. PMID:22519745

  5. Retinoic Acid-Treated Pluripotent Stem Cells Undergoing Neurogenesis Present Increased Aneuploidy and Micronuclei Formation

    PubMed Central

    Sartore, Rafaela C.; Campos, Priscila B.; Trujillo, Cleber A.; Ramalho, Bia L.; Negraes, Priscilla D.; Paulsen, Bruna S.; Meletti, Tamara; Costa, Elaine S.; Chicaybam, Leonardo; Bonamino, Martin H.; Ulrich, Henning; Rehen, Stevens K.

    2011-01-01

    The existence of loss and gain of chromosomes, known as aneuploidy, has been previously described within the central nervous system. During development, at least one-third of neural progenitor cells (NPCs) are aneuploid. Notably, aneuploid NPCs may survive and functionally integrate into the mature neural circuitry. Given the unanswered significance of this phenomenon, we tested the hypothesis that neural differentiation induced by all-trans retinoic acid (RA) in pluripotent stem cells is accompanied by increased levels of aneuploidy, as previously described for cortical NPCs in vivo. In this work we used embryonal carcinoma (EC) cells, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells undergoing differentiation into NPCs. Ploidy analysis revealed a 2-fold increase in the rate of aneuploidy, with the prevalence of chromosome loss in RA primed stem cells when compared to naïve cells. In an attempt to understand the basis of neurogenic aneuploidy, micronuclei formation and survivin expression was assessed in pluripotent stem cells exposed to RA. RA increased micronuclei occurrence by almost 2-fold while decreased survivin expression by 50%, indicating possible mechanisms by which stem cells lose their chromosomes during neural differentiation. DNA fragmentation analysis demonstrated no increase in apoptosis on embryoid bodies treated with RA, indicating that cell death is not the mandatory fate of aneuploid NPCs derived from pluripotent cells. In order to exclude that the increase in aneuploidy was a spurious consequence of RA treatment, not related to neurogenesis, mouse embryonic fibroblasts were treated with RA under the same conditions and no alterations in chromosome gain or loss were observed. These findings indicate a correlation amongst neural differentiation, aneuploidy, micronuclei formation and survivin downregulation in pluripotent stem cells exposed to RA, providing evidence that somatically generated chromosomal variation accompanies

  6. Induced pluripotent stem cells: Mechanisms, achievements and perspectives in farm animals.

    PubMed

    Kumar, Dharmendra; Talluri, Thirumala R; Anand, Taruna; Kues, Wilfried A

    2015-03-26

    Pluripotent stem cells are unspecialized cells with unlimited self-renewal, and they can be triggered to differentiate into desired specialized cell types. These features provide the basis for an unlimited cell source for innovative cell therapies. Pluripotent cells also allow to study developmental pathways, and to employ them or their differentiated cell derivatives in pharmaceutical testing and biotechnological applications. Via blastocyst complementation, pluripotent cells are a favoured tool for the generation of genetically modified mice. The recently established technology to generate an induced pluripotency status by ectopic co-expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc allows to extending these applications to farm animal species, for which the derivation of genuine embryonic stem cells was not successful so far. Most induced pluripotent stem (iPS) cells are generated by retroviral or lentiviral transduction of reprogramming factors. Multiple viral integrations into the genome may cause insertional mutagenesis and may increase the risk of tumour formation. Non-integration methods have been reported to overcome the safety concerns associated with retro and lentiviral-derived iPS cells, such as transient expression of the reprogramming factors using episomal plasmids, and direct delivery of reprogramming mRNAs or proteins. In this review, we focus on the mechanisms of cellular reprogramming and current methods used to induce pluripotency. We also highlight problems associated with the generation of iPS cells. An increased understanding of the fundamental mechanisms underlying pluripotency and refining the methodology of iPS cell generation will have a profound impact on future development and application in regenerative medicine and reproductive biotechnology of farm animals. PMID:25815117

  7. Induced pluripotent stem cells: Mechanisms, achievements and perspectives in farm animals

    PubMed Central

    Kumar, Dharmendra; Talluri, Thirumala R; Anand, Taruna; Kues, Wilfried A

    2015-01-01

    Pluripotent stem cells are unspecialized cells with unlimited self-renewal, and they can be triggered to differentiate into desired specialized cell types. These features provide the basis for an unlimited cell source for innovative cell therapies. Pluripotent cells also allow to study developmental pathways, and to employ them or their differentiated cell derivatives in pharmaceutical testing and biotechnological applications. Via blastocyst complementation, pluripotent cells are a favoured tool for the generation of genetically modified mice. The recently established technology to generate an induced pluripotency status by ectopic co-expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc allows to extending these applications to farm animal species, for which the derivation of genuine embryonic stem cells was not successful so far. Most induced pluripotent stem (iPS) cells are generated by retroviral or lentiviral transduction of reprogramming factors. Multiple viral integrations into the genome may cause insertional mutagenesis and may increase the risk of tumour formation. Non-integration methods have been reported to overcome the safety concerns associated with retro and lentiviral-derived iPS cells, such as transient expression of the reprogramming factors using episomal plasmids, and direct delivery of reprogramming mRNAs or proteins. In this review, we focus on the mechanisms of cellular reprogramming and current methods used to induce pluripotency. We also highlight problems associated with the generation of iPS cells. An increased understanding of the fundamental mechanisms underlying pluripotency and refining the methodology of iPS cell generation will have a profound impact on future development and application in regenerative medicine and reproductive biotechnology of farm animals. PMID:25815117

  8. Human developmental chondrogenesis as a basis for engineering chondrocytes from pluripotent stem cells.

    PubMed

    Wu, Ling; Bluguermann, Carolina; Kyupelyan, Levon; Latour, Brooke; Gonzalez, Stephanie; Shah, Saumya; Galic, Zoran; Ge, Sundi; Zhu, Yuhua; Petrigliano, Frank A; Nsair, Ali; Miriuka, Santiago G; Li, Xinmin; Lyons, Karen M; Crooks, Gay M; McAllister, David R; Van Handel, Ben; Adams, John S; Evseenko, Denis

    2013-01-01

    Joint injury and osteoarthritis affect millions of people worldwide, but attempts to generate articular cartilage using adult stem/progenitor cells have been unsuccessful. We hypothesized that recapitulation of the human developmental chondrogenic program using pluripotent stem cells (PSCs) may represent a superior approach for cartilage restoration. Using laser-capture microdissection followed by microarray analysis, we first defined a surface phenotype (CD166(low/neg)CD146(low/neg)CD73(+)CD44(low)BMPR1B(+)) distinguishing the earliest cartilage committed cells (prechondrocytes) at 5-6 weeks of development. Functional studies confirmed these cells are chondrocyte progenitors. From 12 weeks, only the superficial layers of articular cartilage were enriched in cells with this progenitor phenotype. Isolation of cells with a similar immunophenotype from differentiating human PSCs revealed a population of CD166(low/neg)BMPR1B(+) putative cartilage-committed progenitors. Taken as a whole, these data define a developmental approach for the generation of highly purified functional human chondrocytes from PSCs that could enable substantial progress in cartilage tissue engineering. PMID:24371811

  9. Human Developmental Chondrogenesis as a Basis for Engineering Chondrocytes from Pluripotent Stem Cells

    PubMed Central

    Wu, Ling; Bluguermann, Carolina; Kyupelyan, Levon; Latour, Brooke; Gonzalez, Stephanie; Shah, Saumya; Galic, Zoran; Ge, Sundi; Zhu, Yuhua; Petrigliano, Frank A.; Nsair, Ali; Miriuka, Santiago G.; Li, Xinmin; Lyons, Karen M.; Crooks, Gay M.; McAllister, David R.; Van Handel, Ben; Adams, John S.; Evseenko, Denis

    2013-01-01

    Summary Joint injury and osteoarthritis affect millions of people worldwide, but attempts to generate articular cartilage using adult stem/progenitor cells have been unsuccessful. We hypothesized that recapitulation of the human developmental chondrogenic program using pluripotent stem cells (PSCs) may represent a superior approach for cartilage restoration. Using laser-capture microdissection followed by microarray analysis, we first defined a surface phenotype (CD166low/negCD146low/negCD73+CD44lowBMPR1B+) distinguishing the earliest cartilage committed cells (prechondrocytes) at 5–6 weeks of development. Functional studies confirmed these cells are chondrocyte progenitors. From 12 weeks, only the superficial layers of articular cartilage were enriched in cells with this progenitor phenotype. Isolation of cells with a similar immunophenotype from differentiating human PSCs revealed a population of CD166low/negBMPR1B+ putative cartilage-committed progenitors. Taken as a whole, these data define a developmental approach for the generation of highly purified functional human chondrocytes from PSCs that could enable substantial progress in cartilage tissue engineering. PMID:24371811

  10. Generation of Human Induced Pluripotent Stem Cells from Umbilical Cord Matrix and Amniotic Membrane Mesenchymal Cells*

    PubMed Central

    Cai, Jinglei; Li, Wen; Su, Huanxing; Qin, Dajiang; Yang, Jiayin; Zhu, Fan; Xu, Jianyong; He, Wenzhi; Guo, Xiangpeng; Labuda, Krystyna; Peterbauer, Anja; Wolbank, Susanne; Zhong, Mei; Li, Zhiyuan; Wu, Wutian; So, Kwok-Fai; Redl, Heinz; Zeng, Lingwen; Esteban, Miguel Angel; Pei, Duanqing

    2010-01-01

    The umbilical cord and placenta are extra-embryonic tissues of particular interest for regenerative medicine. They share an early developmental origin and are a source of vast amounts of cells with multilineage differentiation potential that are poorly immunogenic and without controversy. Moreover, these cells are likely exempt from incorporated mutations when compared with juvenile or adult donor cells such as skin fibroblasts or keratinocytes. Here we report the efficient generation of induced pluripotent stem cells (iPSCs) from mesenchymal cells of the umbilical cord matrix (up to 0.4% of the cells became reprogrammed) and the placental amniotic membrane (up to 0.1%) using exogenous factors and a chemical mixture. iPSCs from these 2 tissues homogeneously showed human embryonic stem cell (hESC)-like characteristics including morphology, positive staining for alkaline phosphatase, normal karyotype, and expression of hESC-like markers including Nanog, Rex1, Oct4, TRA-1–60, TRA-1–80, SSEA-3, and SSEA-4. Selected clones also formed embryonic bodies and teratomas containing derivatives of the 3 germ layers, and could as well be readily differentiated into functional motor neurons. Among other things, our cell lines may prove useful for comparisons between iPSCs derived from multiple tissues regarding the extent of the epigenetic reprogramming, differentiation ability, stability of the resulting lineages, and the risk of associated abnormalities. PMID:20139068

  11. An efficient SCNT technology for the establishment of personalized and public human pluripotent stem cell banks.

    PubMed

    Lee, Jeoung Eun; Chung, Young Gie; Eum, Jin Hee; Lee, Yumie; Lee, Dong Ryul

    2016-04-01

    Although three different research groups have reported successful derivations of human somatic cell nuclear transfer-derived embryonic stem cell (SCNT-ESC) lines using fetal, neonatal and adult fibroblasts, the extremely poor development of cloned embryos has hindered its potential applications in regenerative medicine. Recently, however, our group discovered that the severe methylation of lysine 9 in Histone H3 in a human somatic cell genome was a major SCNT reprogramming barrier, and the overexpression of KDM4A, a H3K9me3 demethylase, significantly improved the blastocyst formation of SCNT embryos. In particular, by applying this new approach, we were able to produce multiple SCNT-ES cell lines using oocytes obtained from donors whose eggs previously failed to develop to the blastocyst stage. Moreover, the success rate was closer to 25%, which is comparable to that of IVF embryos, so that our new human SCNT method seems to be a practical approach to establishing a pluripotent stem cell bank for the general public as well as for individual patients. [BMB Reports 2016; 49(4): 197-198]. PMID:26996342

  12. Directed differentiation of definitive hemogenic endothelium and hematopoietic progenitors from human pluripotent stem cells.

    PubMed

    Ditadi, Andrea; Sturgeon, Christopher M

    2016-05-15

    The generation of hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) remains a major goal for regenerative medicine and disease modeling. However, hPSC differentiation cultures produce mostly hematopoietic progenitors belonging to the embryonic HSC-independent hematopoietic program, which may not be relevant or accurate for modeling normal and disease-state adult hematopoietic processes. Through a stage-specific directed differentiation approach, it is now possible to generate exclusively definitive hematopoietic progenitors from hPSCs showing characteristics of the more developmentally advanced fetal hematopoiesis. Here, we summarize recent efforts at generating hPSC-derived definitive hematopoiesis through embryoid body differentiation under defined conditions. Embryoid bodies are generated through enzymatic dissociation of hPSCs from matrigel-coated plasticware, followed by recombinant BMP4, driving mesoderm specification. Definitive hematopoiesis is specified by a GSK3β-inhibitor, followed by recombinant VEGF and supportive hematopoietic cytokines. The CD34+ cells obtained using this method are then suitable for hematopoietic assays for definitive hematopoietic potential. PMID:26439174

  13. An efficient SCNT technology for the establishment of personalized and public human pluripotent stem cell banks

    PubMed Central

    Lee, Jeoung Eun; Chung, Young Gie; Eum, Jin Hee; Lee, Yumie; Lee, Dong Ryul

    2016-01-01

    Although three different research groups have reported successful derivations of human somatic cell nuclear transfer-derived embryonic stem cell (SCNT-ESC) lines using fetal, neonatal and adult fibroblasts, the extremely poor development of cloned embryos has hindered its potential applications in regenerative medicine. Recently, however, our group discovered that the severe methylation of lysine 9 in Histone H3 in a human somatic cell genome was a major SCNT reprogramming barrier, and the overexpression of KDM4A, a H3K9me3 demethylase, significantly improved the blastocyst formation of SCNT embryos. In particular, by applying this new approach, we were able to produce multiple SCNT-ES cell lines using oocytes obtained from donors whose eggs previously failed to develop to the blastocyst stage. Moreover, the success rate was closer to 25%, which is comparable to that of IVF embryos, so that our new human SCNT method seems to be a practical approach to establishing a pluripotent stem cell bank for the general public as well as for individual patients. [BMB Reports 2016; 49(4): 197-198] PMID:26996342

  14. Cortical interneurons from human pluripotent stem cells: prospects for neurological and psychiatric disease

    PubMed Central

    Arber, Charles; Li, Meng

    2012-01-01

    Cortical interneurons represent 20% of the cells in the cortex. These cells are local inhibitory neurons whose function is to modulate the firing activities of the excitatory projection neurons. Cortical interneuron dysfunction is believed to lead to runaway excitation underlying (or implicated in) seizure-based diseases, such as epilepsy, autism, and schizophrenia. The complex development of this cell type and the intricacies involved in defining the relative subtypes are being increasingly well defined. This has led to exciting experimental cell therapy in model organisms, whereby fetal-derived interneuron precursors can reverse seizure severity and reduce mortality in adult epileptic rodents. These proof-of-principle studies raise hope for potential interneuron-based transplantation therapies for treating epilepsy. On the other hand, cortical neurons generated from patient iPSCs serve as a valuable tool to explore genetic influences of interneuron development and function. This is a fundamental step in enhancing our understanding of the molecular basis of neuropsychiatric illnesses and the development of targeted treatments. Protocols are currently being developed for inducing cortical interneuron subtypes from mouse and human pluripotent stem cells. This review sets out to summarize the progress made in cortical interneuron development, fetal tissue transplantation and the recent advance in stem cell differentiation toward interneurons. PMID:23493959

  15. Lrrc34, a novel nucleolar protein, interacts with npm1 and ncl and has an impact on pluripotent stem cells.

    PubMed

    Lührig, Sandra; Siamishi, Iliana; Tesmer-Wolf, Marieke; Zechner, Ulrich; Engel, Wolfgang; Nolte, Jessica

    2014-12-01

    The gene Lrrc34 (leucine rich repeat containing 34) is highly expressed in pluripotent stem cells and its expression is strongly downregulated upon differentiation. These results let us to suggest a role for Lrrc34 in the regulation and maintenance of pluripotency. Expression analyses revealed that Lrrc34 is predominantly expressed in pluripotent stem cells and has an impact on the expression of known pluripotency genes, such as Oct4. Methylation studies of the Lrrc34 promoter showed a hypomethylation in undifferentiated stem cells and chromatin immunoprecipitation-quantitative polymerase chain reaction analyses of histone modifications revealed an enrichment of activating histone modifications on the Lrrc34 promoter region. Further, we could verify the nucleolus-the place of ribosome biogenesis-as the major subcellular localization of the LRRC34 protein. We have verified the interaction of LRRC34 with two major nucleolar proteins, Nucleophosmin and Nucleolin, by two independent methods, suggesting a role for Lrrc34 in ribosome biogenesis of pluripotent stem cells. In conclusion, LRRC34 is a novel nucleolar protein that is predominantly expressed in pluripotent stem cells. Its altered expression has an impact on pluripotency-regulating genes and it interacts with proteins known to be involved in ribosome biogenesis. Therefore we suggest a role for Lrrc34 in ribosome biogenesis of pluripotent stem cells. PMID:24991885

  16. Induction of Human Embryonic and Induced Pluripotent Stem Cells Into Urothelium

    PubMed Central

    Osborn, Stephanie L.; Thangappan, Ravikumar; Luria, Ayala; Lee, Justin H.; Nolta, Jan

    2014-01-01

    In vitro generation of human urothelium from stem cells would be a major advancement in the regenerative medicine field, providing alternate nonurologic and/or nonautologous tissue sources for bladder grafts. Such a model would also help decipher the mechanisms of urothelial differentiation and would facilitate investigation of deviated differentiation of normal progenitors into urothelial cancer stem cells, perhaps elucidating areas of intervention for improved treatments. Thus far, in vitro derivation of urothelium from human embryonic stem cells (hESCs) or human induced pluripotent stem (hiPS) cells has not been reported. The goal of this work was to develop an efficient in vitro protocol for the induction of hESCs into urothelium through an intermediary definitive endoderm step and free of matrices and cell contact. During directed differentiation in a urothelial-specific medium (“Uromedium”), hESCs produced up to 60% urothelium, as determined by uroplakin expression; subsequent propagation selected for 90% urothelium. Alteration of the epithelial and mesenchymal cell signaling contribution through noncell contact coculture or conditioned media did not enhance the production of urothelium. Temporospatial evaluation of transcription factors known to be involved in urothelial specification showed association of IRF1, GET1, and GATA4 with uroplakin expression. Additional hESC and hiPS cell lines could also be induced into urothelium using this in vitro system. These results demonstrate that derivation and propagation of urothelium from hESCs and hiPS cells can be efficiently accomplished in vitro in the absence of matrices, cell contact, or adult cell signaling and that the induction process appears to mimic normal differentiation. PMID:24657961

  17. An improved ScoreCard to assess the differentiation potential of human pluripotent stem cells

    PubMed Central

    Tsankov, Alexander M.; Akopian, Veronika; Pop, Ramona; Chetty, Sundari; Gifford, Casey A.; Daheron, Laurence; Melton, Douglas A.; Tsankova, Nadejda M.; Meissner, Alexander

    2015-01-01

    Research on human pluripotent stem cells has been hampered by the lack of a standardized, quantitative, scalable assay of pluripotency. We have previously described an assay called ScoreCard that used gene expression signatures to quantify differentiation efficiency. Here we report an improved version of the assay based on qPCR that enables faster, more quantitative assessment of functional pluripotency. We provide an in-depth characterization of the revised signature panel through embryoid body and directed differentiation experiments as well as a detailed comparison to the teratoma assay. We also show that the improved ScoreCard enables applications such as screening of small molecules, genetic perturbations and assessment of culture conditions. Beyond stem cell applications, this approach can in principle be extended to other cell types and lineages. PMID:26501952

  18. Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Afford New Opportunities in Inherited Cardiovascular Disease Modeling

    PubMed Central

    Bayzigitov, Daniel R.; Medvedev, Sergey P.; Dementyeva, Elena V.; Bayramova, Sevda A.; Pokushalov, Evgeny A.; Karaskov, Alexander M.; Zakian, Suren M.

    2016-01-01

    Fundamental studies of molecular and cellular mechanisms of cardiovascular disease pathogenesis are required to create more effective and safer methods of their therapy. The studies can be carried out only when model systems that fully recapitulate pathological phenotype seen in patients are used. Application of laboratory animals for cardiovascular disease modeling is limited because of physiological differences with humans. Since discovery of induced pluripotency generating induced pluripotent stem cells has become a breakthrough technology in human disease modeling. In this review, we discuss a progress that has been made in modeling inherited arrhythmias and cardiomyopathies, studying molecular mechanisms of the diseases, and searching for and testing drug compounds using patient-specific induced pluripotent stem cell-derived cardiomyocytes. PMID:27110425

  19. A qPCR ScoreCard quantifies the differentiation potential of human pluripotent stem cells.

    PubMed

    Tsankov, Alexander M; Akopian, Veronika; Pop, Ramona; Chetty, Sundari; Gifford, Casey A; Daheron, Laurence; Tsankova, Nadejda M; Meissner, Alexander

    2015-11-01

    Research on human pluripotent stem cells has been hampered by the lack of a standardized, quantitative, scalable assay of pluripotency. We previously described an assay called ScoreCard that used gene expression signatures to quantify differentiation efficiency. Here we report an improved version of the assay based on qPCR that enables faster, more quantitative assessment of functional pluripotency. We provide an in-depth characterization of the revised signature panel (commercially available as the TaqMan hPSC Scorecard Assay) through embryoid body and directed differentiation experiments as well as a detailed comparison to the teratoma assay. We further show that the improved ScoreCard enables a wider range of applications, such as screening of small molecules, genetic perturbations and assessment of culture conditions. Our approach can be extended beyond stem cell applications to characterize and assess the utility of other cell types and lineages. PMID:26501952

  20. Generation of induced pluripotent stem cells from buffalo (Bubalus bubalis) fetal fibroblasts with buffalo defined factors.

    PubMed

    Deng, Yanfei; Liu, Qingyou; Luo, Chan; Chen, Shibei; Li, Xiangping; Wang, Caizhu; Liu, Zhenzhen; Lei, Xiaocan; Zhang, Huina; Sun, Hongliang; Lu, Fenghua; Jiang, Jianrong; Shi, Deshun

    2012-09-01

    Ectopically, expression of defined factors could reprogram mammalian somatic cells into induced pluripotent stem cells (iPSCs), which initiates a new strategy to obtain pluripotent stem cell lines. Attempts have been made to generate buffalo pluripotent stem cells by culturing primary germ cells or inner cell mass, but the efficiency is extremely low. Here, we report a successful method to reprogram buffalo fetal fibroblasts (BFFs) into pluripotent stem cells [buffalo induced pluripotent stem cell (biPSCs)] by transduction of buffalo defined factors (Oct4, Sox2, Klf4, and c-Myc) using retroviral vectors. The established biPSCs displayed typical morphological characteristics of pluripotent stem cells, normal karyotype, positive staining of alkaline phosphatase, and expressed pluripotent markers including Oct4, Sox2, Nanog, Lin28, E-Cadherin, SSEA-1, SSEA-4, TRA-1-81, STAT3, and FOXD3. They could form embryoid bodies (EBs) in vitro and teratomas after injecting into the nude BALB/C mice, and 3 germ layers were identified in the EBs and teratomas. Methylation assay revealed that the promoters of Oct4 and Nanog were hypomethylated in biPSCs compared with BFFs and pre-biPSCs, while the promoters of Sox2 and E-Cadherin were hypomethylated in both BFFs and biPSCs. Further, inhibiting p53 expression by coexpression of SV40 large T antigen and buffalo defined factors in BFFs or treating BFFs with p53 inhibitor pifithrin-a (PFT) could increase the efficiency of biPSCs generation up to 3-fold, and nuclear transfer embryos reconstructed with biPSCs could develop to blastocysts. These results indicate that BFFs can be reprogrammed into biPSCs by buffalo defined factors, and the generation efficiency of biPSCs can be increased by inhibition of p53 expression. These efforts will provide a feasible approach for investigating buffalo stem cell signal pathways, establishing buffalo stem cell lines, and producing genetic modification buffaloes in the future. PMID:22420535

  1. Isolation and Mechanical Measurements of Myofibrils from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

    PubMed

    Pioner, Josè Manuel; Racca, Alice W; Klaiman, Jordan M; Yang, Kai-Chun; Guan, Xuan; Pabon, Lil; Muskheli, Veronica; Zaunbrecher, Rebecca; Macadangdang, Jesse; Jeong, Mark Y; Mack, David L; Childers, Martin K; Kim, Deok-Ho; Tesi, Chiara; Poggesi, Corrado; Murry, Charles E; Regnier, Michael

    2016-06-14

    Tension production and contractile properties are poorly characterized aspects of excitation-contraction coupling of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Previous approaches have been limited due to the small size and structural immaturity of early-stage hiPSC-CMs. We developed a substrate nanopatterning approach to produce hiPSC-CMs in culture with adult-like dimensions, T-tubule-like structures, and aligned myofibrils. We then isolated myofibrils from hiPSC-CMs and measured the tension and kinetics of activation and relaxation using a custom-built apparatus with fast solution switching. The contractile properties and ultrastructure of myofibrils more closely resembled human fetal myofibrils of similar gestational age than adult preparations. We also demonstrated the ability to study the development of contractile dysfunction of myofibrils from a patient-derived hiPSC-CM cell line carrying the familial cardiomyopathy MYH7 mutation (E848G). These methods can bring new insights to understanding cardiomyocyte maturation and developmental mechanical dysfunction of hiPSC-CMs with cardiomyopathic mutations. PMID:27161364

  2. Human Finger-Prick Induced Pluripotent Stem Cells Facilitate the Development of Stem Cell Banking

    PubMed Central

    Tan, Hong-Kee; Toh, Cheng-Xu Delon; Ma, Dongrui; Yang, Binxia; Liu, Tong Ming; Lu, Jun; Wong, Chee-Wai; Tan, Tze-Kai; Li, Hu; Syn, Christopher; Tan, Eng-Lee; Lim, Bing; Lim, Yoon-Pin; Cook, Stuart A.

    2014-01-01

    Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients can be a good model for studying human diseases and for future therapeutic regenerative medicine. Current initiatives to establish human iPSC (hiPSC) banking face challenges in recruiting large numbers of donors with diverse diseased, genetic, and phenotypic representations. In this study, we describe the efficient derivation of transgene-free hiPSCs from human finger-prick blood. Finger-prick sample collection can be performed on a “do-it-yourself” basis by donors and sent to the hiPSC facility for reprogramming. We show that single-drop volumes of finger-prick samples are sufficient for performing cellular reprogramming, DNA sequencing, and blood serotyping in parallel. Our novel strategy has the potential to facilitate the development of large-scale hiPSC banking worldwide. PMID:24646489

  3. miR-302 regulates pluripotency, teratoma formation and differentiation in stem cells via an AKT1/OCT4-dependent manner

    PubMed Central

    Li, H-L; Wei, J-F; Fan, L-Y; Wang, S-H; Zhu, L; Li, T-P; Lin, G; Sun, Y; Sun, Z-J; Ding, J; Liang, X-L; Li, J; Han, Q; Zhao, R-C-H

    2016-01-01

    Pluripotency makes human pluripotent stem cells (hPSCs) promising for regenerative medicine, but the teratoma formation has been considered to be a major obstacle for their clinical applications. Here, we determined that the downregulation of miR-302 suppresses the teratoma formation, hampers the self-renewal and pluripotency, and promotes hPSC differentiation. The underlying mechanism is that the high endogenous expression of miR-302 suppresses the AKT1 expression by directly targeting its 3'UTR and subsequently maintains the pluripotent factor OCT4 at high level. Our findings reveal that miR-302 regulates OCT4 by suppressing AKT1, which provides hPSCs two characteristics related to their potential for clinical applications: the benefit of pluripotency and the hindrance of teratoma formation. More importantly, we demonstrate that miR-302 upregulation cannot lead OCT4 negative human adult mesenchymal stem cells (hMSCs) to acquire the teratoma formation in vivo. Whether miR-302 upregulation can drive hMSCs to acquire a higher differentiation potential is worthy of deep investigation. PMID:26821070

  4. Robust generation and expansion of skeletal muscle progenitors and myocytes from human pluripotent stem cells.

    PubMed

    Shelton, Michael; Kocharyan, Avetik; Liu, Jun; Skerjanc, Ilona S; Stanford, William L

    2016-05-15

    Human pluripotent stem cells provide a developmental model to study early embryonic and tissue development, tease apart human disease processes, perform drug screens to identify potential molecular effectors of in situ regeneration, and provide a source for cell and tissue based transplantation. Highly efficient differentiation protocols have been established for many cell types and tissues; however, until very recently robust differentiation into skeletal muscle cells had not been possible unless driven by transgenic expression of master regulators of myogenesis. Nevertheless, several breakthrough protocols have been published in the past two years that efficiently generate cells of the skeletal muscle lineage from pluripotent stem cells. Here, we present an updated version of our recently described 50-day protocol in detail, whereby chemically defined media are used to drive and support muscle lineage development from initial CHIR99021-induced mesoderm through to PAX7-expressing skeletal muscle progenitors and mature skeletal myocytes. Furthermore, we report an optional method to passage and expand differentiating skeletal muscle progenitors approximately 3-fold every 2weeks using Collagenase IV and continued FGF2 supplementation. Both protocols have been optimized using a variety of human pluripotent stem cell lines including patient-derived induced pluripotent stem cells. Taken together, our differentiation and expansion protocols provide sufficient quantities of skeletal muscle progenitors and myocytes that could be used for a variety of studies. PMID:26404920

  5. Expand and Regularize Federal Funding for Human Pluripotent Stem Cell Research

    ERIC Educational Resources Information Center

    Owen-Smith, Jason; Scott, Christopher Thomas; McCormick, Jennifer B.

    2012-01-01

    Human embryonic stem cell (hESC) research has sparked incredible scientific and public excitement, as well as significant controversy. hESCs are pluripotent, which means, in theory, that they can be differentiated into any type of cell found in the human body. Thus, they evoke great enthusiasm about potential clinical applications. They are…

  6. Isolation and cultivation of naive-like human pluripotent stem cells based on HERVH expression.

    PubMed

    Wang, Jichang; Singh, Manvendra; Sun, Chuanbo; Besser, Daniel; Prigione, Alessandro; Ivics, Zoltán; Hurst, Laurence D; Izsvák, Zsuzsanna

    2016-02-01

    The ability to derive and stably maintain ground-state human pluripotent stem cells (hPSCs) that resemble the cells seen in vivo in the inner cell mass has the potential to be an invaluable tool for researchers developing stem cell-based therapies. To date, derivation of human naive-like pluripotent stem cell lines has been limited to a small number of lineages, and their long-term culturing remains problematic. We describe a protocol for genetic and phenotypic tagging, selecting and maintaining naive-like hPSCs. We tag hPSCs by GFP, expressed by the long terminal repeat (LTR7) of HERVH endogenous retrovirus. This simple and efficient protocol has been reproduced with multiple hPSC lines, including embryonic and induced pluripotent stem cells, and it takes ∼6 weeks. By using the reporter, homogeneous hPSC cultures can be derived, characterized and maintained for the long term by repeated re-sorting and re-plating steps. The HERVH-expressing cells have a similar, but nonidentical, expression pattern to other naive-like cells, suggesting that alternative pluripotent states might exist. PMID:26797457

  7. MicroRNA-302 switch to identify and eliminate undifferentiated human pluripotent stem cells

    PubMed Central

    Parr, Callum J. C.; Katayama, Shota; Miki, Kenji; Kuang, Yi; Yoshida, Yoshinori; Morizane, Asuka; Takahashi, Jun; Yamanaka, Shinya; Saito, Hirohide

    2016-01-01

    The efficiency of pluripotent stem cell differentiation is highly variable, often resulting in heterogeneous populations that contain undifferentiated cells. Here we developed a sensitive, target-specific, and general method for removing undesired cells before transplantation. MicroRNA-302a-5p (miR-302a) is highly and specifically expressed in human pluripotent stem cells and gradually decreases to basal levels during differentiation. We synthesized a new RNA tool, miR-switch, as a live-cell reporter mRNA for miR-302a activity that can specifically detect human induced pluripotent stem cells (hiPSCs) down to a spiked level of 0.05% of hiPSCs in a heterogeneous population and can prevent teratoma formation in an in vivo tumorigenicity assay. Automated and selective hiPSC-elimination was achieved by controlling puromycin resistance using the miR-302a switch. Our system uniquely provides sensitive detection of pluripotent stem cells and partially differentiated cells. In addition to its ability to eliminate undifferentiated cells, miR-302a switch also holds great potential in investigating the dynamics of differentiation and/or reprograming of live-cells based on intracellular information. PMID:27608814

  8. MicroRNA-302 switch to identify and eliminate undifferentiated human pluripotent stem cells.

    PubMed

    Parr, Callum J C; Katayama, Shota; Miki, Kenji; Kuang, Yi; Yoshida, Yoshinori; Morizane, Asuka; Takahashi, Jun; Yamanaka, Shinya; Saito, Hirohide

    2016-01-01

    The efficiency of pluripotent stem cell differentiation is highly variable, often resulting in heterogeneous populations that contain undifferentiated cells. Here we developed a sensitive, target-specific, and general method for removing undesired cells before transplantation. MicroRNA-302a-5p (miR-302a) is highly and specifically expressed in human pluripotent stem cells and gradually decreases to basal levels during differentiation. We synthesized a new RNA tool, miR-switch, as a live-cell reporter mRNA for miR-302a activity that can specifically detect human induced pluripotent stem cells (hiPSCs) down to a spiked level of 0.05% of hiPSCs in a heterogeneous population and can prevent teratoma formation in an in vivo tumorigenicity assay. Automated and selective hiPSC-elimination was achieved by controlling puromycin resistance using the miR-302a switch. Our system uniquely provides sensitive detection of pluripotent stem cells and partially differentiated cells. In addition to its ability to eliminate undifferentiated cells, miR-302a switch also holds great potential in investigating the dynamics of differentiation and/or reprograming of live-cells based on intracellular information. PMID:27608814

  9. Generation of Induced Pluripotent Stem Cell Lines from Tibetan Miniature Pig*

    PubMed Central

    Esteban, Miguel A.; Xu, Jianyong; Yang, Jiayin; Peng, Meixiu; Qin, Dajiang; Li, Wen; Jiang, Zhuoxin; Chen, Jiekai; Deng, Kang; Zhong, Mei; Cai, Jinglei; Lai, Liangxue; Pei, Duanqing

    2009-01-01

    Induced pluripotent stem cell (iPS) technology appears to be a general strategy to generate pluripotent stem cells from any given mammalian species. So far, iPS cells have been reported for mouse, human, rat, and monkey. These four species have also established embryonic stem cell (ESC) lines that serve as the gold standard for pluripotency comparisons. Attempts have been made to generate porcine ESC by various means without success. Here we report the successful generation of pluripotent stem cells from fibroblasts isolated from the Tibetan miniature pig using a modified iPS protocol. The resulting iPS cell lines more closely resemble human ESC than cells from other species, have normal karyotype, stain positive for alkaline phosphatase, express high levels of ESC-like markers (Nanog, Rex1, Lin28, and SSEA4), and can differentiate into teratomas composed of the three germ layers. Because porcine physiology closely resembles human, the iPS cells reported here provide an attractive model to study certain human diseases or assess therapeutic applications of iPS in a large animal model. PMID:19376775

  10. Identification of polymer surface adsorbed proteins implicated in pluripotent human embryonic stem cell expansion.

    PubMed

    Hammad, Moamen; Rao, Wei; Smith, James G W; Anderson, Daniel G; Langer, Robert; Young, Lorraine E; Barrett, David A; Davies, Martyn C; Denning, Chris; Alexander, Morgan R

    2016-08-16

    Improved biomaterials are required for application in regenerative medicine, biosensing, and as medical devices. The response of cells to the chemistry of polymers cultured in media is generally regarded as being dominated by proteins adsorbed to the surface. Here we use mass spectrometry to identify proteins adsorbed from a complex mouse embryonic fibroblast (MEF) conditioned medium found to support pluripotent human embryonic stem cell (hESC) expansion on a plasma etched tissue culture polystyrene surface. A total of 71 proteins were identified, of which 14 uniquely correlated with the surface on which pluripotent stem cell expansion was achieved. We have developed a microarray combinatorial protein spotting approach to test the potential of these 14 proteins to support expansion of a hESC cell line (HUES-7) and a human induced pluripotent stem cell line (ReBl-PAT) on a novel polymer (N-(4-Hydroxyphenyl) methacrylamide). These proteins were spotted to form a primary array yielding several protein mixture 'hits' that enhanced cell attachment to the polymer. A second array was generated to test the function of a refined set of protein mixtures. We found that a combination of heat shock protein 90 and heat shock protein-1 encourage elevated adherence of pluripotent stem cells at a level comparable to fibronectin pre-treatment. PMID:27466628

  11. The 3R principle: advancing clinical application of human pluripotent stem cells

    PubMed Central

    2013-01-01

    The first derivation of human embryonic stem cells brought with it a clear understanding that animal models of human disease might be replaced by an unlimited supply of human cells for research, drug discovery, and drug development. With the advent of clinical trials using human pluripotent stem cell-based therapies, it is both timely and relevant to reflect on factors that will facilitate future translation of this technology. Human pluripotent cells are increasingly being used to investigate the molecular mechanisms that underpin normal and pathological human development. Their differentiated progeny are also being used to identify novel pharmaceuticals, to screen for toxic effects of known chemicals, and to investigate cell or tissue transplantation strategies. The intrinsic assumption of these research efforts is that the information gained from these studies will be more accurate, and therefore of greater relevance, than traditional investigations based on animal models of human disease and injury. This review will therefore evaluate how animals and animal-derived products are used for human pluripotent stem cell research, and will indicate how efforts to further reduce or remove animals and animal products from this research will increase the clinical translation of human pluripotent stem cell technologies through drug discovery, toxicology screening, and cell replacement therapies. PMID:23510719

  12. Zinc Chloride Transiently Maintains Mouse Embryonic Stem Cell Pluripotency by Activating Stat3 Signaling

    PubMed Central

    Hu, Jing; Yang, Zhiyong; Wang, Jinbo; Yu, Jia; Guo, Jing; Liu, Shiying; Qian, Chunmei; Song, Liwen; Wu, Yi; Cheng, Jiajing

    2016-01-01

    An improved understanding of the pluripotency maintenance of embryonic stem (ES) cells is important for investigations of early embryo development and for cell replacement therapy, but the mechanism behind pluripotency is still incompletely understood. Recent findings show that zinc, an essential trace element in humans, is critically involved in regulating various signaling pathways and genes expression. However, its role in ES cell fate determination remains to be further explored. Here we showed that 2μM zinc chloride (ZnCl2) transiently maintained mouse ES cell pluripotency in vitro. The cultured mouse ES cells remained undifferentiated under 2μM ZnCl2 treatment in leukemia inhibitory factor (LIF) withdrawal, retinoic acid (RA) or embryoid bodies (EBs) differentiation assays. In addition, ZnCl2 increased pluripotency genes expression and inhibited differentiation genes expression. Further mechanistic studies revealed that ZnCl2 transiently activated signal transducers and activators of transcription 3 (Stat3) signaling through promoting Stat3 phosphorylation. Inhibition of Stat3 signaling abrogated the effects of ZnCl2 on mouse ES cell pluripotency. Taken together, this study demonstrated a critical role of zinc in the pluripotency maintenance of mouse ES cells, as well as an important regulator of Stat3 signaling. PMID:26910359

  13. Investigating the functionality of an OCT4-short response element in human induced pluripotent stem cells

    PubMed Central

    Vega-Crespo, Agustin; Truong, Brian; Hermann, Kip J; Awe, Jason P; Chang, Katherine M; Lee, Patrick C; Schoenberg, Benjamen E; Wu, Lily; Byrne, James A; Lipshutz, Gerald S

    2016-01-01

    Pluripotent stem cells offer great therapeutic promise for personalized treatment platforms for numerous injuries, disorders, and diseases. Octamer-binding transcription factor 4 (OCT4) is a key regulatory gene maintaining pluripotency and self-renewal of mammalian cells. With site-specific integration for gene correction in cellular therapeutics, use of the OCT4 promoter may have advantages when expressing a suicide gene if pluripotency remains. However, the human OCT4 promoter region is 4 kb in size, limiting the capacity of therapeutic genes and other regulatory components for viral vectors, and decreasing the efficiency of homologous recombination. The purpose of this investigation was to characterize the functionality of a novel 967bp OCT4-short response element during pluripotency and to examine the OCT4 titer-dependent response during differentiation to human derivatives not expressing OCT4. Our findings demonstrate that the OCT4-short response element is active in pluripotency and this activity is in high correlation with transgene expression in vitro, and the OCT4-short response element is inactivated when pluripotent cells differentiate. These studies demonstrate that this shortened OCT4 regulatory element is functional and may be useful as part of an optimized safety component in a site-specific gene transferring system that could be used as an efficient and clinically applicable safety platform for gene transfer in cellular therapeutics. PMID:27500178

  14. Human pluripotent stem cell-derived limbal epithelial stem cells on bioengineered matrices for corneal reconstruction.

    PubMed

    Mikhailova, Alexandra; Ilmarinen, Tanja; Ratnayake, Anjula; Petrovski, Goran; Uusitalo, Hannu; Skottman, Heli; Rafat, Mehrdad

    2016-05-01

    Corneal epithelium is renewed by limbal epithelial stem cells (LESCs), a type of tissue-specific stem cells located in the limbal palisades of Vogt at the corneo-scleral junction. Acute trauma or inflammatory disorders of the ocular surface can destroy these stem cells, leading to limbal stem cell deficiency (LSCD) - a painful and vision-threatening condition. Treating these disorders is often challenging and complex, especially in bilateral cases with extensive damage. Human pluripotent stem cells (hPSCs) provide new opportunities for corneal reconstruction using cell-based therapy. Here, we investigated the use of hPSC-derived LESC-like cells on bioengineered collagen matrices in serum-free conditions, aiming for clinical applications to reconstruct the corneal epithelium and partially replace the damaged stroma. Differentiation of hPSCs towards LESC-like cells was directed using small-molecule induction followed by maturation in corneal epithelium culture medium. After four to five weeks of culture, differentiated cells were seeded onto bioengineered matrices fabricated as transparent membranes of uniform thickness, using medical-grade porcine collagen type I and a hybrid cross-linking technology. The bioengineered matrices were fully transparent, with high water content and swelling capacity, and parallel lamellar microstructure. Cell proliferation of hPSC-LESCs was significantly higher on bioengineered matrices than on collagen-coated control wells after two weeks of culture, and LESC markers p63 and cytokeratin 15, along with proliferation marker Ki67 were expressed even after 30 days in culture. Overall, hPSC-LESCs retained their capacity to self-renew and proliferate, but were also able to terminally differentiate upon stimulation, as suggested by protein expression of cytokeratins 3 and 12. We propose the use of bioengineered collagen matrices as carriers for the clinically-relevant hPSC-derived LESC-like cells, as a novel tissue engineering approach for

  15. Two Histone Variants TH2A and TH2B Enhance Human Induced Pluripotent Stem Cell Generation.

    PubMed

    Huynh, Linh My; Shinagawa, Toshie; Ishii, Shunsuke

    2016-02-01

    There are two major methods of reprogramming: generation of induced pluripotent stem cells (iPSCs) by overexpressing embryonic stem cell-specific transcription factors (OCT4, SOX2, KLF4, and c-MYC) and somatic cell nuclear transfer by oocyte-specific factors. Previously, we reported oocyte-enriched histone variants TH2A, TH2B, and the histone chaperone nucleoplasmin (NPM2) enhance the reprogramming by OSKM in mice by inducing open chromatin structure. In this study, we showed that human TH2A, TH2B, and NPM2 enhance the OSKM-induced reprogramming of adult and neonatal human dermal fibroblasts and umbilical vein endothelial cells. Pluripotency of iPSCs generated by coexpressing OSKM, TH2A, TH2B, and NPM2 was shown by in vitro and in vivo differentiation assays. These iPSCs gave rise to highly differentiated teratomas compared to iPSCs induced by OSKM alone. Genome-wide analysis suggests a possibility that TH2A, TH2B, and NPM2 might regulate genes that are involved in naïve stem cell stage. Thus, TH2A, TH2B, and NPM2 enhance reprogramming of human somatic cells and improve the quality of human iPSCs. PMID:26649967

  16. Nanog RNA-binding proteins YBX1 and ILF3 affect pluripotency of embryonic stem cells.

    PubMed

    Guo, Chuanliang; Xue, Yan; Yang, Guanheng; Yin, Shang; Shi, Wansheng; Cheng, Yan; Yan, Xiaoshuang; Fan, Shuyue; Zhang, Huijun; Zeng, Fanyi

    2016-08-01

    Nanog is a well-known transcription factor that plays a fundamental role in stem cell self-renewal and the maintenance of their pluripotent cell identity. There remains a large data gap with respect to the spectrum of the key pluripotency transcription factors' interaction partners. Limited information is available concerning Nanog-associated RNA-binding proteins (RBPs), and the intrinsic protein-RNA interactions characteristic of the regulatory activities of Nanog. Herein, we used an improved affinity protocol to purify Nanog-interacting RBPs from mouse embryonic stem cells (ESCs), and 49 RBPs of Nanog were identified. Among them, the interaction of YBX1 and ILF3 with Nanog mRNA was further confirmed by in vitro assays, such as Western blot, RNA immunoprecipitation (RIP), and ex vivo methods, such as immunofluorescence staining and fluorescent in situ hybridization (FISH), MS2 in vivo biotin-tagged RNA affinity purification (MS2-BioTRAP). Interestingly, RNAi studies revealed that YBX1 and ILF3 positively affected the expression of Nanog and other pluripotency-related genes. Particularly, downregulation of YBX1 or ILF3 resulted in high expression of mesoderm markers. Thus, a reduction in the expression of YBX1 and ILF3 controls the expression of pluripotency-related genes in ESCs, suggesting their roles in further regulation of the pluripotent state of ESCs. PMID:26289635

  17. V-Myc Immortalizes Human Neural Stem Cells in the Absence of Pluripotency-Associated Traits

    PubMed Central

    Pino-Barrio, María José; García-García, Elisa; Menéndez, Pablo; Martínez-Serrano, Alberto

    2015-01-01

    A better understanding of the molecular mechanisms governing stem cell self-renewal will foster the use of different types of stem cells in disease modeling and cell therapy strategies. Immortalization, understood as the capacity for indefinite expansion, is needed for the generation of any cell line. In the case of v-myc immortalized multipotent human Neural Stem Cells (hNSCs), we hypothesized that v-myc immortalization could induce a more de-differentiated state in v-myc hNSC lines. To test this, we investigated the expression of surface, biochemical and genetic markers of stemness and pluripotency in v-myc immortalized and control hNSCs (primary precursors, that is, neurospheres) and compared these two cell types to human Embryonic Stem Cells (hESCs) and fibroblasts. Using a Hierarchical Clustering method and a Principal Component Analysis (PCA), the v-myc hNSCs associated with their counterparts hNSCs (in the absence of v-myc) and displayed a differential expression pattern when compared to hESCs. Moreover, the expression analysis of pluripotency markers suggested no evidence supporting a reprogramming-like process despite the increment in telomerase expression. In conclusion, v-myc expression in hNSC lines ensures self-renewal through the activation of some genes involved in the maintenance of stem cell properties in multipotent cells but does not alter the expression of key pluripotency-associated genes. PMID:25764185

  18. Human amniotic epithelial cells are reprogrammed more efficiently by induced pluripotency than adult fibroblasts.

    PubMed

    Easley, Charles A; Miki, Toshio; Castro, Carlos A; Ozolek, John A; Minervini, Crescenzio F; Ben-Yehudah, Ahmi; Schatten, Gerald P

    2012-06-01

    Cellular reprogramming from adult somatic cells into an embryonic cell-like state, termed induced pluripotency, has been achieved in several cell types. However, the ability to reprogram human amniotic epithelial cells (hAECs), an abundant cell source derived from discarded placental tissue, has only recently been investigated. Here we show that not only are hAECs easily reprogrammed into induced pluripotent stem cells (AE-iPSCs), but hAECs reprogram faster and more efficiently than adult and neonatal somatic dermal fibroblasts. Furthermore, AE-iPSCs express higher levels of NANOG and OCT4 compared to human foreskin fibroblast iPSCs (HFF1-iPSCs) and express decreased levels of genes associated with differentiation, including NEUROD1 and SOX17, markers of neuronal differentiation. To elucidate the mechanism behind the higher reprogramming efficiency of hAECs, we analyzed global DNA methylation, global histone acetylation, and the mitochondrial DNA A3243G point mutation. Whereas hAECs show no differences in global histone acetylation or mitochondrial point mutation accumulation compared to adult and neonatal dermal fibroblasts, hAECs demonstrate a decreased global DNA methylation compared to dermal fibroblasts. Likewise, quantitative gene expression analyses show that hAECs endogenously express OCT4, SOX2, KLF4, and c-MYC, all four factors used in cellular reprogramming. Thus, hAECs represent an ideal cell type for testing novel approaches for generating clinically viable iPSCs and offer significant advantages over postnatal cells that more likely may be contaminated by environmental exposures and infectious agents. PMID:22686477

  19. PRMT5 is required for human embryonic stem cell proliferation but not pluripotency

    PubMed Central

    Gkountela, Sofia; Li, Ziwei; Chin, Chee Jia; Lee, Serena A.; Clark, Amander T.

    2014-01-01

    Summary Human pluripotent stem cells (PSCs) are critical in vitro tools for understanding mechanisms that regulate lineage differentiation in the human embryo as well as a potentially unlimited supply of stem cells for regenerative medicine. Pluripotent human and mouse embryonic stem cells (ESCs) derived from the inner cell mass of blastocysts share a similar transcription factor network to maintain pluripotency and self-renewal, yet there are considerable molecular differences reflecting the diverse environments in which mouse and human ESCs are derived. In the current study we evaluated the role of Protein arginine methyltransferase 5 (PRMT5) in human ESC (hESC) self-renewal and pluripotency given its critical role in safeguarding mouse ESC pluripotency. Unlike the mouse, we discovered that PRMT5 has no role in hESC pluripotency. Using microarray analysis we discovered that a significant depletion in PRMT5 RNA and protein from hESCs changed the expression of only 78 genes, with the majority being repressed. Functionally, we discovered that depletion of PRMT5 had no effect on expression of OCT4, NANOG or SOX2, and did not prevent teratoma formation. Instead, we show that PRMT5 functions in hESCs to regulate proliferation in the self-renewing state by regulating the fraction of cells in Gap 1 (G1) of the cell cycle and increasing expression of the G1 cell cycle inhibitor P57. Taken together our data unveils a distinct role for PRMT5 in hESCs and identifies P57 as new target. PMID:24477620

  20. Transcriptome Characteristics and X-Chromosome Inactivation Status in Cultured Rat Pluripotent Stem Cells.

    PubMed

    Vaskova, Evgeniya A; Medvedev, Sergey P; Sorokina, Anastasiya E; Nemudryy, Artem A; Elisaphenko, Evgeniy A; Zakharova, Irina S; Shevchenko, Alexander I; Kizilova, Elena A; Zhelezova, Antonina I; Evshin, Ivan S; Sharipov, Ruslan N; Minina, Julia M; Zhdanova, Natalia S; Khegay, Igor I; Kolpakov, Fedor A; Sukhikh, Gennadiy T; Pokushalov, Evgeniy A; Karaskov, Alexander M; Vlasov, Valentin V; Ivanova, Ludmila N; Zakian, Suren M

    2015-12-15

    Rat pluripotent stem cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) as mouse and human ones have a great potential for studying mammalian early development, disease modeling, and evaluation of regenerative medicine approaches. However, data on pluripotency realization and self-renewal maintenance in rat cells are still very limited, and differentiation protocols of rat ESCs (rESCs) and iPSCs to study development and obtain specific cell types for biomedical applications are poorly developed. In this study, the RNA-Seq technique was first used for detailed transcriptome characterization in rat pluripotent cells. The rESC and iPSC transcriptomes demonstrated a high similarity and were significantly different from those in differentiated cells. Additionally, we have shown that reprogramming of rat somatic cells to a pluripotent state was accompanied by X-chromosome reactivation. There were two active X chromosomes in XX rESCs and iPSCs, which is one of the key attributes of the pluripotent state. Differentiation of both rESCs and iPSCs led to X-chromosome inactivation (XCI). The dynamics of XCI in differentiating rat cells was very similar to that in mice. Two types of facultative heterochromatin described in various mammalian species were revealed on the rat inactive X chromosome. To explore XCI dynamics, we established a new monolayer differentiation protocol for rESCs and iPSCs that may be applied to study different biological processes and optimized for directed derivation of specific cell types. PMID:26418521

  1. Functional vascular smooth muscle cells derived from human induced pluripotent stem cells via mesenchymal stem cell intermediates

    PubMed Central

    Bajpai, Vivek K.; Mistriotis, Panagiotis; Loh, Yuin-Han; Daley, George Q.; Andreadis, Stelios T.

    2012-01-01

    Aims Smooth muscle cells (SMC) play an important role in vascular homeostasis and disease. Although adult mesenchymal stem cells (MSC) have been used as a source of contractile SMC, they suffer from limited proliferation potential and culture senescence, particularly when originating from older donors. By comparison, human induced pluripotent stem cells (hiPSC) can provide an unlimited source of functional SMC for autologous cell-based therapies and for creating models of vascular disease. Our goal was to develop an efficient strategy to derive functional, contractile SMC from hiPSC. Methods and results We developed a robust, stage-wise, feeder-free strategy for hiPSC differentiation into functional SMC through an intermediate stage of multipotent MSC, which could be coaxed to differentiate into fat, bone, cartilage, and muscle. At this stage, the cells were highly proliferative and displayed higher clonogenic potential and reduced senescence when compared with parental hair follicle mesenchymal stem cells. In addition, when exposed to differentiation medium, the myogenic proteins such as α-smooth muscle actin, calponin, and myosin heavy chain were significantly upregulated and displayed robust fibrillar organization, suggesting the development of a contractile phenotype. Indeed, tissue constructs prepared from these cells exhibited high levels of contractility in response to receptor- and non-receptor-mediated agonists. Conclusion We developed an efficient stage-wise strategy that enabled hiPSC differentiation into contractile SMC through an intermediate population of clonogenic and multipotent MSC. The high yield of MSC and SMC derivation suggests that our strategy may facilitate an acquisition of the large numbers of cells required for regenerative medicine or for studying vascular disease pathophysiology. PMID:22941255

  2. Multilineage-differentiating stress-enduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts

    PubMed Central

    Wakao, Shohei; Kitada, Masaaki; Kuroda, Yasumasa; Shigemoto, Taeko; Matsuse, Dai; Akashi, Hideo; Tanimura, Yukihiro; Tsuchiyama, Kenichiro; Kikuchi, Tomohiko; Goda, Makoto; Nakahata, Tatsutoshi; Fujiyoshi, Yoshinori; Dezawa, Mari

    2011-01-01

    The stochastic and elite models have been proposed for the mechanism of induced pluripotent stem (iPS) cell generation. In this study we report a system that supports the elite model. We previously identified multilineage-differentiating stress-enduring (Muse) cells in human dermal fibroblasts that are characterized by stress tolerance, expression of pluripotency markers, self-renewal, and the ability to differentiate into endodermal-, mesodermal-, and ectodermal-lineage cells from a single cell. They can be isolated as stage-specific embryonic antigen-3/CD105 double-positive cells. When human fibroblasts were separated into Muse and non-Muse cells and transduced with Oct3/4, Sox2, Klf4, and c-Myc, iPS cells were generated exclusively from Muse cells but not from non-Muse cells. Although some colonies were formed from non-Muse cells, they were unlike iPS cells. Furthermore, epigenetic alterations were not seen, and some of the major pluripotency markers were not expressed for the entire period during iPS cell generation. These findings were confirmed further using cells transduced with a single polycistronic virus vector encoding all four factors. The results demonstrate that in adult human fibroblasts a subset of preexisting adult stem cells whose properties are similar in some respects to those of iPS cells selectively become iPS cells, but the remaining cells make no contribution to the generation of iPS cells. Therefore this system seems to fit the elite model rather than the stochastic model. PMID:21628574

  3. Multilineage-differentiating stress-enduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts.

    PubMed

    Wakao, Shohei; Kitada, Masaaki; Kuroda, Yasumasa; Shigemoto, Taeko; Matsuse, Dai; Akashi, Hideo; Tanimura, Yukihiro; Tsuchiyama, Kenichiro; Kikuchi, Tomohiko; Goda, Makoto; Nakahata, Tatsutoshi; Fujiyoshi, Yoshinori; Dezawa, Mari

    2011-06-14

    The stochastic and elite models have been proposed for the mechanism of induced pluripotent stem (iPS) cell generation. In this study we report a system that supports the elite model. We previously identified multilineage-differentiating stress-enduring (Muse) cells in human dermal fibroblasts that are characterized by stress tolerance, expression of pluripotency markers, self-renewal, and the ability to differentiate into endodermal-, mesodermal-, and ectodermal-lineage cells from a single cell. They can be isolated as stage-specific embryonic antigen-3/CD105 double-positive cells. When human fibroblasts were separated into Muse and non-Muse cells and transduced with Oct3/4, Sox2, Klf4, and c-Myc, iPS cells were generated exclusively from Muse cells but not from non-Muse cells. Although some colonies were formed from non-Muse cells, they were unlike iPS cells. Furthermore, epigenetic alterations were not seen, and some of the major pluripotency markers were not expressed for the entire period during iPS cell generation. These findings were confirmed further using cells transduced with a single polycistronic virus vector encoding all four factors. The results demonstrate that in adult human fibroblasts a subset of preexisting adult stem cells whose properties are similar in some respects to those of iPS cells selectively become iPS cells, but the remaining cells make no contribution to the generation of iPS cells. Therefore this system seems to fit the elite model rather than the stochastic model. PMID:21628574

  4. Differentiate or Die: 3-Bromopyruvate and Pluripotency in Mouse Embryonic Stem Cells

    PubMed Central

    Rodrigues, Ana Sofia; Pereira, Sandro L.; Correia, Marcelo; Gomes, Andreia; Perestrelo, Tânia; Ramalho-Santos, João

    2015-01-01

    Background Pluripotent embryonic stem cells grown under standard conditions (ESC) have a markedly glycolytic profile, which is shared with many different types of cancer cells. Thus, some therapeutic strategies suggest that pharmacologically shifting cancer cells towards an oxidative phenotype, using glycolysis inhibitors, may reduce cancer aggressiveness. Given the metabolic parallels between cancer and stemness would chemotherapeutical agents have an effect on pluripotency, and could a strategy involving these agents be envisioned to modulate stem cell fate in an accessible manner? In this manuscript we attempted to determine the effects of 3-bromopyruvate (3BrP) in pluripotency. Although it has other intracellular targets, this compound is a potent inhibitor of glycolysis enzymes thought to be important to maintain a glycolytic profile. The goal was also to determine if we could contribute towards a pharmacologically accessible metabolic strategy to influence cell differentiation. Methodology/Principal Findings Mouse embryonic stem cells (mESC) grown under standard pluripotency conditions (in the presence of Leukemia Inducing Factor- LIF) were treated with 3BrP. As a positive control for differentiation other mESCs were grown without LIF. Overall our results demonstrate that 3BrP negatively affects pluripotency, forcing cells to become less glycolytic and with more active mitochondria. These changes in metabolism are correlated with increased differentiation, even under pluripotency conditions (i.e. in the presence of LIF). However, 3BrP also significantly impaired cell function, and may have other roles besides affecting the metabolic profile of mESCs. Conclusions/Findings Treatment of mESCs with 3BrP triggered a metabolic switch and loss of pluripotency, even in the presence of LIF. Interestingly, the positive control for differentiation allowed for a distinction between 3BrP effects and changes associated with spontaneous differentiation/loss of pluripotency

  5. An overview of mammalian pluripotency.

    PubMed

    Wu, Jun; Yamauchi, Takayoshi; Izpisua Belmonte, Juan Carlos

    2016-05-15

    Mammalian pluripotency is the ability to give rise to all somatic cells as well as the germ cells of an adult mammal. It is a unique feature of embryonic epiblast cells, existing only transiently, as cells pass through early developmental stages. By contrast, pluripotency can be captured and stabilized indefinitely in cell culture and can also be reactivated in differentiated cells via nuclear reprogramming. Pluripotent stem cells (PSCs) are the in vitro carriers of pluripotency and they can inhabit discrete pluripotent states depending on the stage at which they were derived and their culture conditions. Here, and in the accompanying poster, we provide a summary of mammalian pluripotency both in vivo and in vitro, and highlight recent and future applications of PSCs for basic and translational research. PMID:27190034

  6. The pluripotency factor Nanog regulates pericentromeric heterochromatin organization in mouse embryonic stem cells.

    PubMed

    Novo, Clara Lopes; Tang, Calvin; Ahmed, Kashif; Djuric, Ugljesa; Fussner, Eden; Mullin, Nicholas P; Morgan, Natasha P; Hayre, Jasvinder; Sienerth, Arnold R; Elderkin, Sarah; Nishinakamura, Ryuichi; Chambers, Ian; Ellis, James; Bazett-Jones, David P; Rugg-Gunn, Peter J

    2016-05-01

    An open and decondensed chromatin organization is a defining property of pluripotency. Several epigenetic regulators have been implicated in maintaining an open chromatin organization, but how these processes are connected to the pluripotency network is unknown. Here, we identified a new role for the transcription factor NANOG as a key regulator connecting the pluripotency network with constitutive heterochromatin organization in mouse embryonic stem cells. Deletion of Nanog leads to chromatin compaction and the remodeling of heterochromatin domains. Forced expression of NANOG in epiblast stem cells is sufficient to decompact chromatin. NANOG associates with satellite repeats within heterochromatin domains, contributing to an architecture characterized by highly dispersed chromatin fibers, low levels of H3K9me3, and high major satellite transcription, and the strong transactivation domain of NANOG is required for this organization. The heterochromatin-associated protein SALL1 is a direct cofactor for NANOG, and loss of Sall1 recapitulates the Nanog-null phenotype, but the loss of Sall1 can be circumvented through direct recruitment of the NANOG transactivation domain to major satellites. These results establish a direct connection between the pluripotency network and chromatin organization and emphasize that maintaining an open heterochromatin architecture is a highly regulated process in embryonic stem cells. PMID:27125671

  7. Patient-Specific Induced Pluripotent Stem Cells for SOD1-Associated Amyotrophic Lateral Sclerosis Pathogenesis Studies

    PubMed Central

    Chestkov, I. V.; Vasilieva, E. A.; Illarioshkin, S. N.; Lagarkova, M. A.; Kiselev, S. L.

    2014-01-01

    The genetic reprogramming technology allows one to generate pluripotent stem cells for individual patients. These cells, called induced pluripotent stem cells (iPSCs), can be an unlimited source of specialized cell types for the body. Thus, autologous somatic cell replacement therapy becomes possible, as well as the generation of in vitro cell models for studying the mechanisms of disease pathogenesis and drug discovery. Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder that leads to a loss of upper and lower motor neurons. About 10% of cases are genetically inherited, and the most common familial form of ALS is associated with mutations in the SOD1 gene. We used the reprogramming technology to generate induced pluripotent stem cells with patients with familial ALS. Patient-specific iPS cells were obtained by both integration and transgene-free delivery methods of reprogramming transcription factors. These iPS cells have the properties of pluripotent cells and are capable of direct differentiation into motor neurons. PMID:24772327

  8. Patient-Specific Induced Pluripotent Stem Cells for SOD1-Associated Amyotrophic Lateral Sclerosis Pathogenesis Studies.

    PubMed

    Chestkov, I V; Vasilieva, E A; Illarioshkin, S N; Lagarkova, M A; Kiselev, S L

    2014-01-01

    The genetic reprogramming technology allows one to generate pluripotent stem cells for individual patients. These cells, called induced pluripotent stem cells (iPSCs), can be an unlimited source of specialized cell types for the body. Thus, autologous somatic cell replacement therapy becomes possible, as well as the generation of in vitro cell models for studying the mechanisms of disease pathogenesis and drug discovery. Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder that leads to a loss of upper and lower motor neurons. About 10% of cases are genetically inherited, and the most common familial form of ALS is associated with mutations in the SOD1 gene. We used the reprogramming technology to generate induced pluripotent stem cells with patients with familial ALS. Patient-specific iPS cells were obtained by both integration and transgene-free delivery methods of reprogramming transcription factors. These iPS cells have the properties of pluripotent cells and are capable of direct differentiation into motor neurons. PMID:24772327

  9. The pluripotency factor Nanog regulates pericentromeric heterochromatin organization in mouse embryonic stem cells

    PubMed Central

    Novo, Clara Lopes; Tang, Calvin; Ahmed, Kashif; Djuric, Ugljesa; Fussner, Eden; Mullin, Nicholas P.; Morgan, Natasha P.; Hayre, Jasvinder; Sienerth, Arnold R.; Elderkin, Sarah; Nishinakamura, Ryuichi; Chambers, Ian; Ellis, James; Bazett-Jones, David P.; Rugg-Gunn, Peter J.

    2016-01-01

    An open and decondensed chromatin organization is a defining property of pluripotency. Several epigenetic regulators have been implicated in maintaining an open chromatin organization, but how these processes are connected to the pluripotency network is unknown. Here, we identified a new role for the transcription factor NANOG as a key regulator connecting the pluripotency network with constitutive heterochromatin organization in mouse embryonic stem cells. Deletion of Nanog leads to chromatin compaction and the remodeling of heterochromatin domains. Forced expression of NANOG in epiblast stem cells is sufficient to decompact chromatin. NANOG associates with satellite repeats within heterochromatin domains, contributing to an architecture characterized by highly dispersed chromatin fibers, low levels of H3K9me3, and high major satellite transcription, and the strong transactivation domain of NANOG is required for this organization. The heterochromatin-associated protein SALL1 is a direct cofactor for NANOG, and loss of Sall1 recapitulates the Nanog-null phenotype, but the loss of Sall1 can be circumvented through direct recruitment of the NANOG transactivation domain to major satellites. These results establish a direct connection between the pluripotency network and chromatin organization and emphasize that maintaining an open heterochromatin architecture is a highly regulated process in embryonic stem cells. PMID:27125671

  10. Human Pluripotent Stem Cell-Derived Cardiomyocytes as Research and Therapeutic Tools

    PubMed Central

    Pesl, Martin; Lacampagne, Alain; Dvorak, Petr; Rotrekl, Vladimir; Meli, Albano C.

    2014-01-01

    Human pluripotent stem cells (hPSCs), namely, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), with their ability of indefinite self-renewal and capability to differentiate into cell types derivatives of all three germ layers, represent a powerful research tool in developmental biology, for drug screening, disease modelling, and potentially cell replacement therapy. Efficient differentiation protocols that would result in the cell type of our interest are needed for maximal exploitation of these cells. In the present work, we aim at focusing on the protocols for differentiation of hPSCs into functional cardiomyocytes in vitro as well as achievements in the heart disease modelling and drug testing on the patient-specific iPSC-derived cardiomyocytes (iPSC-CMs). PMID:24800237

  11. The nucleoporin Nup153 regulates embryonic stem cell pluripotency through gene silencing

    PubMed Central

    Jacinto, Filipe V.; Benner, Chris; Hetzer, Martin W.

    2015-01-01

    Nucleoporins (Nups) are a family of proteins best known as the constituent building blocks of nuclear pore complexes (NPCs), membrane-embedded channels that mediate nuclear transport across the nuclear envelope. Recent evidence suggests that several Nups have additional roles in controlling the activation and silencing of developmental genes; however, the mechanistic details of these functions remain poorly understood. Here, we show that depletion of Nup153 in mouse embryonic stem cells (mESCs) causes the derepression of developmental genes and induction of early differentiation. This loss of stem cell identity is not associated with defects in the nuclear import of key pluripotency factors. Rather, Nup153 binds around the transcriptional start site (TSS) of developmental genes and mediates the recruitment of the polycomb-repressive complex 1 (PRC1) to a subset of its target loci. Our results demonstrate a chromatin-associated role of Nup153 in maintaining stem cell pluripotency by functioning in mammalian epigenetic gene silencing. PMID:26080816

  12. Concise Review: Advances in Generating Hepatocytes from Pluripotent Stem Cells for Translational Medicine

    PubMed Central

    Szkolnicka, Dagmara

    2016-01-01

    Abstract The liver is one of the major organs in the human body. Severe or prolonged exposure of the liver to different factors may cause life‐threatening disease, which necessitates donor organ transplantation. While orthotopic liver transplantation can be used to effectively treat liver failure, it is an invasive procedure, which is severely limited by organ donation. Therefore, alternative sources of liver support have been proposed and studied. This includes the use of pluripotent stem cell‐derived hepatocytes as a renewable source of cells for therapy. In addition to cell‐based therapies, in vitro engineered liver tissue provides powerful models for human drug discovery and disease modeling. This review focuses on the generation of hepatocyte‐like cells from pluripotent stem cells and their application in translational medicine. Stem Cells 2016;34:1421–1426 PMID:27015786

  13. Cell-Penetrating Peptide as a Means of Directing the Differentiation of Induced Pluripotent Stem Cells

    PubMed Central

    Kaitsuka, Taku; Tomizawa, Kazuhito

    2015-01-01

    Protein transduction using cell-penetrating peptides (CPPs) is useful for the delivery of large protein molecules, including some transcription factors. This method is safer than gene transfection methods with a viral vector because there is no risk of genomic integration of the exogenous DNA. Recently, this method was reported as a means for the induction of induced pluripotent stem (iPS) cells, directing the differentiation into specific cell types and supporting gene editing/correction. Furthermore, we developed a direct differentiation method to obtain a pancreatic lineage from mouse and human pluripotent stem cells via the protein transduction of three transcription factors, Pdx1, NeuroD, and MafA. Here, we discuss the possibility of using CPPs as a means of directing the differentiation of iPS cells and other stem cell technologies. PMID:26561805

  14. Long-term maintenance of human induced pluripotent stem cells by automated cell culture system.

    PubMed

    Konagaya, Shuhei; Ando, Takeshi; Yamauchi, Toshiaki; Suemori, Hirofumi; Iwata, Hiroo

    2015-01-01

    Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem (iPS) cells, are regarded as new sources for cell replacement therapy. These cells can unlimitedly expand under undifferentiated conditions and be differentiated into multiple cell types. Automated culture systems enable the large-scale production of cells. In addition to reducing the time and effort of researchers, an automated culture system improves the reproducibility of cell cultures. In the present study, we newly designed a fully automated cell culture system for human iPS maintenance. Using an automated culture system, hiPS cells maintained their undifferentiated state for 60 days. Automatically prepared hiPS cells had a potency of differentiation into three germ layer cells including dopaminergic neurons and pancreatic cells. PMID:26573336

  15. Concise Review: Advances in Generating Hepatocytes from Pluripotent Stem Cells for Translational Medicine.

    PubMed

    Szkolnicka, Dagmara; Hay, David C

    2016-06-01

    The liver is one of the major organs in the human body. Severe or prolonged exposure of the liver to different factors may cause life-threatening disease, which necessitates donor organ transplantation. While orthotopic liver transplantation can be used to effectively treat liver failure, it is an invasive procedure, which is severely limited by organ donation. Therefore, alternative sources of liver support have been proposed and studied. This includes the use of pluripotent stem cell-derived hepatocytes as a renewable source of cells for therapy. In addition to cell-based therapies, in vitro engineered liver tissue provides powerful models for human drug discovery and disease modeling. This review focuses on the generation of hepatocyte-like cells from pluripotent stem cells and their application in translational medicine. Stem Cells 2016;34:1421-1426. PMID:27015786

  16. "The state of the heart": Recent advances in engineering human cardiac tissue from pluripotent stem cells.

    PubMed

    Sirabella, Dario; Cimetta, Elisa; Vunjak-Novakovic, Gordana

    2015-08-01

    The pressing need for effective cell therapy for the heart has led to the investigation of suitable cell sources for tissue replacement. In recent years, human pluripotent stem cell research expanded tremendously, in particular since the derivation of human-induced pluripotent stem cells. In parallel, bioengineering technologies have led to novel approaches for in vitro cell culture. The combination of these two fields holds potential for in vitro generation of high-fidelity heart tissue, both for basic research and for therapeutic applications. However, this new multidisciplinary science is still at an early stage. Many questions need to be answered and improvements need to be made before clinical applications become a reality. Here we discuss the current status of human stem cell differentiation into cardiomyocytes and the combined use of bioengineering approaches for cardiac tissue formation and maturation in developmental studies, disease modeling, drug testing, and regenerative medicine. PMID:26069271

  17. Mammalian genes induce partially reprogrammed pluripotent stem cells in non-mammalian vertebrate and invertebrate species

    PubMed Central

    Rosselló, Ricardo Antonio; Chen, Chun-Chun; Dai, Rui; Howard, Jason T; Hochgeschwender, Ute; Jarvis, Erich D

    2013-01-01

    Cells are fundamental units of life, but little is known about evolution of cell states. Induced pluripotent stem cells (iPSCs) are once differentiated cells that have been re-programmed to an embryonic stem cell-like state, providing a powerful platform for biology and medicine. However, they have been limited to a few mammalian species. Here we found that a set of four mammalian transcription factor genes used to generate iPSCs in mouse and humans can induce a partially reprogrammed pluripotent stem cell (PRPSCs) state in vertebrate and invertebrate model organisms, in mammals, birds, fish, and fly, which span 550 million years from a common ancestor. These findings are one of the first to show cross-lineage stem cell-like induction, and to generate pluripotent-like cells for several of these species with in vivo chimeras. We suggest that the stem-cell state may be highly conserved across a wide phylogenetic range. DOI: http://dx.doi.org/10.7554/eLife.00036.001 PMID:24015354

  18. HNF4A is essential for specification of hepatic progenitors from human pluripotent stem cells

    PubMed Central

    DeLaForest, Ann; Nagaoka, Masato; Si-Tayeb, Karim; Noto, Fallon K.; Konopka, Genevieve; Battle, Michele A.; Duncan, Stephen A.

    2011-01-01

    The availability of pluripotent stem cells offers the possibility of using such cells to model hepatic disease and development. With this in mind, we previously established a protocol that facilitates the differentiation of both human embryonic stem cells and induced pluripotent stem cells into cells that share many characteristics with hepatocytes. The use of highly defined culture conditions and the avoidance of feeder cells or embryoid bodies allowed synchronous and reproducible differentiation to occur. The differentiation towards a hepatocyte-like fate appeared to recapitulate many of the developmental stages normally associated with the formation of hepatocytes in vivo. In the current study, we addressed the feasibility of using human pluripotent stem cells to probe the molecular mechanisms underlying human hepatocyte differentiation. We demonstrate (1) that human embryonic stem cells express a number of mRNAs that characterize each stage in the differentiation process, (2) that gene expression can be efficiently depleted throughout the differentiation time course using shRNAs expressed from lentiviruses and (3) that the nuclear hormone receptor HNF4A is essential for specification of human hepatic progenitor cells by establishing the expression of the network of transcription factors that controls the onset of hepatocyte cell fate. PMID:21852396

  19. Robust pluripotent stem cell expansion and cardiomyocyte differentiation via geometric patterning.

    PubMed

    Myers, Frank B; Silver, Jason S; Zhuge, Yan; Beygui, Ramin E; Zarins, Christopher K; Lee, Luke P; Abilez, Oscar J

    2013-12-01

    Geometric factors including the size, shape, density, and spacing of pluripotent stem cell colonies play a significant role in the maintenance of pluripotency and in cell fate determination. These factors are impossible to control using standard tissue culture methods. As such, there can be substantial batch-to-batch variability in cell line maintenance and differentiation yield. Here, we demonstrate a simple, robust technique for pluripotent stem cell expansion and cardiomyocyte differentiation by patterning cell colonies with a silicone stencil. We have observed that patterning human induced pluripotent stem cell (hiPSC) colonies improves the uniformity and repeatability of their size, density, and shape. Uniformity of colony geometry leads to improved homogeneity in the expression of pluripotency markers SSEA4 and Nanog as compared with conventional clump passaging. Patterned cell colonies are capable of undergoing directed differentiation into spontaneously beating cardiomyocyte clusters with improved yield and repeatability over unpatterned cultures seeded either as cell clumps or uniform single cell suspensions. Circular patterns result in a highly repeatable 3D ring-shaped band of cardiomyocytes which electrically couple and lead to propagating contraction waves around the ring. Because of these advantages, geometrically patterning stem cells using stencils may offer greater repeatability from batch-to-batch and person-to-person, an increase in differentiation yield, a faster experimental workflow, and a simpler protocol to communicate and follow. Furthermore, the ability to control where cardiomyocytes arise across a culture well during differentiation could greatly aid the design of electrophysiological assays for drug-screening. PMID:24141327

  20. Robust Pluripotent Stem Cell Expansion and Cardiomyocyte Differentiation via Geometric Patterning

    PubMed Central

    Myers, Frank B.; Silver, Jason S.; Zhuge, Yan; Beygui, Ramin E.; Zarins, Christopher K.; Lee, Luke P.; Abilez, Oscar J.

    2013-01-01

    Geometric factors including the size, shape, density, and spacing of pluripotent stem cell colonies play a significant role in the maintenance of pluripotency and in cell fate determination. These factors are impossible to control using standard tissue culture methods. As such, there can be substantial batch-to-batch variability in cell line maintenance and differentiation yield. Here, we demonstrate a simple, robust technique for pluripotent stem cell expansion and cardiomyocyte differentiation by patterning cell colonies with a silicone stencil. We have observed that patterning human induced pluripotent stem cell (hiPSC) colonies improves the uniformity and repeatability of their size, density, and shape. Uniformity of colony geometry leads to improved homogeneity in the expression of pluripotency markers SSEA4 and Nanog as compared with conventional clump passaging. Patterned cell colonies are capable of undergoing directed differentiation into spontaneously beating cardiomyocyte clusters with improved yield and repeatability over unpatterned cultures seeded either as cell clumps or uniform single cell suspensions. Circular patterns result in a highly repeatable 3D ring-shaped band of cardiomyocytes which electrically couple and lead to propagating contraction waves around the ring. Because of these advantages, geometrically patterning stem cells using stencils may offer greater repeatability from batch-to-batch and person-to-person, an increase in differentiation yield, a faster experimental workflow, and a simpler protocol to communicate and follow. Furthermore, the ability to control where cardiomyocytes arise across a culture well during differentiation could greatly aid the design of electrophysiological assays for drug-screening. PMID:24141327

  1. Inhibition of pluripotent stem cell-derived teratoma formation by small molecules.

    PubMed

    Lee, Mi-Ok; Moon, Sung Hwan; Jeong, Ho-Chang; Yi, Ji-Yeon; Lee, Tae-Hee; Shim, Sung Han; Rhee, Yong-Hee; Lee, Sang-Hun; Oh, Seok-Jeong; Lee, Moo-Yeol; Han, Min-Joon; Cho, Yee Sook; Chung, Hyung-Min; Kim, Kwang-Soo; Cha, Hyuk-Jin

    2013-08-27

    The future of safe cell-based therapy rests on overcoming teratoma/tumor formation, in particular when using human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Because the presence of a few remaining undifferentiated hPSCs can cause undesirable teratomas after transplantation, complete removal of these cells with no/minimal damage to differentiated cells is a prerequisite for clinical application of hPSC-based therapy. Having identified a unique hESC signature of pro- and antiapoptotic gene expression profile, we hypothesized that targeting hPSC-specific antiapoptotic factor(s) (i.e., survivin or Bcl10) represents an efficient strategy to selectively eliminate pluripotent cells with teratoma potential. Here we report the successful identification of small molecules that can effectively inhibit these antiapoptotic factors, leading to selective and efficient removal of pluripotent stem cells through apoptotic cell death. In particular, a single treatment of hESC-derived mixed population with chemical inhibitors of survivin (e.g., quercetin or YM155) induced selective and complete cell death of undifferentiated hPSCs. In contrast, differentiated cell types (e.g., dopamine neurons and smooth-muscle cells) derived from hPSCs survived well and maintained their functionality. We found that quercetin-induced selective cell death is caused by mitochondrial accumulation of p53 and is sufficient to prevent teratoma formation after transplantation of hESC- or hiPSC-derived cells. Taken together, these results provide the "proof of concept" that small-molecule targeting of hPSC-specific antiapoptotic pathway(s) is a viable strategy to prevent tumor formation by selectively eliminating remaining undifferentiated pluripotent cells for safe hPSC-based therapy. PMID:23918355

  2. Defining an optimal surface chemistry for pluripotent stem cell culture in 2D and 3D

    NASA Astrophysics Data System (ADS)

    Zonca, Michael R., Jr.

    Surface chemistry is critical for growing pluripotent stem cells in an undifferentiated state. There is great potential to engineer the surface chemistry at the nanoscale level to regulate stem cell adhesion. However, the challenge is to identify the optimal surface chemistry of the substrata for ES cell attachment and maintenance. Using a high-throughput polymerization and screening platform, a chemically defined, synthetic polymer grafted coating that supports strong attachment and high expansion capacity of pluripotent stem cells has been discovered using mouse embryonic stem (ES) cells as a model system. This optimal substrate, N-[3-(Dimethylamino)propyl] methacrylamide (DMAPMA) that is grafted on 2D synthetic poly(ether sulfone) (PES) membrane, sustains the self-renewal of ES cells (up to 7 passages). DMAPMA supports cell attachment of ES cells through integrin beta1 in a RGD-independent manner and is similar to another recently reported polymer surface. Next, DMAPMA has been able to be transferred to 3D by grafting to synthetic, polymeric, PES fibrous matrices through both photo-induced and plasma-induced polymerization. These 3D modified fibers exhibited higher cell proliferation and greater expression of pluripotency markers of mouse ES cells than 2D PES membranes. Our results indicated that desirable surfaces in 2D can be scaled to 3D and that both surface chemistry and structural dimension strongly influence the growth and differentiation of pluripotent stem cells. Lastly, the feasibility of incorporating DMAPMA into a widely used natural polymer, alginate, has been tested. Novel adhesive alginate hydrogels have been successfully synthesized by either direct polymerization of DMAPMA and methacrylic acid blended with alginate, or photo-induced DMAPMA polymerization on alginate nanofibrous hydrogels. In particular, DMAPMA-coated alginate hydrogels support strong ES cell attachment, exhibiting a concentration dependency of DMAPMA. This research provides a

  3. Teratoma Formation: A Tool for Monitoring Pluripotency in Stem Cell Research

    PubMed Central

    Nelakanti, Raman V.; Kooreman, Nigel G.; Wu, Joseph C.

    2015-01-01

    This unit describes protocols for evaluating the pluripotency of embryonic and induced pluripotent stem cells by a teratoma formation assay. Cells are prepared for injection and transplanted into immunodeficient mice at the gastrocnemius muscle, a site well suited for teratoma growth and surgical access. Teratomas that form from the cell transplants are explanted, fixed in paraformaldehyde, and embedded in paraffin. These preserved samples are sectioned, stained, and analyzed. Pluripotency of a cell line is confirmed by whether the teratoma contains tissues derived from each of the embryonic germ layers: endoderm, mesoderm, and ectoderm. Alternatively, explanted and fixed teratomas can be cryopreserved for immunohistochemistry. Antibody staining can allow for a more detailed identification of specific tissue types present in teratoma samples. PMID:25640819

  4. Epigenetic regulation in pluripotent stem cells: a key to breaking the epigenetic barrier.

    PubMed

    Watanabe, Akira; Yamada, Yasuhiro; Yamanaka, Shinya

    2013-01-01

    The differentiation and reprogramming of cells are accompanied by drastic changes in the epigenetic profiles of cells. Waddington's classical model clearly describes how differentiating cells acquire their cell identity as the developmental potential of an individual cell population declines towards the terminally differentiated state. The recent discovery of induced pluripotent stem cells as well as of somatic cell nuclear transfer provided evidence that the process of differentiation can be reversed. The identity of somatic cells is strictly protected by an epigenetic barrier, and these cells acquire pluripotency by breaking the epigenetic barrier by reprogramming factors such as Oct3/4, Sox2, Klf4, Myc and LIN28. This review covers the current understanding of the spatio-temporal regulation of epigenetics in pluripotent and differentiated cells, and discusses how cells determine their identity and overcome the epigenetic barrier during the reprogramming process. PMID:23166402

  5. Design and formulation of functional pluripotent stem cell-derived cardiac microtissues

    PubMed Central

    Thavandiran, Nimalan; Dubois, Nicole; Mikryukov, Alexander; Massé, Stéphane; Beca, Bogdan; Simmons, Craig A.; Deshpande, Vikram S.; McGarry, J. Patrick; Chen, Christopher S.; Nanthakumar, Kumaraswamy; Keller, Gordon M.; Radisic, Milica; Zandstra, Peter W.

    2013-01-01

    Access to robust and information-rich human cardiac tissue models would accelerate drug-based strategies for treating heart disease. Despite significant effort, the generation of high-fidelity adult-like human cardiac tissue analogs remains challenging. We used computational modeling of tissue contraction and assembly mechanics in conjunction with microfabricated constraints to guide the design of aligned and functional 3D human pluripotent stem cell (hPSC)-derived cardiac microtissues that we term cardiac microwires (CMWs). Miniaturization of the platform circumvented the need for tissue vascularization and enabled higher-throughput image-based analysis of CMW drug responsiveness. CMW tissue properties could be tuned using electromechanical stimuli and cell composition. Specifically, controlling self-assembly of 3D tissues in aligned collagen, and pacing with point stimulation electrodes, were found to promote cardiac maturation-associated gene expression and in vivo-like electrical signal propagation. Furthermore, screening a range of hPSC-derived cardiac cell ratios identified that 75% NKX2 Homeobox 5 (NKX2-5)+ cardiomyocytes and 25% Cluster of Differentiation 90 OR (CD90)+ nonmyocytes optimized tissue remodeling dynamics and yielded enhanced structural and functional properties. Finally, we demonstrate the utility of the optimized platform in a tachycardic model of arrhythmogenesis, an aspect of cardiac electrophysiology not previously recapitulated in 3D in vitro hPSC-derived cardiac microtissue models. The design criteria identified with our CMW platform should accelerate the development of predictive in vitro assays of human heart tissue function. PMID:24255110

  6. Functional Neurons Generated from T Cell-Derived Induced Pluripotent Stem Cells for Neurological Disease Modeling.

    PubMed

    Matsumoto, Takuya; Fujimori, Koki; Andoh-Noda, Tomoko; Ando, Takayuki; Kuzumaki, Naoko; Toyoshima, Manabu; Tada, Hirobumi; Imaizumi, Kent; Ishikawa, Mitsuru; Yamaguchi, Ryo; Isoda, Miho; Zhou, Zhi; Sato, Shigeto; Kobayashi, Tetsuro; Ohtaka, Manami; Nishimura, Ken; Kurosawa, Hiroshi; Yoshikawa, Takeo; Takahashi, Takuya; Nakanishi, Mahito; Ohyama, Manabu; Hattori, Nobutaka; Akamatsu, Wado; Okano, Hideyuki

    2016-03-01

    Modeling of neurological diseases using induced pluripotent stem cells (iPSCs) derived from the somatic cells of patients has provided a means of elucidating pathogenic mechanisms and performing drug screening. T cells are an ideal source of patient-specific iPSCs because they can be easily obtained from samples. Recent studies indicated that iPSCs retain an epigenetic memory relating to their cell of origin that restricts their differentiation potential. The classical method of differentiation via embryoid body formation was not suitable for T cell-derived iPSCs (TiPSCs). We developed a neurosphere-based robust differentiation protocol, which enabled TiPSCs to differentiate into functional neurons, despite differences in global gene expression between TiPSCs and adult human dermal fibroblast-derived iPSCs. Furthermore, neurons derived from TiPSCs generated from a juvenile patient with Parkinson's disease exhibited several Parkinson's disease phenotypes. Therefore, we conclude that TiPSCs are a useful tool for modeling neurological diseases. PMID:26905201

  7. Using human induced pluripotent stem cells to model cerebellar disease: Hope and hype

    PubMed Central

    Wiethoff, Sarah; Arber, Charles; Li, Abi; Wray, Selina; Houlden, Henry; Patani, Rickie

    2015-01-01

    The cerebellum forms a highly ordered and indispensible component of motor function within the adult neuraxis, consisting of several distinct cellular subtypes. Cerebellar disease, through a variety of genetic and acquired causes, results in the loss of function of defined subclasses of neurons, and remains a significant and untreatable health care burden. The scarcity of therapies in this arena can partially be explained by unresolved disease mechanisms due to inaccessibility of human cerebellar neurons in a relevant experimental context where initiating disease mechanisms could be functionally elucidated, or drug screens conducted. In this review we discuss the potential promise of human induced pluripotent stem cells (hiPSCs) for regenerative neurology, with a particular emphasis on in vitro modelling of cerebellar degeneration. We discuss progress made thus far using hiPSC-based models of neurodegeneration, noting the relatively slower pace of discovery made in modelling cerebellar dysfunction. We conclude by speculating how strategies attempting cerebellar differentiation from hiPSCs can be refined to allow the generation of accurate disease models. This in turn will permit a greater understanding of cerebellar pathophysiology to inform mechanistically rationalised therapies, which are desperately needed in this field. PMID:25985846

  8. Functional Neurons Generated from T Cell-Derived Induced Pluripotent Stem Cells for Neurological Disease Modeling

    PubMed Central

    Matsumoto, Takuya; Fujimori, Koki; Andoh-Noda, Tomoko; Ando, Takayuki; Kuzumaki, Naoko; Toyoshima, Manabu; Tada, Hirobumi; Imaizumi, Kent; Ishikawa, Mitsuru; Yamaguchi, Ryo; Isoda, Miho; Zhou, Zhi; Sato, Shigeto; Kobayashi, Tetsuro; Ohtaka, Manami; Nishimura, Ken; Kurosawa, Hiroshi; Yoshikawa, Takeo; Takahashi, Takuya; Nakanishi, Mahito; Ohyama, Manabu; Hattori, Nobutaka; Akamatsu, Wado; Okano, Hideyuki

    2016-01-01

    Summary Modeling of neurological diseases using induced pluripotent stem cells (iPSCs) derived from the somatic cells of patients has provided a means of elucidating pathogenic mechanisms and performing drug screening. T cells are an ideal source of patient-specific iPSCs because they can be easily obtained from samples. Recent studies indicated that iPSCs retain an epigenetic memory relating to their cell of origin that restricts their differentiation potential. The classical method of differentiation via embryoid body formation was not suitable for T cell-derived iPSCs (TiPSCs). We developed a neurosphere-based robust differentiation protocol, which enabled TiPSCs to differentiate into functional neurons, despite differences in global gene expression between TiPSCs and adult human dermal fibroblast-derived iPSCs. Furthermore, neurons derived from TiPSCs generated from a juvenile patient with Parkinson's disease exhibited several Parkinson's disease phenotypes. Therefore, we conclude that TiPSCs are a useful tool for modeling neurological diseases. PMID:26905201

  9. Cardiomyocytes from human pluripotent stem cells: From laboratory curiosity to industrial biomedical platform.

    PubMed

    Denning, Chris; Borgdorff, Viola; Crutchley, James; Firth, Karl S A; George, Vinoj; Kalra, Spandan; Kondrashov, Alexander; Hoang, Minh Duc; Mosqueira, Diogo; Patel, Asha; Prodanov, Ljupcho; Rajamohan, Divya; Skarnes, William C; Smith, James G W; Young, Lorraine E

    2016-07-01

    Cardiomyocytes from human pluripotent stem cells (hPSCs-CMs) could revolutionise biomedicine. Global burden of heart failure will soon reach USD $90bn, while unexpected cardiotoxicity underlies 28% of drug withdrawals. Advances in hPSC isolation, Cas9/CRISPR genome engineering and hPSC-CM differentiation have improved patient care, progressed drugs to clinic and opened a new era in safety pharmacology. Nevertheless, predictive cardiotoxicity using hPSC-CMs contrasts from failure to almost total success. Since this likely relates to cell immaturity, efforts are underway to use biochemical and biophysical cues to improve many of the ~30 structural and functional properties of hPSC-CMs towards those seen in adult CMs. Other developments needed for widespread hPSC-CM utility include subtype specification, cost reduction of large scale differentiation and elimination of the phenotyping bottleneck. This review will consider these factors in the evolution of hPSC-CM technologies, as well as their integration into high content industrial platforms that assess structure, mitochondrial function, electrophysiology, calcium transients and contractility. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel. PMID:26524115

  10. Robust derivation of epicardium and its differentiated smooth muscle cell progeny from human pluripotent stem cells.

    PubMed

    Iyer, Dharini; Gambardella, Laure; Bernard, William G; Serrano, Felipe; Mascetti, Victoria L; Pedersen, Roger A; Talasila, Amarnath; Sinha, Sanjay

    2015-04-15

    The epicardium has emerged as a multipotent cardiovascular progenitor source with therapeutic potential for coronary smooth muscle cell, cardiac fibroblast (CF) and cardiomyocyte regeneration, owing to its fundamental role in heart development and its potential ability to initiate myocardial repair in injured adult tissues. Here, we describe a chemically defined method for generating epicardium and epicardium-derived smooth muscle cells (EPI-SMCs) and CFs from human pluripotent stem cells (HPSCs) through an intermediate lateral plate mesoderm (LM) stage. HPSCs were initially differentiated to LM in the presence of FGF2 and high levels of BMP4. The LM was robustly differentiated to an epicardial lineage by activation of WNT, BMP and retinoic acid signalling pathways. HPSC-derived epicardium displayed enhanced expression of epithelial- and epicardium-specific markers, exhibited morphological features comparable with human foetal epicardial explants and engrafted in the subepicardial space in vivo. The in vitro-derived epicardial cells underwent an epithelial-to-mesenchymal transition when treated with PDGF-BB and TGFβ1, resulting in vascular SMCs that displayed contractile ability in response to vasoconstrictors. Furthermore, the EPI-SMCs displayed low density lipoprotein uptake and effective lowering of lipoprotein levels upon treatment with statins, similar to primary human coronary artery SMCs. Cumulatively, these findings suggest that HPSC-derived epicardium and EPI-SMCs could serve as important tools for studying human cardiogenesis, and as a platform for vascular disease modelling and drug screening. PMID:25813541

  11. Expansion of Multipotent Stem Cells from the Adult Human Brain

    PubMed Central

    Murrell, Wayne; Palmero, Emily; Bianco, John; Stangeland, Biljana; Joel, Mrinal; Paulson, Linda; Thiede, Bernd; Grieg, Zanina; Ramsnes, Ingunn; Skjellegrind, Håvard K.; Nygård, Ståle; Brandal, Petter; Sandberg, Cecilie; Vik-Mo, Einar; Palmero, Sheryl; Langmoen, Iver A.

    2013-01-01

    The discovery of stem cells in the adult human brain has revealed new possible scenarios for treatment of the sick or injured brain. Both clinical use of and preclinical research on human adult neural stem cells have, however, been seriously hampered by the fact that it has been impossible to passage these cells more than a very few times and with little expansion of cell numbers. Having explored a number of alternative culturing conditions we here present an efficient method for the establishment and propagation of human brain stem cells from whatever brain tissue samples we have tried. We describe virtually unlimited expansion of an authentic stem cell phenotype. Pluripotency proteins Sox2 and Oct4 are expressed without artificial induction. For the first time multipotency of adult human brain-derived stem cells is demonstrated beyond tissue boundaries. We characterize these cells in detail in vitro including microarray and proteomic approaches. Whilst clarification of these cells’ behavior is ongoing, results so far portend well for the future repair of tissues by transplantation of an adult patient’s own-derived stem cells. PMID:23967194

  12. Study of Pluripotency Markers in Zebrafish Embryos and Transient Embryonic Stem Cell Cultures

    PubMed Central

    Robles, Vanesa; Martí, Mercé

    2011-01-01

    Abstract Targeted genomic manipulation using embryonic stem (ES) cells has not yet been achieved in zebrafish, although methods for zebrafish ES cell culture has been described in literature. The knowledge of pluripotency markers in this species is almost nonexistent and this is a very limiting factor in the definition of the ideal culture conditions for ES cells. Here, we studied the expression of several genes associated with pluripotency in zebrafish embryonic cells versus differentiated cells and the expression of some of these genes is recorded throughout embryonic development. Some of the commonly accepted pluripotency markers are also tested in embryonic cells, transient embryonic cell cultures, and differentiated cells. Our results support the hypothesis that stage-specific embryonic antigen 1 (SSEA1) is a marker that precedes the expression of pluripotency genes in a zebrafish embryonic cell colony, in the same way that SOX2 precedes nestin expression in those colonies that have already started differentiation toward neurons. We consider this study a step forward in the knowledge of zebrafish pluripotency markers and, therefore, an important tool for the monitoring of zebrafish embryonic cell cultures. PMID:21563922

  13. Transcriptomic profiling of human embryonic stem cells upon cell cycle manipulation during pluripotent state dissolution

    PubMed Central

    Gonzales, Kevin Andrew Uy; Liang, Hongqing

    2015-01-01

    While distinct cell cycle structures have been known to correlate with pluripotent or differentiated cell states [1], there is no evidence on how the cell cycle machinery directly contributes to human embryonic stem cell (hESC) pluripotency. We established a determinant role of cell cycle machineries on the pluripotent state by demonstrating that the specific perturbation of the S and G2 phases can prevent pluripotent state dissolution (PSD) [2]. Active mechanisms in these phases, such as the DNA damage checkpoint and Cyclin B1, promote the pluripotent state [2]. To understand the mechanisms behind the effect on PSD by these pathways in hESCs, we performed comprehensive gene expression analysis by time-course microarray experiments. From these datasets, we observed expression changes in genes involved in the TGFβ signaling pathway, which has a well-established role in hESC maintenance [3], [4], [5]. The microarray data have been deposited in NCBI's Gene Expression Omnibus (GEO) and can be accessed through GEO Series accession numbers GSE62062 and GSE63215. PMID:26697349

  14. Human induced pluripotent stem cell lines show stress defense mechanisms and mitochondrial regulation similar to those of human embryonic stem cells.

    PubMed

    Armstrong, Lyle; Tilgner, Katarzyna; Saretzki, Gabriele; Atkinson, Stuart P; Stojkovic, Miodrag; Moreno, Ruben; Przyborski, Stefan; Lako, Majlinda

    2010-04-01

    The generation of induced pluripotent stem cells (iPSC) has enormous potential for the development of patient-specific regenerative medicine. Human embryonic stem cells (hESC) are able to defend their genomic integrity by maintaining low levels of reactive oxygen species (ROS) through a combination of enhanced removal capacity and limited production of these molecules. Such limited ROS production stems partly from the small number of mitochondria present in hESC; thus, it was important to determine that human iPSC (hiPSC) generation is able to eliminate the extra mitochondria present in the parental fibroblasts (reminiscent of "bottleneck" situation after fertilization) and to show that hiPSC have antioxidant defenses similar to hESC. We were able to generate seven hiPSC lines from adult human dermal fibroblasts and have fully characterized two of those clones. Both hiPSC clones express pluripotency markers and are able to differentiate in vitro into cells belonging to all three germ layers. One of these clones is able to produce fully differentiated teratoma, whereas the other hiPSC clone is unable to silence the viral expression of OCT4 and c-MYC, produce fully differentiated teratoma, and unable to downregulate the expression of some of the pluripotency genes during the differentiation process. In spite of these differences, both clones show ROS stress defense mechanisms and mitochondrial biogenesis similar to hESC. Together our data suggest that, during the reprogramming process, certain cellular mechanisms are in place to ensure that hiPSC are provided with the same defense mechanisms against accumulation of ROS as the hESC. PMID:20073085

  15. Differentiation patterns of mouse embryonic stem cells and induced pluripotent stem cells into neurons.

    PubMed

    Nakamura, Mai; Kamishibahara, Yu; Kitazawa, Ayako; Kawaguchi, Hideo; Shimizu, Norio

    2016-05-01

    Mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have the ability to differentiate in vitro into various cell lineages including neurons. The differentiation of these cells into neurons has potential applications in regenerative medicine. Previously, we reported that a chick dorsal root ganglion (DRG)-conditioned medium (CM) promoted the differentiation of mouse ES and iPS cells into neurons. Here, we used real-time PCR to investigate the differentiation patterns of ES and iPS cells into neurons when DRG-CM was added. DRG-CM promoted the expression levels of βIII-tubulin gene (a marker of postmitotic neurons) in ES and iPS cells. ES cells differentiated into neurons faster than iPS cells, and the maximum peaks of gene expression involved in motor, sensory, and dopaminergic neurons were different. Rho kinase (ROCK) inhibitors could be very valuable at numerous stages in the production and use of stem cells in basic research and eventual cell-based therapies. Thus, we investigated whether the addition of a ROCK inhibitor Y-27632 and DRG-CM on the basis of the differentiation patterns promotes the neuronal differentiation of ES cells. When the ROCK inhibitor was added to the culture medium at the initial stages of cultivation, it stimulated the neuronal differentiation of ES cells more strongly than that stimulated by DRG-CM. Moreover, the combination of the ROCK inhibitor and DRG-CM promoted the neuronal differentiation of ES cells when the ROCK inhibitor was added to the culture medium at day 3. The ROCK inhibitor may be useful for promoting neuronal differentiation of ES cells. PMID:25354731

  16. Association of expression levels of pluripotency/stem cell markers with the differentiation outcome of Wharton's jelly mesenchymal stem cells into insulin producing cells.

    PubMed

    Kassem, Dina H; Kamal, Mohamed M; El-Kholy, Abd El-Latif G; El-Mesallamy, Hala O

    2016-08-01

    Recently, there has been much attention towards generation of insulin producing cells (IPCs) from stem cells, especially from Wharton's jelly mesenchymal stem cells (WJ-MSCs). However, generation of mature IPCs remains a challenge. Assessment of generation of IPCs was usually done by examining β-cell markers, however, assessment of pluripotency/stem cell markers drew less attention. Therefore, the purpose of this study was to investigate the levels of pluripotency/stem cell markers during differentiation of WJ-MSCs into IPCs and the association of these levels with differentiation outcomes. WJ-MSCs were isolated, characterized then induced to differentiate into IPCs using three different protocols namely A, B and C. Differentiated IPCs were assessed by the expression of pluripotency/stem cell markers, together with β-cell markers using qRT-PCR, and functionally by measuring glucose stimulated insulin secretion. Differentiated cells from protocol A showed lowest expression of pluripotency/stem cell markers and relatively best GSIS. However, protocol B showed concomitant expression of pluripotency/stem cell and β-cell markers with relatively less insulin secretion as compared to protocol A. Protocol C failed to generate glucose-responsive IPCs. In conclusion, sustained expression of pluripotency/stem cell markers could be associated with the incomplete differentiation of WJ-MSCs into IPCs. A novel finding for which further investigations are warranted. PMID:27265786

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

    PubMed Central

    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.

    2016-01-01

    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. PMID:26743714

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

    PubMed

    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

    2016-01-01

    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. PMID:26743714

  19. A Cell Surfaceome Map for Immunophenotyping and Sorting Pluripotent Stem Cells*

    PubMed Central

    Gundry, Rebekah L.; Riordon, Daniel R.; Tarasova, Yelena; Chuppa, Sandra; Bhattacharya, Subarna; Juhasz, Ondrej; Wiedemeier, Olena; Milanovich, Samuel; Noto, Fallon K.; Tchernyshyov, Irina; Raginski, Kimberly; Bausch-Fluck, Damaris; Tae, Hyun-Jin; Marshall, Shannon; Duncan, Stephen A.; Wollscheid, Bernd; Wersto, Robert P.; Rao, Sridhar; Van Eyk, Jennifer E.; Boheler, Kenneth R.

    2012-01-01

    Induction of a pluripotent state in somatic cells through nuclear reprogramming has ushered in a new era of regenerative medicine. Heterogeneity and varied differentiation potentials among induced pluripotent stem cell (iPSC) lines are, however, complicating factors that limit their usefulness for disease modeling, drug discovery, and patient therapies. Thus, there is an urgent need to develop nonmutagenic rapid throughput methods capable of distinguishing among putative iPSC lines of variable quality. To address this issue, we have applied a highly specific chemoproteomic targeting strategy for de novo discovery of cell surface N-glycoproteins to increase the knowledge-base of surface exposed proteins and accessible epitopes of pluripotent stem cells. We report the identification of 500 cell surface proteins on four embryonic stem cell and iPSCs lines and demonstrate the biological significance of this resource on mouse fibroblasts containing an oct4-GFP expression cassette that is active in reprogrammed cells. These results together with immunophenotyping, cell sorting, and functional analyses demonstrate that these newly identified surface marker panels are useful for isolating iPSCs from heterogeneous reprogrammed cultures and for isolating functionally distinct stem cell subpopulations. PMID:22493178

  20. Site-specific recombinase strategy to create induced pluripotent stem cells efficiently with plasmid DNA.

    PubMed

    Karow, Marisa; Chavez, Christopher L; Farruggio, Alfonso P; Geisinger, Jonathan M; Keravala, Annahita; Jung, W Edward; Lan, Feng; Wu, Joseph C; Chen-Tsai, Yanru; Calos, Michele P

    2011-11-01

    Induced pluripotent stem cells (iPSCs) have revolutionized the stem cell field. iPSCs are most often produced by using retroviruses. However, the resulting cells may be ill-suited for clinical applications. Many alternative strategies to make iPSCs have been developed, but the nonintegrating strategies tend to be inefficient, while the integrating strategies involve random integration. Here, we report a facile strategy to create murine iPSCs that uses plasmid DNA and single transfection with sequence-specific recombinases. PhiC31 integrase was used to insert the reprogramming cassette into the genome, producing iPSCs. Cre recombinase was then used for excision of the reprogramming genes. The iPSCs were demonstrated to be pluripotent by in vitro and in vivo criteria, both before and after excision of the reprogramming cassette. This strategy is comparable with retroviral approaches in efficiency, but is nonhazardous for the user, simple to perform, and results in nonrandom integration of a reprogramming cassette that can be readily deleted. We demonstrated the efficiency of this reprogramming and excision strategy in two accessible cell types, fibroblasts and adipose stem cells. This simple strategy produces pluripotent stem cells that have the potential to be used in a clinical setting. PMID:21898697

  1. Letter to the Editor: Human Pluripotent Stem Cells Release Oncogenic Soluble E-Cadherin.

    PubMed

    Rosner, Margit; Hengstschläger, Markus

    2016-09-01

    Since their discovery, human pluripotent stem cells (hPSCs) including embryonic and induced pluripotent stem cells hold great promise in disease modeling and regenerative medicine. Despite intensive research and remarkable progress, it is becoming increasingly acknowledged that their yet incomplete, biological characterisation represents one of the major drawbacks to their successful translation into the clinics. The expression of the transmembrane protein E-cadherin in hPSCs is well defined to be pivotal to the maintenance of the pluripotent state by mediating intercellular adhesion and intracellular signaling. Next to these canonical functions, were here report for the first time that hPSCs are subject to matrix metalloproteinase-dependent E-cadherin ectodomain shedding. This generates a ∼80-kD, soluble E-cadherin fragment which is released into the extracellular space, and which is well described to exert paracrine signaling activity and classified as being oncogenic. Collectively, this finding does not only improve our knowledge on the signaling crosstalk between hPSCs and their cellular environment and the type and nature of the paracrine signals produced by these cells, but also has clear implications for the development of efficient and safe stem cell-based therapies. Stem Cells 2016;34:2443-2446. PMID:27399873

  2. Full biological characterization of human pluripotent stem cells will open the door to translational research.

    PubMed

    Kramer, Nina; Rosner, Margit; Kovacic, Boris; Hengstschläger, Markus

    2016-09-01

    Since the discovery of human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC), great hopes were held for their therapeutic application including disease modeling, drug discovery screenings, toxicological screenings and regenerative therapy. hESC and hiPSC have the advantage of indefinite self-renewal, thereby generating an inexhaustible pool of cells with, e.g., specific genotype for developing putative treatments; they can differentiate into derivatives of all three germ layers enabling autologous transplantation, and via donor-selection they can express various genotypes of interest for better disease modeling. Furthermore, drug screenings and toxicological screenings in hESC and hiPSC are more pertinent to identify drugs or chemical compounds that are harmful for human, than a mouse model could predict. Despite continuing research in the wide field of therapeutic applications, further understanding of the underlying basic mechanisms of stem cell function is necessary. Here, we summarize current knowledge concerning pluripotency, self-renewal, apoptosis, motility, epithelial-to-mesenchymal transition and differentiation of pluripotent stem cells. PMID:27325309

  3. Cardiac tumorgenic potential of induced pluripotent stem cells in an immunocompetent host with myocardial infarction

    PubMed Central

    Ahmed, Rafeeq PH; Ashraf, Muhammad; Buccini, Stephanie; Shujia, Jiang; Haider, Husnain Kh

    2011-01-01

    Aim Genetic reprogramming of somatic cells with stemness genes to restore their pluripotent status is being studied extensively to generate pluripotent stem cells as an alternative to embryonic stem cells. This study was designed to examine the effectiveness of skeletal myoblast-derived induced pluripotent stem cells (SkiPS) from young male Oct4/GFP transgenic mice for regeneration of the infarcted heart. Methods & results A mouse model of permanent coronary artery ligation was developed in young female immunocompetent C57BL/6J or C57BL/6x129S4 SV/jae Oct4/GFP mice. SkiPS labeled with Q-dots (3 × 105 in 10 μl basal Dulbecco’s modified Eagle’s medium) were transplanted in and around the area of infarct immediately after coronary artery ligation (n = 16) under direct vision. Control mice (n = 12) were injected with the same number of skeletal myoblasts. Histological studies documented successful engraftment of SkiPS in all the surviving animals 4 weeks later. However, six of the 16 SkiPS-transplanted (37.5%) animal hearts showed intramural teratomas, whereas no tumor growth was observed in the control mice. Q-dot-labeled donor cells were also observed at the site of tumors. Histological studies revealed that teratomas were composed of cells from all of the three embryonic germ layers. Ultra-structure studies confirmed the histological findings and showed regions with well-organized myofibrillar structures in the tumors. Conclusion Undifferentiated induced pluripotent stem cells should not be recommended for cardiac transplantation unless screened for specific teratogenic precursors or predifferentiated into cardiac lineage prior to transplantation. PMID:21391851

  4. Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo

    PubMed Central

    Xue, Binghua; Li, Yan; He, Yilong; Wei, Renyue; Sun, Ruizhen; Yin, Zhi; Bou, Gerelchimeg; Liu, Zhonghua

    2016-01-01

    Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC) line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP) positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs) could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs. PMID:26991423

  5. Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo.

    PubMed

    Xue, Binghua; Li, Yan; He, Yilong; Wei, Renyue; Sun, Ruizhen; Yin, Zhi; Bou, Gerelchimeg; Liu, Zhonghua

    2016-01-01

    Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC) line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP) positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs) could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs. PMID:26991423

  6. Comparative Analysis of Mouse-Induced Pluripotent Stem Cells and Mesenchymal Stem Cells During Osteogenic Differentiation In Vitro

    PubMed Central

    Kayashima, Hiroki; Miura, Jiro; Uraguchi, Shinya; Wang, Fangfang; Okawa, Hiroko; Sasaki, Jun-Ichi; Saeki, Makio; Matsumoto, Takuya; Yatani, Hirofumi

    2014-01-01

    Induced pluripotent stem cells (iPSCs) can differentiate into mineralizing cells and are, therefore, expected to be useful for bone regenerative medicine; however, the characteristics of iPSC-derived osteogenic cells remain unclear. Here, we provide a direct in vitro comparison of the osteogenic differentiation process in mesenchymal stem cells (MSCs) and iPSCs from adult C57BL/6J mice. After 30 days of culture in osteogenic medium, both MSCs and iPSCs produced robustly mineralized bone nodules that contained abundant calcium phosphate with hydroxyapatite crystal formation. Mineral deposition was significantly higher in iPSC cultures than in MSC cultures. Scanning electron microscopy revealed budding matrix vesicles in early osteogenic iPSCs; subsequently, the vesicles propagated to exhibit robust mineralization without rich fibrous structures. Early osteogenic MSCs showed deposition of many matrix vesicles in abundant collagen fibrils that became solid mineralized structures. Both cell types demonstrated increased expression of osteogenic marker genes, such as runx2, osterix, dlx5, bone sialoprotein (BSP), and osteocalcin, during osteogenesis; however, real-time reverse transcription–polymerase chain reaction array analysis revealed that osteogenesis-related genes encoding mineralization-associated molecules, bone morphogenetic proteins, and extracellular matrix collagens were differentially expressed between iPSCs and MSCs. These data suggest that iPSCs are capable of differentiation into mature osteoblasts whose associated hydroxyapatite has a crystal structure similar to that of MSC-associated hydroxyapatite; however, the transcriptional differences between iPSCs and MSCs could result in differences in the mineral and matrix environments of the bone nodules. Determining the biological mechanisms underlying cell-specific differences in mineralization during in vitro iPSC osteogenesis may facilitate the development of clinically effective engineered bone. PMID

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

    PubMed Central

    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

  8. Effect of different feeding schedules on the survival and neural differentiation of human embryonic and induced pluripotent stem cells

    PubMed Central

    Jensen, Matthew B.; Jager, Lindsey D.; Cohen, Laura K.; Kwok, Susanna S.; Kwon, Jin M.; Hall, Crystal A.

    2014-01-01

    Neural culture of human pluripotent stem cells is useful for neuroscience research, but the optimal feeding schedule for these in vitro systems is unclear. We evaluated the survival and neural differentiation profiles of human embryonic and induced pluripotent stem cells cultured with medium exchange schedules of five, six, or seven days weekly through two months of differentiation. No significant differences were seen in cell numbers or neural differentiation markers through this culture interval with either human pluripotent cell type. We conclude that there is unlikely to be an advantage of feeding more than five days weekly for this culture system. PMID:25328422

  9. Generation and characterization of integration-free induced pluripotent stem cells from patients with autoimmune disease.

    PubMed

    Son, Mi-Young; Lee, Mi-Ok; Jeon, Hyejin; Seol, Binna; Kim, Jung Hwa; Chang, Jae-Suk; Cho, Yee Sook

    2016-01-01

    Autoimmune diseases (AIDs), a heterogeneous group of immune-mediated disorders, are a major and growing health problem. Although AIDs are currently treated primarily with anti-inflammatory and immunosuppressive drugs, the use of stem cell transplantation in patients with AIDs is becoming increasingly common. However, stem cell transplantation therapy has limitations, including a shortage of available stem cells and immune rejection of cells from nonautologous sources. Induced pluripotent stem cell (iPSC) technology, which allows the generation of patient-specific pluripotent stem cells, could offer an alternative source for clinical applications of stem cell therapies in AID patients. We used nonintegrating oriP/EBNA-1-based episomal vectors to reprogram dermal fibroblasts from patients with AIDs such as ankylosing spondylitis (AS), Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). The pluripotency and multilineage differentiation capacity of each patient-specific iPSC line was validated. The safety of these iPSCs for use in stem cell transplantation is indicated by the fact that all AID-specific iPSCs are integrated transgene free. Finally, all AID-specific iPSCs derived in this study could be differentiated into cells of hematopoietic and mesenchymal lineages in vitro as shown by flow cytometric analysis and induction of terminal differentiation potential. Our results demonstrate the successful generation of integration-free iPSCs from patients with AS, SS and SLE. These findings support the possibility of using iPSC technology in autologous and allogeneic cell replacement therapy for various AIDs, including AS, SS and SLE. PMID:27174201

  10. Generation and characterization of integration-free induced pluripotent stem cells from patients with autoimmune disease

    PubMed Central

    Son, Mi-Young; Lee, Mi-Ok; Jeon, Hyejin; Seol, Binna; Kim, Jung Hwa; Chang, Jae-Suk; Cho, Yee Sook

    2016-01-01

    Autoimmune diseases (AIDs), a heterogeneous group of immune-mediated disorders, are a major and growing health problem. Although AIDs are currently treated primarily with anti-inflammatory and immunosuppressive drugs, the use of stem cell transplantation in patients with AIDs is becoming increasingly common. However, stem cell transplantation therapy has limitations, including a shortage of available stem cells and immune rejection of cells from nonautologous sources. Induced pluripotent stem cell (iPSC) technology, which allows the generation of patient-specific pluripotent stem cells, could offer an alternative source for clinical applications of stem cell therapies in AID patients. We used nonintegrating oriP/EBNA-1-based episomal vectors to reprogram dermal fibroblasts from patients with AIDs such as ankylosing spondylitis (AS), Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). The pluripotency and multilineage differentiation capacity of each patient-specific iPSC line was validated. The safety of these iPSCs for use in stem cell transplantation is indicated by the fact that all AID-specific iPSCs are integrated transgene free. Finally, all AID-specific iPSCs derived in this study could be differentiated into cells of hematopoietic and mesenchymal lineages in vitro as shown by flow cytometric analysis and induction of terminal differentiation potential. Our results demonstrate the successful generation of integration-free iPSCs from patients with AS, SS and SLE. These findings support the possibility of using iPSC technology in autologous and allogeneic cell replacement therapy for various AIDs, including AS, SS and SLE. PMID:27174201

  11. Generation of induced pluripotent stem cells from the pig

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The value of stem cells has become increasingly evident in recent years with the advent of genetic engineering tools that allow site-specific modifications to the genome. The use of stem cells to induce modifications has several potential benefits for the livestock industry including improving anim...

  12. Directed differentiation of induced pluripotent stem cells into chondrogenic lineages for articular cartilage treatment

    PubMed Central

    Lach, Michał; Richter, Magdalena; Pawlicz, Jarosław; Suchorska, Wiktoria M

    2014-01-01

    In recent years, increases in the number of articular cartilage injuries caused by environmental factors or pathological conditions have led to a notable rise in the incidence of premature osteoarthritis. Osteoarthritis, considered a disease of civilization, is the leading cause of disability. At present, standard methods for treating damaged articular cartilage, including autologous chondrocyte implantation or microfracture, are short-term solutions with important side effects. Emerging treatments include the use of induced pluripotent stem cells, a technique that could provide a new tool for treatment of joint damage. However, research in this area is still early, and no optimal protocol for transforming induced pluripotent stem cells into chondrocytes has yet been established. Developments in our understanding of cartilage developmental biology, together with the use of modern technologies in the field of tissue engineering, provide an opportunity to create a complete functional model of articular cartilage. PMID:25383175

  13. Minireview: Genome Editing of Human Pluripotent Stem Cells for Modeling Metabolic Disease.

    PubMed

    Yu, Haojie; Cowan, Chad A

    2016-06-01

    The pathophysiology of metabolic diseases such as coronary artery disease, diabetes, and obesity is complex and multifactorial. Developing new strategies to prevent or treat these diseases requires in vitro models with which researchers can extensively study the molecular mechanisms that lead to disease. Human pluripotent stem cells and their differentiated derivatives have the potential to provide an unlimited source of disease-relevant cell types and, when combined with recent advances in genome editing, make the goal of generating functional metabolic disease models, for the first time, consistently attainable. However, this approach still has certain limitations including lack of robust differentiation methods and potential off-target effects. This review describes the current progress in human pluripotent stem cell-based metabolic disease research using genome-editing technology. PMID:27075706

  14. [The role of induced pluripotent stem cells in modeling of neurological diseases].

    PubMed

    Balogh, Zoltán; Réthelyi, János; Molnár, Mária

    2015-06-28

    The longitudinal follow-up of the development and course of central nervous system related diseases on a molecular level was unsolved for decades. Direct examination of the pathological state on organ or tissue levels was feasible in the late stage of the disease. Modeling diseases has an important role in studying the pathophysiological mechanism underlying central nervous system disorders but animals used as model organism due to species specific nervous system differences can lead to less valid conclusions in translational research. The model of induced pluripotent stem cells may help to solve partially these types of problems. In recent years this model had a strong effect on understanding the pathogenesis of neurodegenerative and neurodevelopmental disorders. Although induced pluripotent stem cells have a low impact on clinical research studies, they have a prominent role in the field of cell physiology and molecular biology research. PMID:26104665

  15. Optimizing neuronal differentiation from induced pluripotent stem cells to model ASD

    PubMed Central

    Kim, Dae-Sung; Ross, P. Joel; Zaslavsky, Kirill; Ellis, James

    2014-01-01

    Autism spectrum disorder (ASD) is an early-onset neurodevelopmental disorder characterized by deficits in social communication, and restricted and repetitive patterns of behavior. Despite its high prevalence, discovery of pathophysiological mechanisms underlying ASD has lagged due to a lack of appropriate model systems. Recent advances in induced pluripotent stem cell (iPSC) technology and neural differentiation techniques allow for detailed functional analyses of neurons generated from living individuals with ASD. Refinement of cortical neuron differentiation methods from iPSCs will enable mechanistic studies of specific neuronal subpopulations that may be preferentially impaired in ASD. In this review, we summarize recent accomplishments in differentiation of cortical neurons from human pluripotent stems cells and efforts to establish in vitro model systems to study ASD using personalized neurons. PMID:24782713

  16. The potential of induced pluripotent stem cells in models of neurological disorders: implications on future therapy.

    PubMed

    Crook, Jeremy Micah; Wallace, Gordon; Tomaskovic-Crook, Eva

    2015-03-01

    There is an urgent need for new and advanced approaches to modeling the pathological mechanisms of complex human neurological disorders. This is underscored by the decline in pharmaceutical research and development efficiency resulting in a relative decrease in new drug launches in the last several decades. Induced pluripotent stem cells represent a new tool to overcome many of the shortcomings of conventional methods, enabling live human neural cell modeling of complex conditions relating to aberrant neurodevelopment, such as schizophrenia, epilepsy and autism as well as age-associated neurodegeneration. This review considers the current status of induced pluripotent stem cell-based modeling of neurological disorders, canvassing proven and putative advantages, current constraints, and future prospects of next-generation culture systems for biomedical research and translation. PMID:25664599

  17. Cardiovascular Disease Modeling Using Patient-Specific Induced Pluripotent Stem Cells

    PubMed Central

    Tanaka, Atsushi; Yuasa, Shinsuke; Node, Koichi; Fukuda, Keiichi

    2015-01-01

    The generation of induced pluripotent stem cells (iPSCs) has opened up a new scientific frontier in medicine. This technology has made it possible to obtain pluripotent stem cells from individuals with genetic disorders. Because iPSCs carry the identical genetic anomalies related to those disorders, iPSCs are an ideal platform for medical research. The pathophysiological cellular phenotypes of genetically heritable heart diseases such as arrhythmias and cardiomyopathies, have been modeled on cell culture dishes using disease-specific iPSC-derived cardiomyocytes. These model systems can potentially provide new insights into disease mechanisms and drug discoveries. This review focuses on recent progress in cardiovascular disease modeling using iPSCs, and discusses problems and future perspectives concerning their use. PMID:26274955

  18. Targeted Correction and Restored Function of the CFTR Gene in Cystic Fibrosis Induced Pluripotent Stem Cells

    PubMed Central

    Crane, Ana M.; Kramer, Philipp; Bui, Jacquelin H.; Chung, Wook Joon; Li, Xuan Shirley; Gonzalez-Garay, Manuel L.; Hawkins, Finn; Liao, Wei; Mora, Daniela; Choi, Sangbum; Wang, Jianbin; Sun, Helena C.; Paschon, David E.; Guschin, Dmitry Y.; Gregory, Philip D.; Kotton, Darrell N.; Holmes, Michael C.; Sorscher, Eric J.; Davis, Brian R.

    2015-01-01

    Summary Recently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources—potentially useful for the development of new therapeutic approaches. Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc-finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology-directed repair (HDR). We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other. The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells. PMID:25772471

  19. Targeted correction and restored function of the CFTR gene in cystic fibrosis induced pluripotent stem cells.

    PubMed

    Crane, Ana M; Kramer, Philipp; Bui, Jacquelin H; Chung, Wook Joon; Li, Xuan Shirley; Gonzalez-Garay, Manuel L; Hawkins, Finn; Liao, Wei; Mora, Daniela; Choi, Sangbum; Wang, Jianbin; Sun, Helena C; Paschon, David E; Guschin, Dmitry Y; Gregory, Philip D; Kotton, Darrell N; Holmes, Michael C; Sorscher, Eric J; Davis, Brian R

    2015-04-14

    Recently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources-potentially useful for the development of new therapeutic approaches. Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc-finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology-directed repair (HDR). We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other. The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells. PMID:25772471

  20. Stepwise Differentiation of Retinal Ganglion Cells from Human Pluripotent Stem Cells Enables Analysis of Glaucomatous Neurodegeneration.

    PubMed

    Ohlemacher, Sarah K; Sridhar, Akshayalakshmi; Xiao, Yucheng; Hochstetler, Alexandra E; Sarfarazi, Mansoor; Cummins, Theodore R; Meyer, Jason S

    2016-06-01

    Human pluripotent stem cells (hPSCs), including both embryonic and induced pluripotent stem cells, possess the unique ability to readily differentiate into any cell type of the body, including cells of the retina. Although previous studies have demonstrated the ability to differentiate hPSCs to a retinal lineage, the ability to derive retinal ganglion cells (RGCs) from hPSCs has been complicated by the lack of specific markers with which to identify these cells from a pluripotent source. In the current study, the definitive identification of hPSC-derived RGCs was accomplished by their directed, stepwise differentiation through an enriched retinal progenitor intermediary, with resultant RGCs expressing a full complement of associated features and proper functional characteristics. These results served as the basis for the establishment of induced pluripotent stem cells (iPSCs) from a patient with a genetically inherited form of glaucoma, which results in damage and loss of RGCs. Patient-derived RGCs specifically exhibited a dramatic increase in apoptosis, similar to the targeted loss of RGCs in glaucoma, which was significantly rescued by the addition of candidate neuroprotective factors. Thus, the current study serves to establish a method by which to definitively acquire and identify RGCs from hPSCs and demonstrates the ability of hPSCs to serve as an effective in vitro model of disease progression. Moreover, iPSC-derived RGCs can be utilized for future drug screening approaches to identify targets for the treatment of glaucoma and other optic neuropathies. Stem Cells 2016;34:1553-1562. PMID:26996528

  1. The Characterisation of Pluripotent and Multipotent Stem Cells Using Fourier Transform Infrared Microspectroscopy

    PubMed Central

    Cao, Julie; Ng, Elizabeth S.; McNaughton, Donald; Stanley, Edouard G.; Elefanty, Andrew G.; Tobin, Mark J.; Heraud, Philip

    2013-01-01

    Fourier transform infrared (FTIR) microspectroscopy shows potential as a benign, objective and rapid tool to screen pluripotent and multipotent stem cells for clinical use. It offers a new experimental approach that provides a holistic measurement of macromolecular composition such that a signature representing the internal cellular phenotype is obtained. The use of this technique therefore contributes information that is complementary to that acquired by conventional genetic and immunohistochemical methods. PMID:24065090

  2. Looking to the future following 10 years of induced pluripotent stem cell technologies.

    PubMed

    Li, Mo; Izpisua Belmonte, Juan Carlos

    2016-09-01

    The development of induced pluripotent stem cells (iPSCs) has fundamentally changed our view on developmental cell-fate determination and led to a cascade of technological innovations in regenerative medicine. Here we provide an overview of the progress in the field over the past decade, as well as our perspective on future directions and clinical implications of iPSC technology. PMID:27490631

  3. Combinatorial development of biomaterials for clonal growth of human pluripotent stem cells

    NASA Astrophysics Data System (ADS)

    Mei, Ying; Saha, Krishanu; Bogatyrev, Said R.; Yang, Jing; Hook, Andrew L.; Kalcioglu, Z. Ilke; Cho, Seung-Woo; Mitalipova, Maisam; Pyzocha, Neena; Rojas, Fredrick; van Vliet, Krystyn J.; Davies, Martyn C.; Alexander, Morgan R.; Langer, Robert; Jaenisch, Rudolf; Anderson, Daniel G.

    2010-09-01

    Both human embryonic stem cells and induced pluripotent stem cells can self-renew indefinitely in culture; however, present methods to clonally grow them are inefficient and poorly defined for genetic manipulation and therapeutic purposes. Here we develop the first chemically defined, xeno-free, feeder-free synthetic substrates to support robust self-renewal of fully dissociated human embryonic stem and induced pluripotent stem cells. Material properties including wettability, surface topography, surface chemistry and indentation elastic modulus of all polymeric substrates were quantified using high-throughput methods to develop structure-function relationships between material properties and biological performance. These analyses show that optimal human embryonic stem cell substrates are generated from monomers with high acrylate content, have a moderate wettability and employ integrin αvβ3 and αvβ5 engagement with adsorbed vitronectin to promote colony formation. The structure-function methodology employed herein provides a general framework for the combinatorial development of synthetic substrates for stem cell culture.

  4. Differentiation of murine embryonic stem and induced pluripotent stem cells to renal lineage in vitro

    SciTech Connect

    Morizane, Ryuji; Monkawa, Toshiaki; Itoh, Hiroshi

    2009-12-25

    Embryonic stem (ES) cells which have the unlimited proliferative capacity and extensive differentiation potency can be an attractive source for kidney regeneration therapies. Recent breakthroughs in the generation of induced pluripotent stem (iPS) cells have provided with another potential source for the artificially-generated kidney. The purpose of this study is to know how to differentiate mouse ES and iPS cells into renal lineage. We used iPS cells from mouse fibroblasts by transfection of four transcription factors, namely Oct4, Sox2, c-Myc and Klf4. Real-time PCR showed that renal lineage markers were expressed in both ES and iPS cells after the induction of differentiation. It also showed that a tubular specific marker, KSP progressively increased to day 18, although the differentiation of iPS cells was slower than ES cells. The results indicated that renal lineage cells can be differentiated from both murine ES and iPS cells. Several inducing factors were tested whether they influenced on cell differentiation. In ES cells, both of GDNF and BMP7 enhanced the differentiation to metanephric mesenchyme, and Activin enhanced the differentiation of ES cells to tubular cells. Activin also enhanced the differentiation of iPS cells to tubular cells, although the enhancement was lower than in ES cells. ES and iPS cells have a potential to differentiate to renal lineage cells, and they will be an attractive resource of kidney regeneration therapy. This differentiation is enhanced by Activin in both ES and iPS cells.

  5. In vitro pathological modelling using patient-specific induced pluripotent stem cells: the case of progeria.

    PubMed

    Nissan, Xavier; Blondel, Sophie; Peschanski, Marc

    2011-12-01

    Progeria, also known as HGPS (Hutchinson-Gilford progeria syndrome), is a rare fatal genetic disease characterized by an appearance of accelerated aging in children. This syndrome is typically caused by mutations in codon 608 (C1804T) of the gene encoding lamins A and C, LMNA, leading to the production of a truncated form of the protein called progerin. Owing to their unique potential to self-renew and to differentiate into any cell types of the organism, pluripotent stem cells offer a unique tool to study molecular and cellular mechanisms related to this global and systemic disease. Recent studies have exploited this potential by generating human induced pluripotent stem cells from HGPS patients' fibroblasts displaying several phenotypic defects characteristic of HGPS such as nuclear abnormalities, progerin expression, altered DNA-repair mechanisms and premature senescence. Altogether, these findings provide new insights on the use of pluripotent stem cells for pathological modelling and may open original therapeutic perspectives for diseases that lack pre-clinical in vitro human models, such as HGPS. PMID:22103524

  6. Enhancement of Arsenic Trioxide-Mediated Changes in Human Induced Pluripotent Stem Cells (IPS)

    PubMed Central

    Graham, Barbara; Stevens, Jacqueline; Wells, Phatia; Sims, Jennifer; Rogers, Christian; Leggett, Sophia S.; Ekunwe, Stephen; Ndebele, Kenneth

    2014-01-01

    Induced pluripotent stem cells (IPS) are an artificially derived type of pluripotent stem cell, showing many of the same characteristics as natural pluripotent stem cells. IPS are a hopeful therapeutic model; however there is a critical need to determine their response to environmental toxins. Effects of arsenic on cells have been studied extensively; however, its effect on IPS is yet to be elucidated. Arsenic trioxide (ATO) has been shown to inhibit cell proliferation, induce apoptosis and genotoxicity in many cells. Based on ATOs action in other cells, we hypothesize that it will induce alterations in morphology, inhibit cell viability and induce a genotoxic effect on IPS. Cells were treated for 24 hours with ATO (0–9 µg/mL). Cell morphology, viability and DNA damage were documented. Results indicated sufficient changes in morphology of cell colonies mainly in cell ability to maintain grouping and ability to remain adherent. Cell viability decreased in a dose dependent manner. There were significant increases in tail length and moment as well as destruction of intact DNA as concentration increased. Exposure to ATO resulted in a reproducible dose dependent sequence of events marked by changes in morphology, decrease of cell viability, and induction of genotoxicity in IPS. PMID:25054231

  7. Humanized Murine Model for HBV and HCV Using Human Induced Pluripotent Stem Cells

    PubMed Central

    Zhou, Xiao-Ling; Sullivan, Gareth J.; Sun, Pingnan; Park, In-Hyun

    2013-01-01

    Infection of hepatitis B virus (HBV) and hepatitis C virus (HCV) results in heterogeneous outcomes from acute asymptomatic infection to chronic infection leading to cirrhosis and hepatocellular carcinoma (HCC). In vitro models using animal hepatocytes, human HCC cell lines, or in vivo transgenic mouse models have contributed invaluably to understanding the pathogenesis of HBV and HCV. A humanized mouse model made by reconstitution of human primary hepatocytes in the liver of the immunodeficient mouse provides a novel experimental opportunity which mimics the in vivo growth of the human hepatocytes. The limited access to primary human hepatocytes necessitated the search for other cellular sources, such as pluripotent stem cells. Human embryonic stem cells (hESCs) have the features of self-renewal and pluripotency and differentiate into cells of all three germ layers, including hepatocytes. Humaninduced pluripotent stem cells (iPSCs) derived from the patient’s or individual’s own cells provide a novel opportunity to generate hepatocyte-like cells with the defined genetic composition. Here, we will review the current perspective of the models used for HBV and HCV study, and introduce the personalized mouse model using human iPSCs. This novel mouse model will facilitate the direct investigation of HBV and HCV in human hepatocytes as well as probing the genetic influence on the susceptibility of hepatocytes to HBV and HCV. PMID:22370780

  8. Direct generation of induced pluripotent stem cells from human nonmobilized blood.

    PubMed

    Kunisato, Atsushi; Wakatsuki, Mariko; Shinba, Haruna; Ota, Toshio; Ishida, Isao; Nagao, Kenji

    2011-01-01

    The use of induced pluripotent stem cells (iPSCs) is an exciting frontier in the study and treatment of human diseases through the generation of specific cell types. Here we show the derivation of iPSCs from human nonmobilized peripheral blood (PB) and bone marrow (BM) mononuclear cells (MNCs) by retroviral transduction of OCT3/4, SOX2, KLF4, and c-MYC. The PB- and BM-derived iPSCs were quite similar to human embryonic stem cells with regard to morphology, expression of surface antigens and pluripotency-associated transcription factors, global gene expression profiles, and differentiation potential in vitro and in vivo. Infected PB and BM MNCs gave rise to iPSCs in the presence of several cytokines, although transduction efficiencies were not high. We found that 5 × 10(5) PB MNCs, which corresponds to less than 1 mL of PB, was enough for the generation of several iPSC colonies. Generation of iPSCs from MNCs of nonmobilized PB, with its relative efficiency and ease of harvesting, could enable the therapeutic use of patient-specific pluripotent stem cells. PMID:20497033

  9. Muse cells, newly found non-tumorigenic pluripotent stem cells, reside in human mesenchymal tissues.

    PubMed

    Wakao, Shohei; Akashi, Hideo; Kushida, Yoshihiro; Dezawa, Mari

    2014-01-01

    Mesenchymal stem cells (MSCs) have been presumed to include a subpopulation of pluripotent-like cells as they differentiate not only into the same mesodermal-lineage cells but also into ectodermal- and endodermal-lineage cells and exert tissue regenerative effects in a wide variety of tissues. A novel type of pluripotent stem cell, Multilineage-differentiating stress enduring (Muse) cells, was recently discovered in mesenchymal tissues such as the bone marrow, adipose tissue, dermis and connective tissue of organs, as well as in cultured fibroblasts and bone marrow-MSCs. Muse cells are able to differentiate into all three germ layers from a single cell and to self-renew, and yet exhibit non-tumorigenic and low telomerase activities. They can migrate to and target damaged sites in vivo, spontaneously differentiate into cells compatible with the targeted tissue, and contribute to tissue repair. Thus, Muse cells may account for the wide variety of differentiation abilities and tissue repair effects that have been observed in MSCs. Muse cells are unique in that they are pluripotent stem cells that belong in the living body, and are thus assumed to play an important role in 'regenerative homeostasis' in vivo. PMID:24471964

  10. Derivation and characterization of bovine induced pluripotent stem cells by transposon-mediated reprogramming.

    PubMed

    Talluri, Thirumala R; Kumar, Dharmendra; Glage, Silke; Garrels, Wiebke; Ivics, Zoltan; Debowski, Katharina; Behr, Rüdiger; Niemann, Heiner; Kues, Wilfried A

    2015-04-01

    Induced pluripotent stem cells (iPSCs) are a seminal breakthrough in stem cell research and are promising tools for advanced regenerative therapies in humans and reproductive biotechnology in farm animals. iPSCs are particularly valuable in species in which authentic embryonic stem cell (ESC) lines are yet not available. Here, we describe a nonviral method for the derivation of bovine iPSCs employing Sleeping Beauty (SB) and piggyBac (PB) transposon systems encoding different combinations of reprogramming factors, each separated by self-cleaving peptide sequences and driven by the chimeric CAGGS promoter. One bovine iPSC line (biPS-1) generated by a PB vector containing six reprogramming genes was analyzed in detail, including morphology, alkaline phosphatase expression, and typical hallmarks of pluripotency, such as expression of pluripotency markers and formation of mature teratomas in immunodeficient mice. Moreover, the biPS-1 line allowed a second round of SB transposon-mediated gene transfer. These results are promising for derivation of germ line-competent bovine iPSCs and will facilitate genetic modification of the bovine genome. PMID:25826726

  11. Myocardial commitment from human pluripotent stem cells: Rapid production of human heart grafts.

    PubMed

    Garreta, Elena; de Oñate, Lorena; Fernández-Santos, M Eugenia; Oria, Roger; Tarantino, Carolina; Climent, Andreu M; Marco, Andrés; Samitier, Mireia; Martínez, Elena; Valls-Margarit, Maria; Matesanz, Rafael; Taylor, Doris A; Fernández-Avilés, Francisco; Izpisua Belmonte, Juan Carlos; Montserrat, Nuria

    2016-08-01

    Genome editing on human pluripotent stem cells (hPSCs) together with the development of protocols for organ decellularization opens the door to the generation of autologous bioartificial hearts. Here we sought to generate for the first time a fluorescent reporter human embryonic stem cell (hESC) line by means of Transcription activator-like effector nucleases (TALENs) to efficiently produce cardiomyocyte-like cells (CLCs) from hPSCs and repopulate decellularized human heart ventricles for heart engineering. In our hands, targeting myosin heavy chain locus (MYH6) with mCherry fluorescent reporter by TALEN technology in hESCs did not alter major pluripotent-related features, and allowed for the definition of a robust protocol for CLCs production also from human induced pluripotent stem cells (hiPSCs) in 14 days. hPSCs-derived CLCs (hPSCs-CLCs) were next used to recellularize acellular cardiac scaffolds. Electrophysiological responses encountered when hPSCs-CLCs were cultured on ventricular decellularized extracellular matrix (vdECM) correlated with significant increases in the levels of expression of different ion channels determinant for calcium homeostasis and heart contractile function. Overall, the approach described here allows for the rapid generation of human cardiac grafts from hPSCs, in a total of 24 days, providing a suitable platform for cardiac engineering and disease modeling in the human setting. PMID:27179434

  12. Human hepatocytes derived from pluripotent stem cells: a promising cell model for drug hepatotoxicity screening.

    PubMed

    Gómez-Lechón, María José; Tolosa, Laia

    2016-09-01

    Drug-induced liver injury (DILI) is a frequent cause of failure in both clinical and post-approval stages of drug development, and poses a key challenge to the pharmaceutical industry. Current animal models offer poor prediction of human DILI. Although several human cell-based models have been proposed for the detection of human DILI, human primary hepatocytes remain the gold standard for preclinical toxicological screening. However, their use is hindered by their limited availability, variability and phenotypic instability. In contrast, pluripotent stem cells, which include embryonic and induced pluripotent stem cells (iPSCs), proliferate extensively in vitro and can be differentiated into hepatocytes by the addition of soluble factors. This provides a stable source of hepatocytes for multiple applications, including early preclinical hepatotoxicity screening. In addition, iPSCs also have the potential to establish genotype-specific cells from different individuals, which would increase the predictivity of toxicity assays allowing more successful clinical trials. Therefore, the generation of human hepatocyte-like cells derived from pluripotent stem cells seems to be promising for overcoming limitations of hepatocyte preparations, and it is expected to have a substantial repercussion in preclinical hepatotoxicity risk assessment in early drug development stages. PMID:27325232

  13. Evaluating Cell Processes, Quality, and Biomarkers in Pluripotent Stem Cells Using Video Bioinformatics.

    PubMed

    Zahedi, Atena; On, Vincent; Lin, Sabrina C; Bays, Brett C; Omaiye, Esther; Bhanu, Bir; Talbot, Prue

    2016-01-01

    There is a foundational need for quality control tools in stem cell laboratories engaged in basic research, regenerative therapies, and toxicological studies. These tools require automated methods for evaluating cell processes and quality during in vitro passaging, expansion, maintenance, and differentiation. In this paper, an unbiased, automated high-content profiling toolkit, StemCellQC, is presented that non-invasively extracts information on cell quality and cellular processes from time-lapse phase-contrast videos. Twenty four (24) morphological and dynamic features were analyzed in healthy, unhealthy, and dying human embryonic stem cell (hESC) colonies to identify those features that were affected in each group. Multiple features differed in the healthy versus unhealthy/dying groups, and these features were linked to growth, motility, and death. Biomarkers were discovered that predicted cell processes before they were detectable by manual observation. StemCellQC distinguished healthy and unhealthy/dying hESC colonies with 96% accuracy by non-invasively measuring and tracking dynamic and morphological features over 48 hours. Changes in cellular processes can be monitored by StemCellQC and predictions can be made about the quality of pluripotent stem cell colonies. This toolkit reduced the time and resources required to track multiple pluripotent stem cell colonies and eliminated handling errors and false classifications due to human bias. StemCellQC provided both user-specified and classifier-determined analysis in cases where the affected features are not intuitive or anticipated. Video analysis algorithms allowed assessment of biological phenomena using automatic detection analysis, which can aid facilities where maintaining stem cell quality and/or monitoring changes in cellular processes are essential. In the future StemCellQC can be expanded to include other features, cell types, treatments, and differentiating cells. PMID:26848582

  14. Evaluating Cell Processes, Quality, and Biomarkers in Pluripotent Stem Cells Using Video Bioinformatics

    PubMed Central

    Lin, Sabrina C.; Bays, Brett C.; Omaiye, Esther; Bhanu, Bir; Talbot, Prue

    2016-01-01

    There is a foundational need for quality control tools in stem cell laboratories engaged in basic research, regenerative therapies, and toxicological studies. These tools require automated methods for evaluating cell processes and quality during in vitro passaging, expansion, maintenance, and differentiation. In this paper, an unbiased, automated high-content profiling toolkit, StemCellQC, is presented that non-invasively extracts information on cell quality and cellular processes from time-lapse phase-contrast videos. Twenty four (24) morphological and dynamic features were analyzed in healthy, unhealthy, and dying human embryonic stem cell (hESC) colonies to identify those features that were affected in each group. Multiple features differed in the healthy versus unhealthy/dying groups, and these features were linked to growth, motility, and death. Biomarkers were discovered that predicted cell processes before they were detectable by manual observation. StemCellQC distinguished healthy and unhealthy/dying hESC colonies with 96% accuracy by non-invasively measuring and tracking dynamic and morphological features over 48 hours. Changes in cellular processes can be monitored by StemCellQC and predictions can be made about the quality of pluripotent stem cell colonies. This toolkit reduced the time and resources required to track multiple pluripotent stem cell colonies and eliminated handling errors and false classifications due to human bias. StemCellQC provided both user-specified and classifier-determined analysis in cases where the affected features are not intuitive or anticipated. Video analysis algorithms allowed assessment of biological phenomena using automatic detection analysis, which can aid facilities where maintaining stem cell quality and/or monitoring changes in cellular processes are essential. In the future StemCellQC can be expanded to include other features, cell types, treatments, and differentiating cells. PMID:26848582

  15. All the adult stem cells, where do they all come from? An external source for organ-specific stem cell pools.

    PubMed

    Nardi, N B

    2005-01-01

    Stem cells can self-renew and maintain the ability to differentiate into mature lineages. Whereas the "stemness" of embryonic stem cells is not discussed, the primitiveness of a stem cell type within adult organisms is not well determined. Data presently available are either inconclusive or controversial regarding two main topics: maintenance or senescente of the adult stem cell pool; and pluripotentiality of the cells. While programmed senescence or apoptosis following uncorrected mutations represent no problem for mature cells, the maintenance of the stem cell pool itself must be assured. Two different mechanisms can be envisaged for that. In the first mechanism, which is generally accepted, stem cells originate during ontogeny along with the organ which they are responsible for, and remain there during all the lifespan of the organism. Several observations derived from recent reports allow the suggestion of a second mechanism. These observations include: organ-specific stem cells are senescent; adult stem cells circulate in the organism; stem cell niches are essential for the existence and function of stem cells; adult stem cells can present lineage markers; embryo-like, pluripotent stem cells are present in adult organisms, as shown by the development of teratomas, tumors composed of derivatives of the three germ layers; and the fact that the gonads may be a reservoir of embryo-like, pluripotent stem cells in adult organisms. The second mechanism for the maintenance of adult stem cells compartments implies a source external to the organ they belong, consisting of pluripotent, embryo-like cells of unrestricted life span, presenting efficient mechanisms for avoiding or correcting mutations and capable to circulate in the organism. According to this model, primitive stem cells exist in a specific organ in adult organisms. They undergo asymmetrical divisions, which originate one "true" stem cell and another one which enters the pool of adult stem cells, circulating

  16. Efficient and Rapid Derivation of Primitive Neural Stem Cells and Generation of Brain Subtype Neurons From Human Pluripotent Stem Cells

    PubMed Central

    Yan, Yiping; Shin, Soojung; Jha, Balendu Shekhar; Liu, Qiuyue; Sheng, Jianting; Li, Fuhai; Zhan, Ming; Davis, Janine; Bharti, Kapil; Zeng, Xianmin; Rao, Mahendra; Malik, Nasir

    2013-01-01

    Human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, are unique cell sources for disease modeling, drug discovery screens, and cell therapy applications. The first step in producing neural lineages from hPSCs is the generation of neural stem cells (NSCs). Current methods of NSC derivation involve the time-consuming, labor-intensive steps of an embryoid body generation or coculture with stromal cell lines that result in low-efficiency derivation of NSCs. In this study, we report a highly efficient serum-free pluripotent stem cell neural induction medium that can induce hPSCs into primitive NSCs (pNSCs) in 7 days, obviating the need for time-consuming, laborious embryoid body generation or rosette picking. The pNSCs expressed the neural stem cell markers Pax6, Sox1, Sox2, and Nestin; were negative for Oct4; could be expanded for multiple passages; and could be differentiated into neurons, astrocytes, and oligodendrocytes, in addition to the brain region-specific neuronal subtypes GABAergic, dopaminergic, and motor neurons. Global gene expression of the transcripts of pNSCs was comparable to that of rosette-derived and human fetal-derived NSCs. This work demonstrates an efficient method to generate expandable pNSCs, which can be further differentiated into central nervous system neurons and glia with temporal, spatial, and positional cues of brain regional heterogeneity. This method of pNSC derivation sets the stage for the scalable production of clinically relevant neural cells for cell therapy applications in good manufacturing practice conditions. PMID:24113065

  17. Thermoresponsive hydrogel maintains the mouse embryonic stem cell "naïve" pluripotency phenotype.

    PubMed

    Mangani, Christian; Lilienkampf, Annamaria; Roy, Marcia; de Sousa, Paul A; Bradley, Mark

    2015-10-15

    A chemically defined thermoresponsive hydrogel, poly(AEtMA-Cl-co-DEAEA) cross-linked with N,N'-methylenebisacrylamide, which allows enzyme-free passaging, was used as a substrate to culture murine embryonic stem cells (mESCs) under defined and undefined conditions. Analysis of 14 stem cell markers showed that the mESCs remained in a "naïve" state of pluripotency with differentiation potential to form endoderm, mesoderm, and ectoderm derived lineages. These results validate the use of a chemically defined hydrogel for standardised and inexpensive mESC culture. PMID:26372076

  18. Progress toward the clinical application of patient-specific pluripotent stem cells

    PubMed Central

    Kiskinis, Evangelos; Eggan, Kevin

    2010-01-01

    Induced pluripotent stem (iPS) cells are generated by epigenetic reprogramming of somatic cells through the exogenous expression of transcription factors. These cells, just like embryonic stem cells, are likely to have a major impact on regenerative medicine, because they self-renew and retain the potential to be differentiated into all cell types of the human body. In this Review, we describe the current state of iPS cell technology, including approaches by which they are generated and what is known about their biology, and discuss the potential applications of these cells for disease modeling, drug discovery, and, eventually, cell replacement therapy. PMID:20051636

  19. Scalable topographies to support proliferation and Oct4 expression by human induced pluripotent stem cells

    PubMed Central

    Reimer, Andreas; Vasilevich, Aliaksei; Hulshof, Frits; Viswanathan, Priyalakshmi; van Blitterswijk, Clemens A.; de Boer, Jan; Watt, Fiona M.

    2016-01-01

    It is well established that topographical features modulate cell behaviour, including cell morphology, proliferation and differentiation. To define the effects of topography on human induced pluripotent stem cells (iPSC), we plated cells on a topographical library containing over 1000 different features in medium lacking animal products (xeno-free). Using high content imaging, we determined the effect of each topography on cell proliferation and expression of the pluripotency marker Oct4 24 h after seeding. Features that maintained Oct4 expression also supported proliferation and cell-cell adhesion at 24 h, and by 4 days colonies of Oct4-positive, Sox2-positive cells had formed. Computational analysis revealed that small feature size was the most important determinant of pluripotency, followed by high wave number and high feature density. Using this information we correctly predicted whether any given topography within our library would support the pluripotent state at 24 h. This approach not only facilitates the design of substrates for optimal human iPSC expansion, but also, potentially, identification of topographies with other desirable characteristics, such as promoting differentiation. PMID:26757610

  20. Induction of pluripotency in human umbilical cord mesenchymal stem cells in feeder layer-free condition.

    PubMed

    Daneshvar, Nasibeh; Rasedee, Abdullah; Shamsabadi, Fatemeh Tash; Moeini, Hassan; Mehrboud, Parvaneh; Rahman, Heshu Sulaiman; Boroojerdi, Mohadeseh Hashem; Vellasamy, Shalini

    2015-12-01

    Induced Pluripotent Stem Cells (iPSCs) has been produced by the reprogramming of several types of somatic cells through the expression of different sets of transcription factors. This study consists of a technique to obtain iPSCs from human umbilical cord mesenchymal stem cells (UC-MSCs) in a feeder layer-free process using a mini-circle vector containing defined reprogramming genes, Lin28, Nanog, Oct4 and Sox2. The human MSCs transfected with the minicircle vector were cultured in iPSCs medium. Human embryonic stem cell (ESC)-like colonies with tightly packed domelike structures appeared 7-10 days after the second transfection. In the earliest stages, the colonies were green fluorescence protein (GFP)-positive, while upon continuous culture and passage, genuine hiPSC clones expressing GFP were observed. The induced cells, based on the ectopic expression of the pluripotent markers, exhibited characteristics similar to the embryonic stem cells. These iPSCs demonstrated in vitro capabilities for differentiation into the three main embryonic germ layers by embryoid bodies formation. There was no evidence of transgenes integration into the genome of the iPSCs in this study. In conclusion, this method offers a means of producing iPSCs without viral delivery that could possibly overcome ethical concerns and immune rejection in the use of stem cells in medical applications. PMID:26471847

  1. Heightened potency of human pluripotent stem cell lines created by transient BMP4 exposure

    PubMed Central

    Yang, Ying; Adachi, Katsuyuki; Sheridan, Megan A.; Alexenko, Andrei P.; Schust, Danny J.; Schulz, Laura C.; Ezashi, Toshihiko; Roberts, R. Michael

    2015-01-01

    Human pluripotent stem cells (PSCs) show epiblast-type pluripotency that is maintained with ACTIVIN/FGF2 signaling. Here, we report the acquisition of a unique stem cell phenotype by both human ES cells (hESCs) and induced pluripotent stem cells (iPSCs) in response to transient (24–36 h) exposure to bone morphogenetic protein 4 (BMP4) plus inhibitors of ACTIVIN signaling (A83-01) and FGF2 (PD173074), followed by trypsin dissociation and recovery of colonies capable of growing on a gelatin substratum in standard medium for human PSCs at low but not high FGF2 concentrations. The self-renewing cell lines stain weakly for CDX2 and strongly for NANOG, can be propagated clonally on either Matrigel or gelatin, and are morphologically distinct from human PSC progenitors on either substratum but still meet standard in vitro criteria for pluripotency. They form well-differentiated teratomas in immune-compromised mice that secrete human chorionic gonadotropin (hCG) into the host mouse and include small areas of trophoblast-like cells. The cells have a distinct transcriptome profile from the human PSCs from which they were derived (including higher expression of NANOG, LEFTY1, and LEFTY2). In nonconditioned medium lacking FGF2, the colonies spontaneously differentiated along multiple lineages, including trophoblast. They responded to PD173074 in the absence of both FGF2 and BMP4 by conversion to trophoblast, and especially syncytiotrophoblast, whereas an A83-01/PD173074 combination favored increased expression of HLA-G, a marker of extravillous trophoblast. Together, these data suggest that the cell lines exhibit totipotent potential and that BMP4 can prime human PSCs to a self-renewing alternative state permissive for trophoblast development. The results may have implications for regulation of lineage decisions in the early embryo. PMID:25870291

  2. [Generation of functional organs from pluripotent stem cells].

    PubMed

    Miyamoto, Tatsuyuki; Nakauchi, Hiromitsu

    2015-10-01

    Hematopoietic stem cells (HSCs) have played a major role in stem cell biology, providing many conceptual ideas and models. Among them is the concept of the "niche", a special bone-marrow microenvironment that by exchanging cues regulates stem-cell fate. The HSC niche also plays an important role in HSC transplantation. Successful engraftment of donor HSCs depends on myeloablative pretreatment to empty the niche. The concept of the stem-cell niche has now been extended to the generation of organs. We postulated that an empty "organ niche" exists in a developing animal when development of an organ is genetically disabled. This organ niche should be developmentally compensated by blastocyst complementation using wild-type primary stem cells (PSCs). We proved the principle of organogenesis from xenogeneic PSCs in an embryo unable to form a specific organ, demonstrating the generation of functionally normal rat pancreas by injecting rat PSCs into pancreatogenesis-disabled mouse embryos. This principle has held in pigs. When pancreatogenesis-disabled pig embryos underwent complementation with blastomeres from wild-type pig embryos to produce chimeric pigs, the chimeras had normal pancreata and survived to adulthood. Demonstration of the generation of a functional organ from PSCs in pigs is a very important step toward generation of human cells, tissues, and organs from individual patients' own PSCs in large animals. PMID:26458462

  3. Controversies in Cardiovascular Research: Induced pluripotent stem cell-derived cardiomyocytes – boutique science or valuable arrhythmia model?

    PubMed Central

    Knollmann, Björn C

    2013-01-01

    As part of the series on Controversies in Cardiovascular Research, the article reviews the strengths and limitations of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) as models of cardiac arrhythmias. Specifically, the article attempts to answer the following questions: Which clinical arrhythmias can be modeled by iPSC-CM? How well can iPSC-CM model adult ventricular myocytes? What are the strengths and limitations of published iPSC-CM arrhythmia models? What new mechanistic insight has been gained? What is the evidence that would support using iPSC-CM to personalize anti-arrhythmic drug therapy? The review also discusses the pros and cons of using the iPSC-CM technology for modeling specific genetic arrhythmia disorders such as long QT syndrome, Brugada Syndrome or Catecholaminergic Polymorphic Ventricular Tachycardia. PMID:23569106

  4. Hydrogel microfluidics for the patterning of pluripotent stem cells

    NASA Astrophysics Data System (ADS)

    Cosson, S.; Lutolf, M. P.

    2014-03-01

    Biomolecular signaling is of utmost importance in governing many biological processes such as the patterning of the developing embryo where biomolecules regulate key cell-fate decisions. In vivo, these factors are presented in a spatiotemporally tightly controlled fashion. Although state-of-the-art microfluidic technologies allow precise biomolecule delivery in time and space, long-term (stem) cell culture at the micro-scale is often far from ideal due to medium evaporation, limited space for cell growth or shear stress. To overcome these challenges, we here introduce a concept based on hydrogel microfluidics for decoupling conventional, macro-scale cell culture from precise biomolecule delivery through a gel layer. We demonstrate the spatiotemporally controlled neuronal commitment of mouse embryonic stem cells via delivery of retinoic acid gradients. This technique should be useful for testing the effect of dose and timing of biomolecules, singly or in combination, on stem cell fate.

  5. Regulation of c-Myc Expression by Ahnak Promotes Induced Pluripotent Stem Cell Generation.

    PubMed

    Lim, Hee Jung; Kim, Jusong; Park, Chang-Hwan; Lee, Sang A; Lee, Man Ryul; Kim, Kye-Seong; Kim, Jaesang; Bae, Yun Soo

    2016-01-01

    We have previously reported that Ahnak-mediated TGFβ signaling leads to down-regulation of c-Myc expression. Here, we show that inhibition of Ahnak can promote generation of induced pluripotent stem cells (iPSC) via up-regulation of endogenous c-Myc. Consistent with the c-Myc inhibitory role of Ahnak, mouse embryonic fibroblasts from Ahnak-deficient mouse (Ahnak(-/-) MEF) show an increased level of c-Myc expression compared with wild type MEF. Generation of iPSC with just three of the four Yamanaka factors, Oct4, Sox2, and Klf4 (hereafter 3F), was significantly enhanced in Ahnak(-/-) MEF. Similar results were obtained when Ahnak-specific shRNA was applied to wild type MEF. Of note, expressionof Ahnak was significantly induced during the formation of embryoid bodies from embryonic stem cells, suggesting that Ahnak-mediated c-Myc inhibition is involved in embryoid body formation and the initial differentiation of pluripotent stem cells. The iPSC from 3F-infected Ahnak(-/-) MEF cells (Ahnak(-/-)-iPSC-3F) showed expression of all stem cell markers examined and the capability to form three primary germ layers. Moreover, injection of Ahnak(-/-)-iPSC-3F into athymic nude mice led to development of teratoma containing tissues from all three primary germ layers, indicating that iPSC from Ahnak(-/-) MEF are bona fide pluripotent stem cells. Taken together, these data provide evidence for a new role for Ahnak in cell fate determination during development and suggest that manipulation of Ahnak and the associated signaling pathway may provide a means to regulate iPSC generation. PMID:26598518

  6. Early maturation and distinct tau pathology in induced pluripotent stem cell-derived neurons from patients with MAPT mutations.

    PubMed

    Iovino, Mariangela; Agathou, Sylvia; González-Rueda, Ana; Del Castillo Velasco-Herrera, Martin; Borroni, Barbara; Alberici, Antonella; Lynch, Timothy; O'Dowd, Sean; Geti, Imbisaat; Gaffney, Daniel; Vallier, Ludovic; Paulsen, Ole; Káradóttir, Ragnhildur Thóra; Spillantini, Maria Grazia

    2015-11-01

    Tauopathies, such as Alzheimer's disease, some cases of frontotemporal dementia, corticobasal degeneration and progressive supranuclear palsy, are characterized by aggregates of the microtubule-associated protein tau, which are linked to neuronal death and disease development and can be caused by mutations in the MAPT gene. Six tau isoforms are present in the adult human brain and they differ by the presence of 3(3R) or 4(4R) C-terminal repeats. Only the shortest 3R isoform is present in foetal brain. MAPT mutations found in human disease affect tau binding to microtubules or the 3R:4R isoform ratio by altering exon 10 splicing. We have differentiated neurons from induced pluripotent stem cells derived from fibroblasts of controls and patients with N279K and P301L MAPT mutations. Induced pluripotent stem cell-derived neurons recapitulate developmental tau expression, showing the adult brain tau isoforms after several months in culture. Both N279K and P301L neurons exhibit earlier electrophysiological maturation and altered mitochondrial transport compared to controls. Specifically, the N279K neurons show abnormally premature developmental 4R tau expression, including changes in the 3R:4R isoform ratio and AT100-hyperphosphorylated tau aggregates, while P301L neurons are characterized by contorted processes with varicosity-like structures, some containing both alpha-synuclein and 4R tau. The previously unreported faster maturation of MAPT mutant human neurons, the developmental expression of 4R tau and the morphological alterations may contribute to disease development. PMID:26220942

  7. Early maturation and distinct tau pathology in induced pluripotent stem cell-derived neurons from patients with MAPT mutations

    PubMed Central

    Iovino, Mariangela; Agathou, Sylvia; González-Rueda, Ana; Del Castillo Velasco-Herrera, Martin; Borroni, Barbara; Alberici, Antonella; Lynch, Timothy; O’Dowd, Sean; Geti, Imbisaat; Gaffney, Daniel; Vallier, Ludovic; Paulsen, Ole; Káradóttir, Ragnhildur Thóra

    2015-01-01

    Tauopathies, such as Alzheimer’s disease, some cases of frontotemporal dementia, corticobasal degeneration and progressive supranuclear palsy, are characterized by aggregates of the microtubule-associated protein tau, which are linked to neuronal death and disease development and can be caused by mutations in the MAPT gene. Six tau isoforms are present in the adult human brain and they differ by the presence of 3(3R) or 4(4R) C-terminal repeats. Only the shortest 3R isoform is present in foetal brain. MAPT mutations found in human disease affect tau binding to microtubules or the 3R:4R isoform ratio by altering exon 10 splicing. We have differentiated neurons from induced pluripotent stem cells derived from fibroblasts of controls and patients with N279K and P301L MAPT mutations. Induced pluripotent stem cell-derived neurons recapitulate developmental tau expression, showing the adult brain tau isoforms after several months in culture. Both N279K and P301L neurons exhibit earlier electrophysiological maturation and altered mitochondrial transport compared to controls. Specifically, the N279K neurons show abnormally premature developmental 4R tau expression, including changes in the 3R:4R isoform ratio and AT100-hyperphosphorylated tau aggregates, while P301L neurons are characterized by contorted processes with varicosity-like structures, some containing both alpha-synuclein and 4R tau. The previously unreported faster maturation of MAPT mutant human neurons, the developmental expression of 4R tau and the morphological alterations may contribute to disease development. PMID:26220942

  8. Scalable Stirred-Suspension Bioreactor Culture of Human Pluripotent Stem Cells

    PubMed Central

    Kehoe, Daniel E.; Jing, Donghui; Lock, Lye T.

    2010-01-01

    Advances in stem cell biology have afforded promising results for the generation of various cell types for therapies against devastating diseases. However, a prerequisite for realizing the therapeutic potential of stem cells is the development of bioprocesses for the production of stem cell progeny in quantities that satisfy clinical demands. Recent reports on the expansion and directed differentiation of human embryonic stem cells (hESCs) in scalable stirred-suspension bioreactors (SSBs) demonstrated that large-scale production of therapeutically useful hESC progeny is feasible with current state-of-the-art culture technologies. Stem cells have been cultured in SSBs as aggregates, in microcarrier suspension and after encapsulation. The various modes in which SSBs can be employed for the cultivation of hESCs and human induced pluripotent stem cells (hiPSCs) are described. To that end, this is the first account of hiPSC cultivation in a microcarrier stirred-suspension system. Given that cultured stem cells and their differentiated progeny are the actual products used in tissue engineering and cell therapies, the impact of bioreactor's operating conditions on stem cell self-renewal and commitment should be considered. The effects of variables specific to SSB operation on stem cell physiology are discussed. Finally, major challenges are presented which remain to be addressed before the mainstream use of SSBs for the large-scale culture of hESCs and hiPSCs. PMID:19739936

  9. GABA's Control of Stem and Cancer Cell Proliferation in Adult Neural and Peripheral Niches

    PubMed Central

    Young, Stephanie Z.; Bordey, Angélique

    2010-01-01

    Aside from traditional neurotransmission and regulation of secretion, γ-amino butyric acid (GABA) through GABAA receptors negatively regulates proliferation of pluripotent and neural stem cells in embryonic and adult tissue. There has also been evidence that GABAergic signaling and its control over proliferation is not only limited to the nervous system, but is widespread through peripheral organs containing adult stem cells. GABA has emerged as a tumor signaling molecule in the periphery that controls the proliferation of tumor cells and perhaps tumor stem cells. Here, we will discuss GABA's presence as a near-universal signal that may be altered in tumor cells resulting in modified mitotic activity. PMID:19509127

  10. Generation and characterization of induced pluripotent stem cells from Aid-deficient mice.

    PubMed

    Shimamoto, Ren; Amano, Naoki; Ichisaka, Tomoko; Watanabe, Akira; Yamanaka, Shinya; Okita, Keisuke

    2014-01-01

    It has been shown that DNA demethylation plays a pivotal role in the generation of induced pluripotent stem (iPS) cells. However, the underlying mechanism of this action is still unclear. Previous reports indicated that activation-induced cytidine deaminase (Aid, also known as Aicda) is involved in DNA demethylation in several developmental processes, as well as cell fusion-mediated reprogramming. Based on these reports, we hypothesized that Aid may be involved in the DNA demethylation that occurs during the generation of iPS cells. In this study, we examined the function of Aid in iPS cell generation using Aid knockout (Aid⁻/⁻) mice expressing a GFP reporter under the control of a pluripotent stem cell marker, Nanog. By introducing Oct3/4, Sox2, Klf4 and c-Myc, Nanog-GFP-positive iPS cells could be generated from the fibroblasts and primary B cells of Aid⁻/⁻ mice. Their induction efficiency was similar to that of wild-type (Aid⁺/⁺) iPS cells. The Aid⁻/⁻ iPS cells showed normal proliferation and gave rise to chimeras, indicating their capacity for self-renewal and pluripotency. A comprehensive DNA methylation analysis showed only a few differences between Aid⁺/⁺ and Aid⁻/⁻ iPS cells. These data suggest that Aid does not have crucial functions in DNA demethylation during iPS cell generation. PMID:24718089

  11. Intracellular α-ketoglutarate maintains the pluripotency of embryonic stem cells.

    PubMed

    Carey, Bryce W; Finley, Lydia W S; Cross, Justin R; Allis, C David; Thompson, Craig B

    2015-02-19

    The role of cellular metabolism in regulating cell proliferation and differentiation remains poorly understood. For example, most mammalian cells cannot proliferate without exogenous glutamine supplementation even though glutamine is a non-essential amino acid. Here we show that mouse embryonic stem (ES) cells grown under conditions that maintain naive pluripotency are capable of proliferation in the absence of exogenous glutamine. Despite this, ES cells consume high levels of exogenous glutamine when the metabolite is available. In comparison to more differentiated cells, naive ES cells utilize both glucose and glutamine catabolism to maintain a high level of intracellular α-ketoglutarate (αKG). Consequently, naive ES cells exhibit an elevated αKG to succinate ratio that promotes histone/DNA demethylation and maintains pluripotency. Direct manipulation of the intracellular αKG/succinate ratio is sufficient to regulate multiple chromatin modifications, including H3K27me3 and ten-eleven translocation (Tet)-dependent DNA demethylation, which contribute to the regulation of pluripotency-associated gene expression. In vitro, supplementation with cell-permeable αKG directly supports ES-cell self-renewal while cell-permeable succinate promotes differentiation. This work reveals that intracellular αKG/succinate levels can contribute to the maintenance of cellular identity and have a mechanistic role in the transcriptional and epigenetic state of stem cells. PMID:25487152

  12. BMP-SMAD signaling: From pluripotent stem cells to cardiovascular commitment.

    PubMed

    Orlova, Valeria V; Chuva de Sousa Lopes, Susana; Valdimarsdottir, Gudrun

    2016-02-01

    Human pluripotent stem cells (hPSCs) can form all somatic cells of the body. They thus offer opportunities for understanding (i) the basic steps of early human development, (ii) the pathophysiology in human degenerative diseases and (iii) approaches to regenerative medicine and drug development. Methods for improving their differentiation to defined mesodermal derivatives in particular will benefit their use in all of these areas but most particularly applications that require cardiac and vascular tissue. However, the molecular mechanisms that regulate mesodermal development in humans are still poorly understood. Gene ablation studies in mice have shown that the signaling pathways activated by the transforming growth factor beta (TGFβ) superfamily, including the bone morphogenetic proteins (BMP), play crucial roles in mesoderm differentiation and patterning the early embryo. Understanding their interplay and interaction with other signaling pathways, how they activate and inhibit transcription factors and epigenetic regulators during self-renewal, maintenance and exit from pluripotency and differentiation could provide vital information for a range of applications. This includes disease modeling when the hPSCs are derived from patients or drug screens for diseases of mesodermal organs. Here, we review the role of the BMP-SMAD signaling pathway in pluripotent stem cells and during mesoderm differentiation with focus on the cells that make up the cardiovascular system. PMID:26651597

  13. High-Efficient Generation of Induced Pluripotent Stem Cells from Human Astrocytes

    PubMed Central

    Ruiz, Sergio; Brennand, Kristen; Panopoulos, Athanasia D.; Herrerías, Aída; Gage, Fred H.; Izpisua-Belmonte, Juan Carlos

    2010-01-01

    The reprogramming of human somatic cells to induced pluripotent stem (hiPS) cells enables the possibility of generating patient-specific autologous cells for regenerative medicine. A number of human somatic cell types have been reported to generate hiPS cells, including fibroblasts, keratinocytes and peripheral blood cells, with variable reprogramming efficiencies and kinetics. Here, we show that human astrocytes can also be reprogrammed into hiPS (ASThiPS) cells, with similar efficiencies to keratinocytes, which are currently reported to have one of the highest somatic reprogramming efficiencies. ASThiPS lines were indistinguishable from human embryonic stem (ES) cells based on the expression of pluripotent markers and the ability to differentiate into the three embryonic germ layers in vitro by embryoid body generation and in vivo by teratoma formation after injection into immunodeficient mice. Our data demonstrates that a human differentiated neural cell type can be reprogrammed to pluripotency and is consistent with the universality of the somatic reprogramming procedure. PMID:21170306

  14. A reproducible and versatile system for the dynamic expansion of human pluripotent stem cells in suspension.

    PubMed

    Elanzew, Andreas; Sommer, Annika; Pusch-Klein, Annette; Brüstle, Oliver; Haupt, Simone

    2015-10-01

    Reprogramming of patient cells to human induced pluripotent stem cells (hiPSC) has facilitated in vitro disease modeling studies aiming at deciphering the molecular and cellular mechanisms that contribute to disease pathogenesis and progression. To fully exploit the potential of hiPSC for biomedical applications, technologies that enable the standardized generation and expansion of hiPSC from large numbers of donors are required. Paralleled automated processes for the expansion of hiPSC could provide an opportunity to maximize the generation of hiPSC collections from patient cohorts while minimizing hands-on time and costs. In order to develop a simple method for the parallel expansion of human pluripotent stem cells (hPSC) we established a protocol for their cultivation as undifferentiated aggregates in a bench-top bioreactor system (BioLevitator™). We show that long-term expansion (10 passages) of hPSCs either in mTeSR or E8 medium preserved a normal karyotype, three-germ-layer differentiation potential and high expression of pluripotency-associated markers. The system enables the expansion from low inoculation densities (0.3 × 10(5) cells/mL) and provides a simplified, cost-efficient and time-saving method for the provision of hiPSC at midi-scale. Implementation of this protocol in cell production schemes has the potential to advance cell manufacturing in many areas of hiPSC-based medical research. PMID:26110829

  15. Tetraploid Embryonic Stem Cells Maintain Pluripotency and Differentiation Potency into Three Germ Layers

    PubMed Central

    Imai, Hiroyuki; Kano, Kiyoshi; Fujii, Wataru; Takasawa, Ken; Wakitani, Shoichi; Hiyama, Masato; Nishino, Koichiro; Kusakabe, Ken Takeshi; Kiso, Yasuo

    2015-01-01

    Polyploid amphibians and fishes occur naturally in nature, while polyploid mammals do not. For example, tetraploid mouse embryos normally develop into blastocysts, but exhibit abnormalities and die soon after implantation. Thus, polyploidization is thought to be harmful during early mammalian development. However, the mechanisms through which polyploidization disrupts development are still poorly understood. In this study, we aimed to elucidate how genome duplication affects early mammalian development. To this end, we established tetraploid embryonic stem cells (TESCs) produced from the inner cell masses of tetraploid blastocysts using electrofusion of two-cell embryos in mice and studied the developmental potential of TESCs. We demonstrated that TESCs possessed essential pluripotency and differentiation potency to form teratomas, which differentiated into the three germ layers, including diploid embryonic stem cells. TESCs also contributed to the inner cell masses in aggregated chimeric blastocysts, despite the observation that tetraploid embryos fail in normal development soon after implantation in mice. In TESCs, stability after several passages, colony morphology, and alkaline phosphatase activity were similar to those of diploid ESCs. TESCs also exhibited sufficient expression and localization of pluripotent markers and retained the normal epigenetic status of relevant reprogramming factors. TESCs proliferated at a slower rate than ESCs, indicating that the difference in genomic dosage was responsible for the different growth rates. Thus, our findings suggested that mouse ESCs maintained intrinsic pluripotency and differentiation potential despite tetraploidization, providing insights into our understanding of developmental elimination in polyploid mammals. PMID:26091100

  16. Patents on Technologies of Human Tissue and Organ Regeneration from Pluripotent Human Embryonic Stem Cells

    PubMed Central

    Parsons, Xuejun H; Teng, Yang D; Moore, Dennis A; Snyder, Evan Y

    2011-01-01

    Human embryonic stem cells (hESCs) are genetically stable with unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of a large supply of disease-targeted human somatic cells that are restricted to the lineage in need of repair. There is a large healthcare need to develop hESC-based therapeutic solutions to provide optimal regeneration and reconstruction treatment options for the damaged or lost tissue or organ that have been lacking. In spite of controversy surrounding the ownership of hESCs, the number of patent applications related to hESCs is growing rapidly. This review gives an overview of different patent applications on technologies of derivation, maintenance, differentiation, and manipulation of hESCs for therapies. Many of the published patent applications have been based on previously established methods in the animal systems and multi-lineage inclination of pluripotent cells through spontaneous germ-layer differentiation. Innovative human stem cell technologies that are safe and effective for human tissue and organ regeneration in the clinical setting remain to be developed. Our overall view on the current patent situation of hESC technologies suggests a trend towards hESC patent filings on novel therapeutic strategies of direct control and modulation of hESC pluripotent fate, particularly in a 3-dimensional context, when deriving clinically-relevant lineages for regenerative therapies. PMID:23355961

  17. Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells

    PubMed Central

    Raya, Ángel; Rodríguez-Pizà, Ignasi; Guenechea, Guillermo; Vassena, Rita; Navarro, Susana; Barrero, María José; Consiglio, Antonella; Castellà, Maria; Río, Paula; Sleep, Eduard; González, Federico; Tiscornia, Gustavo; Garreta, Elena; Aasen, Trond; Veiga, Anna; Verma, Inder M.; Surrallés, Jordi; Bueren, Juan; Belmonte, Juan Carlos Izpisúa

    2009-01-01

    The generation of induced pluripotent stem (iPS) cells has enabled the derivation of patient-specific pluripotent cells and provided valuable experimental platforms to model human disease. Patient-specific iPS cells are also thought to hold great therapeutic potential, although direct evidence for this is still lacking. Here we show that, on correction of the genetic defect, somatic cells from Fanconi anaemia patients can be reprogrammed to pluripotency to generate patient-specific iPS cells. These cell lines appear indistinguishable from human embryonic stem cells and iPS cells from healthy individuals. Most importantly, we show that corrected Fanconi-anaemia-specific iPS cells can give rise to haematopoietic progenitors of the myeloid and erythroid lineages that are phenotypically normal, that is, disease-free. These data offer proof-of-concept that iPS cell technology can be used for the generation of disease-corrected, patient-specific cells with potential value for cell therapy applications. PMID:19483674

  18. Generation and characterization of human cryptorchid-specific induced pluripotent stem cells from urine.

    PubMed

    Zhou, Junmei; Wang, Xue; Zhang, Shengli; Gu, Yijun; Yu, Ling; Wu, Jing; Gao, Tongbin; Chen, Fang

    2013-03-01

    Cryptorchidism is a common congenital birth defect in human beings with the possible complication of infertility. An in vitro model of cryptorchidism might be valuable due to the inaccessibility of human embryos for research purposes. In this study, we reprogrammed urine cells containing genetic variations in insulin-like factor 3, zinc finger (ZNF) 214, and ZNF215 from a cryptorchid patient by introducing human OCT4, SOX2, C-MYC, and KLF4 with lentivirus. The cells were then replated on irradiated mouse embryonic fibroblasts and cultured with the human embryonic stem (ES) cell medium. The compact colonies with well-defined borders were manually picked, and 2 induced pluripotent cell lines were fully characterized. Our results demonstrated that these 2 cell lines were similar to human ES cells in morphological appearance, marker expression, and epigenetic status of the pluripotent cell-specific gene, OCT4. These cells could be differentiated into cells of all 3 germ layers in teratomas and in vitro, including into the VASA-positive germ cell lineage. Both parental urine cells and the reprogrammed cells possessed the normal karyotype and the same short tandem repeat loci, indicating that these 2 cell population share the same genetic identity. This establishment and characterization of human urine-derived cryptorchid-specific induced pluripotent stem cells could present a good human genetic system for future studies investigating the molecular mechanism of cryptorchidism. PMID:23025704

  19. Rapid and Highly Efficient Generation of Induced Pluripotent Stem Cells from Human Umbilical Vein Endothelial Cells

    PubMed Central

    Yi, Fei; Herrerías, Aída; Batchelder, Erika M.; Belmonte, Juan Carlos Izpisua

    2011-01-01

    The ability to induce somatic cells to pluripotency by ectopic expression of defined transcription factors (e.g. KLF-4, OCT4, SOX2, c-MYC, or KOSM) has transformed the future of regenerative medicine. Here we report somatic cell reprogramming of human umbilical vein endothelial cells (HUVECs), yielding induced pluripotent stem (iPS) cells with the fastest kinetics, and one of the highest reprogramming efficiencies for a human somatic cell to date. HUVEC-derived iPS (Huv-iPS) cell colonies appeared as early as 6 days after a single KOSM infection, and were generated with a 2.5–3% reprogramming efficiency. Furthermore, when HUVEC reprogramming was performed under hypoxic conditions in the presence of a TGF-beta family signaling inhibitor, colony formation increased an additional ∼2.5-fold over standard conditions. Huv-iPS cells were indistinguishable from human embryonic stem (ES) cells with regards to morphology, pluripotent marker expression, and their ability to generate all embryonic germ layers in vitro and in vivo. The high efficiency and rapid kinetics of Huv-iPS cell formation, coupled with the ease by which HUVECs can be collected, expanded and stored, make these cells an attractive somatic source for therapeutic application, and for studying the reprogramming process. PMID:21603572

  20. Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells

    PubMed Central

    Green, Michael D; Chen, Antonia; Nostro, Maria-Cristina; d'Souza, Sunita L; Schaniel, Christoph; Lemischka, Ihor R; Gouon-Evans, Valerie; Keller, Gordon; Snoeck, Hans-Willem

    2016-01-01

    Directed differentiation of human embryonic stem (hES) cells and human induced pluripotent stem (hiPS) cells captures in vivo developmental pathways for specifying lineages in vitro, thus avoiding perturbation of the genome with exogenous genetic material. Thus far, derivation of endodermal lineages has focused predominantly on hepatocytes, pancreatic endocrine cells and intestinal cells1–5. The ability to differentiate pluripotent cells into anterior foregut endoderm (AFE) derivatives would expand their utility for cell therapy and basic research to tissues important for immune function, such as the thymus; for metabolism, such as thyroid and parathyroid; and for respiratory function, such as trachea and lung. We find that dual inhibition of transforming growth factor (TGF)-β and bone morphogenic protein (BMP) signaling after specification of definitive endoderm from pluripotent cells results in a highly enriched AFE population that is competent to be patterned along dorsoventral and anteroposterior axes. These findings provide an approach for the generation of AFE derivatives. PMID:21358635

  1. Coupling of X-Chromosome reactivation with the pluripotent stem cell state

    PubMed Central

    Payer, Bernhard; Lee, Jeannie T

    2014-01-01

    X-chromosome inactivation (XCI) in female mammals is a dramatic example of epigenetic gene regulation, which entails the silencing of an entire chromosome through a wide range of mechanisms involving noncoding RNAs, chromatin-modifications, and DNA-methylation. While XCI is associated with the differentiated cell state, it is reversed by X-chromosome reactivation (XCR) ex vivo in pluripotent stem cells and in vivo in the early mouse embryo and the germline. Critical in the regulation of XCI vs. XCR is the X-inactivation center, a multigene locus on the X-chromosome harboring several long noncoding RNA genes including, most prominently, Xist and Tsix. These genes, which sit at the top of the XCI hierarchy, are by themselves controlled by pluripotency factors, coupling XCR with the naïve pluripotent stem cell state. In this point-of-view article we review the latest findings regarding this intricate relationship between cell differentiation state and epigenetic control of the X-chromosome. In particular, we discuss the emerging picture of complex multifactorial regulatory mechanisms, ensuring both a fine-tuned and robust X-reactivation process. PMID:25137047

  2. Modeling human development and disease in pluripotent stem cell-derived gastric organoids

    PubMed Central

    McCracken, Kyle W.; Catá, Emily M.; Crawford, Calyn M.; Sinagoga, Katie L.; Schumacher, Michael; Rockich, Briana E.; Tsai, Yu-Hwai; Mayhew, Christopher N.; Spence, Jason R.; Zavros, Yana; Wells, James M.

    2014-01-01

    Gastric diseases, including peptic ulcer disease and gastric cancer, affect 10% of the world’s population and are largely due to chronic H. pylori infection1–3. Species differences in embryonic development and architecture of the adult stomach make animal models suboptimal for studying human stomach organogenesis and pathogenesis4, and there is no experimental model of normal human gastric mucosa. Here we report the de novo generation of three-dimensional human gastric tissue in vitro through the directed differentiation of human pluripotent stem cells (hPSCs). We identified that temporal manipulation of the FGF, WNT, BMP, retinoic acid and EGF signaling pathways and three-dimensional growth are sufficient to generate human gastric organoids (hGOs). Developing hGOs progressed through molecular and morphogenetic stages that were nearly identical to the developing antrum of the mouse stomach. Organoids formed primitive gastric gland- and pit-like domains, proliferative zones containing LGR5-expressing cells, surface and antral mucous cells, and a diversity of gastric endocrine cells. We used hGO cultures to identify novel signaling mechanisms that regulate early endoderm patterning and gastric endocrine cell differentiation upstream of the transcription factor NEUROG3. Using hGOs to model pathogenesis of human disease, we found that H. pylori infection resulted in rapid association of the virulence factor CagA with the c-Met receptor, activation of signaling and induction of epithelial proliferation. Together, these studies describe a novel and robust in vitro system for elucidating the mechanisms underlying human stomach development and disease. PMID:25363776

  3. Stimulating progress in regenerative medicine: improving the cloning and recovery of cryopreserved human pluripotent stem cells with ROCK inhibitors

    PubMed Central

    Rizzino, Angie

    2011-01-01

    Until recently, culturing human pluripotent stem cells was hampered by three prominent technical problems: a high degree of unwanted cellular stress when the cells are passaged, unacceptably low cloning efficiency and poor recovery of cryopreserved stocks. This review discusses recent developments that address these problems. A major focus of the review is the use of p160 Rho-associated coiled-coil kinase inhibitors for improving both the cloning efficiency and the recovery of cryopreserved human embryonic stem cells and human induced pluripotent stem cells. An underlying theme of this review is that the three problems have a common cause: separation of human pluripotent stem cells from one another increases cellular stress, which greatly decreases their viability unless special steps are taken. PMID:20868334

  4. Efficient generation of rat induced pluripotent stem cells using a non-viral inducible vector.

    PubMed

    Merkl, Claudia; Saalfrank, Anja; Riesen, Nathalie; Kühn, Ralf; Pertek, Anna; Eser, Stefan; Hardt, Markus Sebastian; Kind, Alexander; Saur, Dieter; Wurst, Wolfgang; Iglesias, Antonio; Schnieke, Angelika

    2013-01-01

    Current methods of generating rat induced pluripotent stem cells are based on viral transduction of pluripotency inducing genes (Oct4, Sox2, c-myc and Klf4) into somatic cells. These activate endogenous pluripotency genes and reprogram the identity of the cell to an undifferentiated state. Epigenetic silencing of exogenous genes has to occur to allow normal iPS cell differentiation. To gain more control over the expression of exogenous reprogramming factors, we used a novel doxycycline-inducible plasmid vector encoding Oct4, Sox2, c-Myc and Klf4. To ensure efficient and controlled generation of iPS cells by plasmid transfection we equipped the reprogramming vector with a bacteriophage φC31 attB site and used a φC31 integrase expression vector to enhance vector integration. A series of doxycycline-independent rat iPS cell lines were established. These were characterized by immunocytochemical detection of Oct4, SSEA1 and SSEA4, alkaline phosphatase staining, methylation analysis of the endogenous Oct4 promoter and RT-PCR analysis of endogenous rat pluripotency genes. We also determined the number of vector integrations and the extent to which reprogramming factor gene expression was controlled. Protocols were developed to generate embryoid bodies and rat iPS cells demonstrated as pluripotent by generating derivatives of all three embryonic germ layers in vitro, and teratoma formation in vivo. All data suggest that our rat iPS cells, generated by plasmid based reprogramming, are similar to rat ES cells. Methods of DNA transfection, protein transduction and feeder-free monolayer culture of rat iPS cells were established to enable future applications. PMID:23383095

  5. Regenerative Effects of Mesenchymal Stem Cells: Contribution of Muse Cells, a Novel Pluripotent Stem Cell Type that Resides in Mesenchymal Cells.

    PubMed

    Wakao, Shohei; Kuroda, Yasumasa; Ogura, Fumitaka; Shigemoto, Taeko; Dezawa, Mari

    2012-01-01

    Mesenchymal stem cells (MSCs) are easily accessible and safe for regenerative medicine. MSCs exert trophic, immunomodulatory, anti-apoptotic, and tissue regeneration effects in a variety of tissues and organs, but their entity remains an enigma. Because MSCs are generally harvested from mesenchymal tissues, such as bone marrow, adipose tissue, or umbilical cord as adherent cells, MSCs comprise crude cell populations and are heterogeneous. The specific cells responsible for each effect have not been clarified. The most interesting property of MSCs is that, despite being adult stem cells that belong to the mesenchymal tissue lineage, they are able to differentiate into a broad spectrum of cells beyond the boundary of mesodermal lineage cells into ectodermal or endodermal lineages, and repair tissues. The broad spectrum of differentiation ability and tissue-repairing effects of MSCs might be mediated in part by the presence of a novel pluripotent stem cell type recently found in adult human mesenchymal tissues, termed multilineage-differentiating stress enduring (Muse) cells. Here we review recently updated studies of the regenerative effects of MSCs and discuss their potential in regenerative medicine. PMID:24710542

  6. Regenerative Effects of Mesenchymal Stem Cells: Contribution of Muse Cells, a Novel Pluripotent Stem Cell Type that Resides in Mesenchymal Cells

    PubMed Central

    Wakao, Shohei; Kuroda, Yasumasa; Ogura, Fumitaka; Shigemoto, Taeko; Dezawa, Mari

    2012-01-01

    Mesenchymal stem cells (MSCs) are easily accessible and safe for regenerative medicine. MSCs exert trophic, immunomodulatory, anti-apoptotic, and tissue regeneration effects in a variety of tissues and organs, but their entity remains an enigma. Because MSCs are generally harvested from mesenchymal tissues, such as bone marrow, adipose tissue, or umbilical cord as adherent cells, MSCs comprise crude cell populations and are heterogeneous. The specific cells responsible for each effect have not been clarified. The most interesting property of MSCs is that, despite being adult stem cells that belong to the mesenchymal tissue lineage, they are able to differentiate into a broad spectrum of cells beyond the boundary of mesodermal lineage cells into ectodermal or endodermal lineages, and repair tissues. The broad spectrum of differentiation ability and tissue-repairing effects of MSCs might be mediated in part by the presence of a novel pluripotent stem cell type recently found in adult human mesenchymal tissues, termed multilineage-differentiating stress enduring (Muse) cells. Here we review recently updated studies of the regenerative effects of MSCs and discuss their potential in regenerative medicine. PMID:24710542

  7. Efficient passage of human pluripotent stem cells on spider silk matrices under xeno-free conditions.

    PubMed

    Wu, Siqin; Johansson, Jan; Hovatta, Outi; Rising, Anna

    2016-04-01

    Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine and pharmaceutical development. Such applications require cell culture methods and reagents that are chemically defined, xeno-free, scalable, and low-cost. Herein, we describe non-mechanical passaging of hPSCs on spider silk films under chemically defined and xeno-free conditions. The cells were dissociated into single cells or small aggregates using Accutase or enzyme-free dissociation buffer and then passaged to spider silk films, where they expanded in monolayers until they covered the surface. Cells cultured over 10 passages on spider silk film remained karyotypically normal and pluripotent. In conclusion, a novel method for passaging dissociated hPSCs under conditions that are compatible with clinical applications is presented. The method is cost-efficient and may be useful for both research and clinical applications. PMID:26427704

  8. Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates.

    PubMed

    Sahni, Geetika; Yuan, Jun; Toh, Yi-Chin

    2016-01-01

    Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into all three germ layers. This makes them an attractive cell source for regenerative medicine and experimental modeling of normal and diseased organogenesis. However, the differentiation of hPSCs in vitro is heterogeneous and spatially disordered. Cell micropatterning technologies potentially offer the means to spatially control stem cell microenvironments and organize the resultant differentiation fates. Micropatterning hPSCs needs to take into account the stringent requirements for hPSC survival and maintenance. Here, we describe stencil micropatterning as a method that is highly compatible with hPSCs. hPSC micropatterns are specified by the geometries of the cell stencil through-holes, which physically confine the locations where hPSCs can access and attach to the underlying extracellular matrix-coated substrate. Due to this mode of operation, there is greater flexibility to use substrates that can adequately support hPSCs as compared to other cell micropatterning methods. We also highlight critical steps for the successful generation of hPSC micropatterns. As an example, we demonstrate that stencil micropatterning of hPSCs can be used to modulate spatial polarization of cell-cell and cell-matrix adhesions, which in turn determines mesoendoderm differentiation patterns. This simple and robust method to micropattern hPSCs widens the prospects of establishing experimental models to investigate tissue organization and patterning during early embryonic development. PMID:27340925

  9. Pluripotent Stem Cells Derived From Mouse and Human White Mature Adipocytes

    PubMed Central

    Abdmaulen, Raushan; Ly, Albert; Cubberly, Mark R.; Shahmirian, Laurine J.; Heydarkhan-Hagvall, Sepideh; Dumesic, Daniel A.; Yao, Yucheng

    2014-01-01

    White mature adipocytes give rise to so-called dedifferentiated fat (DFAT) cells that spontaneously undergo multilineage differentiation. In this study, we defined stem cell characteristics of DFAT cells as they are generated from adipocytes and the relationship between these characteristics and lineage differentiation. Both mouse and human DFAT cells, prepared from adipose tissue and lipoaspirate, respectively, showed evidence of pluripotency, with a maximum 5–7 days after adipocyte isolation. The DFAT cells spontaneously formed clusters in culture, which transiently expressed multiple stem cell markers, including stage-specific embryonic antigens, and Sca-1 (mouse) and CD105 (human), as determined by real-time polymerase chain reaction, fluorescence-activated cell sorting, and immunostaining. As the stem cell markers decreased, markers characteristic of the three germ layers and specific lineage differentiation, such as α-fetoprotein (endoderm, hepatic), Neurofilament-66 (ectoderm, neurogenic), and Troponin I (mesoderm, cardiomyogenic), increased. However, no teratoma formation was detected after injection in immunodeficient mice. A novel modification of the adipocyte isolation aimed at ensuring the initial purity of the adipocytes and avoiding ceiling culture allowed isolation of DFAT cells with pluripotent characteristics. Thus, the adipocyte-derived DFAT cells represent a plastic stem cell population that is highly responsive to changes in culture conditions and may benefit cell-based therapies. PMID:24396033

  10. Concise Review: Pluripotent Stem Cell-Based Regenerative Applications for Failing β-Cell Function

    PubMed Central

    Holditch, Sara J.; Terzic, Andre

    2014-01-01

    Diabetes engenders the loss of pancreatic β-cell mass and/or function, resulting in insulin deficiency relative to the metabolic needs of the body. Diabetic care has traditionally relied on pharmacotherapy, exemplified by insulin replacement to target peripheral actions of the hormone. With growing understanding of the pathogenesis of diabetic disease, alternative approaches aiming at repair and restoration of failing β-cell function are increasingly considered as complements to current diabetes therapy regimens. To this end, emphasis is placed on transplantation of exogenous pancreas/islets or artificial islets, enhanced proliferation and maturation of endogenous β cells, prevention of β-cell loss, or fortified renewal of β-like-cell populations from stem cell pools and non-β-cell sources. In light of emerging clinical experiences with human embryonic stem cells and approval of the first in-human trial with induced pluripotent stem cells, in this study we highlight advances in β-cell regeneration strategies with a focus on pluripotent stem cell platforms in the context of translational applications. PMID:24646490

  11. Angiogenic activity mediates bone repair from human pluripotent stem cell-derived osteogenic cells

    PubMed Central

    Zou, Li; Chen, Qingshan; Quanbeck, Zachary; Bechtold, Joan E.; Kaufman, Dan S.

    2016-01-01

    Human pluripotent stem cells provide a standardized resource for bone repair. However, criteria to determine which exogenous cells best heal orthopedic injuries remain poorly defined. We evaluated osteogenic progenitor cells derived from both human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs). Phenotypic and genotypic analyses demonstrated that these hESCs/hiPSCs are similar in their osteogenic differentiation efficiency and they generate osteogenic cells comparable to osteogenic cells derived from mesenchymal stromal cells (BM-MSCs). However, expression of angiogenic factors, such as vascular endothelial growth factor and basic fibroblast growth factor in these osteogenic progenitor cells are markedly different, suggesting distinct pro-angiogenic potential of these stem cell derivatives. Studies to repair a femur non-union fracture demonstrate only osteogenic progenitor cells with higher pro-angiogenic potential significantly enhance bone repair in vivo. Together, these studies highlight a key role of pro-angiogenic potential of transplanted osteogenic cells for effective cell-mediated bone repair. PMID:26980556

  12. Vitamin C stimulates human gingival stem cell proliferation and expression of pluripotent markers.

    PubMed

    Van Pham, Phuc; Tran, Nga Yen; Phan, Nhan Lu-Chinh; Vu, Ngoc Bich; Phan, Ngoc Kim

    2016-02-01

    Gingival stem cells (GSCs) are a novel source of mesenchymal stem cells (MSCs) that are easily accessed from the oral cavity. GSCs were considered valuable autograft MSCs with particular characteristics. However, the limitation in the number of available GSCs remains an obstacle. Therefore, this study aimed to stimulate GSC proliferation by ascorbic acid (AA) and determined the effects of AA on GSC pluripotent potential-related gene expression. GSCs were isolated from gum tissue by explant culture and continuously subcultured before analysis of stemness and effects of AA on pluripotent-related gene expression. GSCs cultured with various concentrations of AA showed increased proliferation in a dose-dependent manner. AA-treated GSCs showed significantly higher expression of SSEA-3, Sox-2, Oct-3/4, Nanog, and TRA-1-60 compared with control cells. More importantly, GSCs also maintained their stemness with MSC phenotypes and failed to cause tumors in nude athymic mice. Our results show that AA is a suitable factor to stimulate GSC proliferation. PMID:26487430

  13. Labeling pluripotent stem cell-derived neural progenitors with iron oxide particles for magnetic resonance imaging.

    PubMed

    Sart, Sébastien; Bejarano, Fabian Calixto; Yan, Yuanwei; Grant, Samuel C; Li, Yan

    2015-01-01

    Due to the unlimited proliferation capacity and the unique differentiation ability of pluripotent stem cells (PSCs), including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), large numbers of PSC-derived cell products are in demand for applications in drug screening, disease modeling, and especially cell therapy. In stem cell-based therapy, tracking transplanted cells with magnetic resonance imaging (MRI) has emerged as a powerful technique to reveal cell survival and distribution. This chapter illustrated the basic steps of labeling PSC-derived neural progenitors (NPs) with micron-sized particles of iron oxide (MPIO, 0.86 μm) for MRI analysis. The protocol described PSC expansion and differentiation into NPs, and the labeling of the derived cells either after replating on adherent surface or in suspension. The labeled cells can be analyzed using in vitro MRI analysis. The methods presented here can be easily adapted for cell labeling in cell processing facilities under current Good Manufacturing Practices (cGMP). The iron oxide-labeled NPs can be used for cellular monitoring of in vitro cultures and in vivo transplantation. PMID:25304204

  14. Modeling diseases of noncoding unstable repeat expansions using mutant pluripotent stem cells

    PubMed Central

    Yanovsky-Dagan, Shira; Mor-Shaked, Hagar; Eiges, Rachel

    2015-01-01

    Pathogenic mutations involving DNA repeat expansions are responsible for over 20 different neuronal and neuromuscular diseases. All result from expanded tracts of repetitive DNA sequences (mostly microsatellites) that become unstable beyond a critical length when transmitted across generations. Nearly all are inherited as autosomal dominant conditions and are typically associated with anticipation. Pathologic unstable repeat expansions can be classified according to their length, repeat sequence, gene location and underlying pathologic mechanisms. This review summarizes the current contribution of mutant pluripotent stem cells (diseased human embryonic stem cells and patient-derived induced pluripotent stem cells) to the research of unstable repeat pathologies by focusing on particularly large unstable noncoding expansions. Among this class of disorders are Fragile X syndrome and Fragile X-associated tremor/ataxia syndrome, myotonic dystrophy type 1 and myotonic dystrophy type 2, Friedreich ataxia and C9 related amyotrophic lateral sclerosis and/or frontotemporal dementia, Facioscapulohumeral Muscular Dystrophy and potentially more. Common features that are typical to this subclass of conditions are RNA toxic gain-of-function, epigenetic loss-of-function, toxic repeat-associated non-ATG translation and somatic instability. For each mechanism we summarize the currently available stem cell based models, highlight how they contributed to better understanding of the related mechanism, and discuss how they may be utilized in future investigations. PMID:26131313

  15. Induced Pluripotent Stem Cells and Outer Retinal Disease

    PubMed Central

    Yang, Jin; Cai, Bingcui; Glencer, Patrick; Li, Zhiqing; Zhang, Xiaomin; Li, Xiaorong

    2016-01-01

    The retina, which is composed of multiple layers of differing cell types, has been considered the first choice for gene therapy, disease modeling, and stem cell-derived retinal cell transplant therapy. Because of its special characteristics, the retina, located in the posterior part of the eye, can be well observed directly after gene therapy or transplantation. The blood-retinal barrier is part of a specialized ocular microenvironment that is immune privileged. This protects transplanted cells and tissue. Having two eyes makes perfect natural control possible after a single eye receives gene or stem cell therapy. For this reason, research about exploring retinal diseases' underlying molecular mechanisms and potential therapeutic approach using stem cell technique has been developing rapidly. This review is to present an up-to-date summary of the iPSC's sources, variations, differentiation methods, and the wide-ranging application of iPSCs-RPCS or iPSCs-RPE on retinal disease modeling, diagnostics, and therapeutics. PMID:26880948

  16. Biphasic modulation of Wnt signaling supports efficient foregut endoderm formation from human pluripotent stem cells.

    PubMed

    Hoepfner, Jeannine; Kleinsorge, Mandy; Papp, Oliver; Ackermann, Mania; Alfken, Susanne; Rinas, Ursula; Solodenko, Wladimir; Kirschning, Andreas; Sgodda, Malte; Cantz, Tobias

    2016-05-01

    Pluripotent stem cells (embryonic stem cells and induced pluripotent stem cells) are of great promise in regenerative medicine, including molecular studies of disease mechanisms, if the affected cell type can be authentically generated during in vitro differentiation. Most existing protocols aim to mimic embryonic development steps by the supplementation of specific cytokines and small molecules, but the involved signaling pathways need further exploration. In this study, we investigated enhanced initial activation of Wnt signaling for definitive endoderm formation and subsequent rapid shutdown of Wnt signaling for proper foregut endoderm specification using 3 μM CHIR99021 and 0.5 μg/mL of secreted frizzled-related protein 5 (sFRP-5) for biphasic modulation of the Wnt pathway. The definitive endoderm and foregut endoderm differentiation capabilities of Wnt pathway-modulated cells were determined based on the expression levels of the endodermal transcription factors SOX17 and FOXA2 and those of the transcription activator GATA4 and the α-fetoprotein (AFP) gene, respectively. Furthermore, the resulting biphasic Wnt pathway modulation was investigated at the protein level by analyzing phosphorylation of glycogen synthase kinase 3 beta (GSK3β) and β-catenin. Finally, Wnt target gene expression was determined using an improved lentiviral reporter construct that enabled robust T-cell transcription factor 4 (TCF4)/lymphoid enhancer-binding factor 1 (LEF1)-mediated luciferase expression in differentiating pluripotent stem cells. In conclusion, we demonstrated robust, homogeneous, and efficient derivation of foregut endodermal cells by inducing a biphasic modulation of the Wnt signaling pathway. PMID:26861571

  17. Disease modeling using human induced pluripotent stem cells: Lessons from the liver☆

    PubMed Central

    Gieseck, Richard L.; Colquhoun, Jennifer; Hannan, Nicholas R.F.

    2014-01-01

    Human pluripotent stem cells (hPSCs) have the capacity to differentiate into any of the hundreds of distinct cell types that comprise the human body. This unique characteristic has resulted in considerable interest in the field of regenerative medicine, given the potential for these cells to be used to protect, repair, or replace diseased, injured, and aged cells within the human body. In addition to their potential in therapeutics, hPSCs can be used to study the earliest stages of human development and to provide a platform for both drug screening and disease modeling using human cells. Recently, the description of human induced pluripotent stem cells (hIPSCs) has allowed the field of disease modeling to become far more accessible and physiologically relevant, as pluripotent cells can be generated from patients of any genetic background. Disease models derived from hIPSCs that manifest cellular disease phenotypes have been established to study several monogenic diseases; furthermore, hIPSCs can be used for phenotype-based drug screens to investigate complex diseases for which the underlying genetic mechanism is unknown. As a result, the use of stem cells as research tools has seen an unprecedented growth within the last decade as researchers look for in vitro disease models which closely mimic in vivo responses in humans. Here, we discuss the beginnings of hPSCs, starting with isolation of human embryonic stem cells, moving into the development and optimization of hIPSC technology, and ending with the application of hIPSCs towards disease modeling and drug screening applications, with specific examples highlighting the modeling of inherited metabolic disorders of the liver. This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics. PMID:24943800

  18. Disease modeling using human induced pluripotent stem cells: lessons from the liver.

    PubMed

    Gieseck, Richard L; Colquhoun, Jennifer; Hannan, Nicholas R F

    2015-01-01

    Human pluripotent stem cells (hPSCs) have the capacity to differentiate into any of the hundreds of distinct cell types that comprise the human body. This unique characteristic has resulted in considerable interest in the field of regenerative medicine, given the potential for these cells to be used to protect, repair, or replace diseased, injured, and aged cells within the human body. In addition to their potential in therapeutics, hPSCs can be used to study the earliest stages of human development and to provide a platform for both drug screening and disease modeling using human cells. Recently, the description of human induced pluripotent stem cells (hIPSCs) has allowed the field of disease modeling to become far more accessible and physiologically relevant, as pluripotent cells can be generated from patients of any genetic background. Disease models derived from hIPSCs that manifest cellular disease phenotypes have been established to study several monogenic diseases; furthermore, hIPSCs can be used for phenotype-based drug screens to investigate complex diseases for which the underlying genetic mechanism is unknown. As a result, the use of stem cells as research tools has seen an unprecedented growth within the last decade as researchers look for in vitro disease models which closely mimic in vivo responses in humans. Here, we discuss the beginnings of hPSCs, starting with isolation of human embryonic stem cells, moving into the development and optimization of hIPSC technology, and ending with the application of hIPSCs towards disease modeling and drug screening applications, with specific examples highlighting the modeling of inherited metabolic disorders of the liver. This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics. PMID:24943800

  19. The molecular nature of very small embryonic-like stem cells in adult tissues.

    PubMed

    Kim, YongHwan; Jeong, Jaeho; Kang, Hyunsook; Lim, Jisun; Heo, Jinbeom; Ratajczak, Janina; Ratajczak, Mariusz Z; Shin, Dong-Myung

    2014-11-01

    Pluripotent stem cells (PSCs) have been considered as the most important cells in regenerative medicine as they are able to differentiate into all types of cells in the human body. PSCs have been established from several sources of embryo tissue or by reprogramming of terminally differentiated adult tissue by transduction of so-called Yamanaka factors (Oct4, Sox2, Klf4, and cMyc). Interestingly, accumulating evidence has demonstrated the residence of PSCs in adult tissue and with the ability to differentiate into multiple types of tissue-committed stem cells (TCSCs). We also recently demonstrated that a population of pluripotent Oct4(+) SSEA-1(+)Sca-1(+)Lin(-)CD45(-) very small embryonic-like stem cells (VSELs) resides in the adult murine bone marrow (BM) and in other murine tissue. These very small (∼3-6 μm) cells express pluripotent markers such as Oct4, Nanog, and SSEA-1. VSELs could be specified into several tissue-residing TCSCs in response to tissue/organ injury, and thus suggesting that these cells have a physiological role in the rejuvenation of a pool of TCSCs under steady-state conditions. In this review article, we discuss the molecular nature of the rare population of VSELs which have a crucial role in regulating the pluripotency, proliferation, differentiation, and aging of these cells. PMID:25473442

  20. The Molecular Nature of Very Small Embryonic-Like Stem Cells in Adult Tissues

    PubMed Central

    Kim, YongHwan; Jeong, Jaeho; Kang, Hyunsook; Lim, Jisun; Heo, Jinbeom; Ratajczak, Janina; Ratajczak, Mariusz Z.; Shin, Dong-Myung

    2014-01-01

    Pluripotent stem cells (PSCs) have been considered as the most important cells in regenerative medicine as they are able to differentiate into all types of cells in the human body. PSCs have been established from several sources of embryo tissue or by reprogramming of terminally differentiated adult tissue by transduction of so-called Yamanaka factors (Oct4, Sox2, Klf4, and cMyc). Interestingly, accumulating evidence has demonstrated the residence of PSCs in adult tissue and with the ability to differentiate into multiple types of tissue-committed stem cells (TCSCs). We also recently demonstrated that a population of pluripotent Oct4+ SSEA-1+Sca-1+Lin−CD45− very small embryonic-like stem cells (VSELs) resides in the adult murine bone marrow (BM) and in other murine tissue. These very small (∼3–6 μm) cells express pluripotent markers such as Oct4, Nanog, and SSEA-1. VSELs could be specified into several tissue-residing TCSCs in response to tissue/organ injury, and thus suggesting that these cells have a physiological role in the rejuvenation of a pool of TCSCs under steady-state conditions. In this review article, we discuss the molecular nature of the rare population of VSELs which have a crucial role in regulating the pluripotency, proliferation, differentiation, and aging of these cells. PMID:25473442

  1. Tumourigenicity and Immunogenicity of Induced Neural Stem Cell Grafts Versus Induced Pluripotent Stem Cell Grafts in Syngeneic Mouse Brain.

    PubMed

    Gao, Mou; Yao, Hui; Dong, Qin; Zhang, Hongtian; Yang, Zhijun; Yang, Yang; Zhu, Jianwei; Xu, Minhui; Xu, Ruxiang

    2016-01-01

    Along with the development of stem cell-based therapies for central nervous system (CNS) disease, the safety of stem cell grafts in the CNS, such as induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs), should be of primary concern. To provide scientific basis for evaluating the safety of these stem cells, we determined their tumourigenicity and immunogenicity in syngeneic mouse brain. Both iPSCs and embryonic stem cells (ESCs) were able to form tumours in the mouse brain, leading to tissue destruction along with immune cell infiltration. In contrast, no evidence of tumour formation, brain injury or immune rejection was observed with iNSCs, neural stem cells (NSCs) or mesenchymal stem cells (MSCs). With the help of gene ontology (GO) enrichment analysis, we detected significantly elevated levels of chemokines in the brain tissue and serum of mice that developed tumours after ESC or iPSC transplantation. Moreover, we also investigated the interactions between chemokines and NF-κB signalling and found that NF-κB activation was positively correlated with the constantly rising levels of chemokines, and vice versa. In short, iNSC grafts, which lacked any resulting tumourigenicity or immunogenicity, are safer than iPSC grafts. PMID:27417157

  2. Tumourigenicity and Immunogenicity of Induced Neural Stem Cell Grafts Versus Induced Pluripotent Stem Cell Grafts in Syngeneic Mouse Brain

    PubMed Central

    Gao, Mou; Yao, Hui; Dong, Qin; Zhang, Hongtian; Yang, Zhijun; Yang, Yang; Zhu, Jianwei; Xu, Minhui; Xu, Ruxiang

    2016-01-01

    Along with the development of stem cell-based therapies for central nervous system (CNS) disease, the safety of stem cell grafts in the CNS, such as induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs), should be of primary concern. To provide scientific basis for evaluating the safety of these stem cells, we determined their tumourigenicity and immunogenicity in syngeneic mouse brain. Both iPSCs and embryonic stem cells (ESCs) were able to form tumours in the mouse brain, leading to tissue destruction along with immune cell infiltration. In contrast, no evidence of tumour formation, brain injury or immune rejection was observed with iNSCs, neural stem cells (NSCs) or mesenchymal stem cells (MSCs). With the help of gene ontology (GO) enrichment analysis, we detected significantly elevated levels of chemokines in the brain tissue and serum of mice that developed tumours after ESC or iPSC transplantation. Moreover, we also investigated the interactions between chemokines and NF-κB signalling and found that NF-κB activation was positively correlated with the constantly rising levels of chemokines, and vice versa. In short, iNSC grafts, which lacked any resulting tumourigenicity or immunogenicity, are safer than iPSC grafts. PMID:27417157

  3. Collagen-graft mixed cellulose esters membrane maintains undifferentiated morphology and markers of potential pluripotency in feeder-free culture of induced pluripotent stem cells.

    PubMed

    Lotfalah Moradi, Sadegh; Hajishafieeha, Zahra; Nojedehi, Shahrzad; Dinarvand, Vida; Hesami Tackallou, Saeed; Roy, Ram V; Ardeshirylajimi, Abdolreza; Soleimani, Masoud

    2016-09-01

    Induced pluripotent stem cells (iPSCs) are unique and unlimited clinical sources of stem cell therapy for the regenerative medicine. Feeder layer preparation is an important step for iPSCs production, which is expensive, time-consuming and requires conversance. In the present study, we investigated the maintenance of pluripotency, and stemness of the iPSCs through feeder-free culture on a collagen-grafted Mixed Cellulose Esters membrane (MCE-COL) after three passages during twelve days. Results have demonstrated that the iPSCs cultured on MCE-COL membrane had a fine, typical undifferentiated morphology, increased proliferation rate and significant multi-lineage differentiation potential. Alkaline phosphatase (ALP) staining and pluripotency associated gene markers expression further confirmed that iPSCs cultured on the surface of MCE-COL had more ALP positive colonies and enhanced expression of Oct-4, Nanog, Sox-2 and ALP in comparison with MCE and control groups. Since MCE-COL membrane has three dimensional structure and bioactivity, it has the potential for usage in the feeder-free culture of iPSCs, and could be a suitable candidate to use as a feeder layer in stem cells preparation. PMID:27449919

  4. Brown-like adipose progenitors derived from human induced pluripotent stem cells: Identification of critical pathways governing their adipogenic capacity.

    PubMed

    Hafner, Anne-Laure; Contet, Julian; Ravaud, Christophe; Yao, Xi; Villageois, Phi; Suknuntha, Kran; Annab, Karima; Peraldi, Pascal; Binetruy, Bernard; Slukvin, Igor I; Ladoux, Annie; Dani, Christian

    2016-01-01

    Human induced pluripotent stem cells (hiPSCs) show great promise for obesity treatment as they represent an unlimited source of brown/brite adipose progenitors (BAPs). However, hiPSC-BAPs display a low adipogenic capacity compared to adult-BAPs when maintained in a traditional adipogenic cocktail. The reasons of this feature are unknown and hamper their use both in cell-based therapy and basic research. Here we show that treatment with TGFβ pathway inhibitor SB431542 together with ascorbic acid and EGF were required to promote hiPSCs-BAP differentiation at a level similar to adult-BAP differentiation. hiPSC-BAPs expressed the molecular identity of adult-UCP1 expressing cells (PAX3, CIDEA, DIO2) with both brown (ZIC1) and brite (CD137) adipocyte markers. Altogether, these data highlighted the critical role of TGFβ pathway in switching off hiPSC-brown adipogenesis and revealed novel factors to unlock their differentiation. As hiPSC-BAPs display similarities with adult-BAPs, it opens new opportunities to develop alternative strategies to counteract obesity. PMID:27577850

  5. Brown-like adipose progenitors derived from human induced pluripotent stem cells: Identification of critical pathways governing their adipogenic capacity

    PubMed Central

    Hafner, Anne-Laure; Contet, Julian; Ravaud, Christophe; Yao, Xi; Villageois, Phi; Suknuntha, Kran; Annab, Karima; Peraldi, Pascal; Binetruy, Bernard; Slukvin, Igor I.; Ladoux, Annie; Dani, Christian

    2016-01-01

    Human induced pluripotent stem cells (hiPSCs) show great promise for obesity treatment as they represent an unlimited source of brown/brite adipose progenitors (BAPs). However, hiPSC-BAPs display a low adipogenic capacity compared to adult-BAPs when maintained in a traditional adipogenic cocktail. The reasons of this feature are unknown and hamper their use both in cell-based therapy and basic research. Here we show that treatment with TGFβ pathway inhibitor SB431542 together with ascorbic acid and EGF were required to promote hiPSCs-BAP differentiation at a level similar to adult-BAP differentiation. hiPSC-BAPs expressed the molecular identity of adult-UCP1 expressing cells (PAX3, CIDEA, DIO2) with both brown (ZIC1) and brite (CD137) adipocyte markers. Altogether, these data highlighted the critical role of TGFβ pathway in switching off hiPSC-brown adipogenesis and revealed novel factors to unlock their differentiation. As hiPSC-BAPs display similarities with adult-BAPs, it opens new opportunities to develop alternative strategies to counteract obesity. PMID:27577850

  6. Potential Role of Induced Pluripotent Stem Cells (IPSCs) for Cell-Based Therapy of the Ocular Surface.

    PubMed

    Casaroli-Marano, Ricardo P; Nieto-Nicolau, Núria; Martínez-Conesa, Eva M; Edel, Michael; B Álvarez-Palomo, Ana

    2015-01-01

    The integrity and normal function of the corneal epithelium are crucial for maintaining the cornea's transparency and vision. The existence of a cell population with progenitor characteristics in the limbus maintains a dynamic of constant epithelial repair and renewal. Currently, cell-based therapies for bio replacement-cultured limbal epithelial transplantation (CLET) and cultured oral mucosal epithelial transplantation (COMET)-present very encouraging clinical results for treating limbal stem cell deficiency (LSCD) and restoring vision. Another emerging therapeutic approach consists of obtaining and implementing human progenitor cells of different origins in association with tissue engineering methods. The development of cell-based therapies using stem cells, such as human adult mesenchymal or induced pluripotent stem cells (IPSCs), represent a significant breakthrough in the treatment of certain eye diseases, offering a more rational, less invasive, and better physiological treatment option in regenerative medicine for the ocular surface. This review will focus on the main concepts of cell-based therapies for the ocular surface and the future use of IPSCs to treat LSCD. PMID:26239129

  7. Potential Role of Induced Pluripotent Stem Cells (IPSCs) for Cell-Based Therapy of the Ocular Surface

    PubMed Central

    Casaroli-Marano, Ricardo P.; Nieto-Nicolau, Núria; Martínez-Conesa, Eva M.; Edel, Michael; Álvarez-Palomo, Ana B.

    2015-01-01

    The integrity and normal function of the corneal epithelium are crucial for maintaining the cornea’s transparency and vision. The existence of a cell population with progenitor characteristics in the limbus maintains a dynamic of constant epithelial repair and renewal. Currently, cell-based therapies for bio replacement—cultured limbal epithelial transplantation (CLET) and cultured oral mucosal epithelial transplantation (COMET)—present very encouraging clinical results for treating limbal stem cell deficiency (LSCD) and restoring vision. Another emerging therapeutic approach consists of obtaining and implementing human progenitor cells of different origins in association with tissue engineering methods. The development of cell-based therapies using stem cells, such as human adult mesenchymal or induced pluripotent stem cells (IPSCs), represent a significant breakthrough in the treatment of certain eye diseases, offering a more rational, less invasive, and better physiological treatment option in regenerative medicine for the ocular surface. This review will focus on the main concepts of cell-based therapies for the ocular surface and the future use of IPSCs to treat LSCD. PMID:26239129

  8. Generation of Human Melanocytes from Induced Pluripotent Stem Cells

    PubMed Central

    Okada, Yohei; Akamatsu, Wado; Kuwahara, Reiko; Ohyama, Manabu; Amagai, Masayuki; Matsuzaki, Yumi; Yamanaka, Shinya; Okano, Hideyuki; Kawakami, Yutaka

    2011-01-01

    Epidermal melanocytes play an important role in protecting the skin from UV rays, and their functional impairment results in pigment disorders. Additionally, melanomas are considered to arise from mutations that accumulate in melanocyte stem cells. The mechanisms underlying melanocyte differentiation and the defining characteristics of melanocyte stem cells in humans are, however, largely unknown. In the present study, we set out to generate melanocytes from human iPS cells in vitro, leading to a preliminary investigation of the mechanisms of human melanocyte differentiation. We generated iPS cell lines from human dermal fibroblasts using the Yamanaka factors (SOX2, OCT3/4, and KLF4, with or without c-MYC). These iPS cell lines were subsequently used to form embryoid bodies (EBs) and then differentiated into melanocytes via culture supplementation with Wnt3a, SCF, and ET-3. Seven weeks after inducing differentiation, pigmented cells expressing melanocyte markers such as MITF, tyrosinase, SILV, and TYRP1, were detected. Melanosomes were identified in these pigmented cells by electron microscopy, and global gene expression profiling of the pigmented cells showed a high similarity to that of human primary foreskin-derived melanocytes, suggesting the successful generation of melanocytes from iPS cells. This in vitro differentiation system should prove useful for understanding human melanocyte biology and revealing the mechanism of various pigment cell disorders, including melanoma. PMID:21249204

  9. Induction of Germ Cell-like Cells from Porcine Induced Pluripotent Stem Cells

    PubMed Central

    Wang, Hanning; Xiang, Jinzhu; Zhang, Wei; Li, Junhong; Wei, Qingqing; Zhong, Liang; Ouyang, Hongsheng; Han, Jianyong

    2016-01-01

    The ability to generate germ cells from pluripotent stem cells (PSCs) is valuable for human regenerative medicine and animal breeding. Germ cell-like cells (GCLCs) have been differentiated from mouse and human PSCs, but not from porcine PSCs, which are considered an ideal model for stem cell applications. Here, we developed a defined culture system for the induction of primordial germ cell-like cells (PGCLCs) from porcine induced PSCs (piPSCs). The identity of the PGCLCs was characterized by observing cell morphology, detecting germ cell marker gene expression and evaluating epigenetic properties. PGCLCs could further differentiate into spermatogonial stem cell-like cells (SSCLCs) in vitro. Importantly, meiosis occurred during SSCLC induction. Xenotransplantation of GCLCs into seminiferous tubules of infertile immunodeficient mice resulted in immunohistochemically identifiable germ cells in vivo. Overall, our study provides a feasible strategy for directing piPSCs to the germ cell fate and lays a foundation for exploring germ cell development mechanisms. PMID:27264660

  10. Use of Differentiated Pluripotent Stem Cells in Replacement Therapy for Treating Disease

    PubMed Central

    Fox, Ira J.; Daley, George Q.; Goldman, Steven A.; Huard, Johnny; Kamp, Timothy J.; Trucco, Massimo

    2015-01-01

    Patient-derived pluripotent stem cells (PSC) directed to various cell fates holds promise as source material for treating numerous disorders. The availability of precisely differentiated PSC-derived cells will dramatically impact blood component and hematopoietic stem cell therapies, and should facilitate treatment of diabetes, some forms of liver disease and neurologic disorders, retinal diseases, and possibly heart disease. Although an unlimited supply of specific cell types are needed, other barriers must be overcome. This review of the state of cell therapies highlights important challenges. Successful cell transplantation will require optimizing the best cell type and site for engraftment, overcoming limitations to cell migration and tissue integration, and occasionally needing to control immunologic reactivity. Collaboration among scientists, clinicians, and industry is critical for generating new stem cell-based therapies. PMID:25146295

  11. Key anticipated regulatory issues for clinical use of human induced pluripotent stem cells

    PubMed Central

    Knoepfler, Paul S

    2013-01-01

    The production of human induced pluripotent stem cells (hiPSCs) has greatly expanded the realm of possible stem cell-based regenerative medicine therapies and has particularly exciting potential for autologous therapies. However, future therapies based on hiPSCs will first have to address not only similar regulatory issues as those facing human embryonic stem cells with the US FDA and international regulatory agencies, but also hiPSCs have raised unique concerns as well. While the first possible clinical use of hiPSCs remains down the road, as a field it would be wise for us to anticipate potential roadblocks and begin formulating solutions. In this article, I discuss the potential regulatory issues facing hiPSCs and propose some potential changes in the direction of the field in response. PMID:22830621

  12. Induction of Germ Cell-like Cells from Porcine Induced Pluripotent Stem Cells.

    PubMed

    Wang, Hanning; Xiang, Jinzhu; Zhang, Wei; Li, Junhong; Wei, Qingqing; Zhong, Liang; Ouyang, Hongsheng; Han, Jianyong

    2016-01-01

    The ability to generate germ cells from pluripotent stem cells (PSCs) is valuable for human regenerative medicine and animal breeding. Germ cell-like cells (GCLCs) have been differentiated from mouse and human PSCs, but not from porcine PSCs, which are considered an ideal model for stem cell applications. Here, we developed a defined culture system for the induction of primordial germ cell-like cells (PGCLCs) from porcine induced PSCs (piPSCs). The identity of the PGCLCs was characterized by observing cell morphology, detecting germ cell marker gene expression and evaluating epigenetic properties. PGCLCs could further differentiate into spermatogonial stem cell-like cells (SSCLCs) in vitro. Importantly, meiosis occurred during SSCLC induction. Xenotransplantation of GCLCs into seminiferous tubules of infertile immunodeficient mice resulted in immunohistochemically identifiable germ cells in vivo. Overall, our study provides a feasible strategy for directing piPSCs to the germ cell fate and lays a foundation for exploring germ cell development mechanisms. PMID:27264660

  13. Microfabric Vessels for Embryoid Body Formation and Rapid Differentiation of Pluripotent Stem Cells

    PubMed Central

    Sato, Hiroki; Idiris, Alimjan; Miwa, Tatsuaki; Kumagai, Hiromichi

    2016-01-01

    Various scalable three-dimensional culture systems for regenerative medicine using human induced pluripotent stem cells (hiPSCs) have been developed to date. However, stable production of hiPSCs with homogeneous qualities still remains a challenge. Here, we describe a novel and simple embryoid body (EB) formation system using unique microfabricated culture vessels. Furthermore, this culture system is useful for high throughput EB formation and rapid generation of differentiated cells such as neural stem cells (NSCs) from hiPSCs. The period of NSC differentiation was significantly shortened under high EB density culture conditions. Simultaneous mass production of a pure population of NSCs was possible within 4 days. These results indicate that the novel culture system might not only become a unique tool to obtain new insights into developmental biology based on human stem cells, but also provide an important tractable platform for efficient and stable production of NSCs for clinical applications. PMID:27507707

  14. Microfabric Vessels for Embryoid Body Formation and Rapid Differentiation of Pluripotent Stem Cells.

    PubMed

    Sato, Hiroki; Idiris, Alimjan; Miwa, Tatsuaki; Kumagai, Hiromichi

    2016-01-01

    Various scalable three-dimensional culture systems for regenerative medicine using human induced pluripotent stem cells (hiPSCs) have been developed to date. However, stable production of hiPSCs with homogeneous qualities still remains a challenge. Here, we describe a novel and simple embryoid body (EB) formation system using unique microfabricated culture vessels. Furthermore, this culture system is useful for high throughput EB formation and rapid generation of differentiated cells such as neural stem cells (NSCs) from hiPSCs. The period of NSC differentiation was significantly shortened under high EB density culture conditions. Simultaneous mass production of a pure population of NSCs was possible within 4 days. These results indicate that the novel culture system might not only become a unique tool to obtain new insights into developmental biology based on human stem cells, but also provide an important tractable platform for efficient and stable production of NSCs for clinical applications. PMID:27507707

  15. Pluripotent stem cells, a potential source of beta-cells for diabetes therapy.

    PubMed

    Hansson, Mattias; Madsen, Ole D

    2010-04-01

    Although the reconstitution of a functional beta-cell mass by transplantation of isolated islets can restore euglycemia in the absence of insulin treatment, a shortage of donor material is one of the factors preventing the general use of cell replacement therapy for the treatment of type 1 diabetes mellitus (T1DM). Advances in the directed differentiation of pluripotent stem cells toward beta-cells via the stepwise recapitulation of embryonic development have generated proof of concept demonstrating that stem cells may be an appropriate source of cells for the generation of therapeutic beta-cells. However, progress toward a clinical application of this technology is slow and challenging. This review highlights some of the critical issues impeding the translation of stem cell-based diabetes therapies to the clinic. PMID:20336589

  16. Open chromatin in pluripotency and reprogramming

    PubMed Central

    Meshorer, Eran; Ramalho-Santos, Miguel

    2013-01-01

    Pluripotent stem cells can be derived from embryos or induced from adult cells by reprogramming. They are unique from any other stem cell in that they can give rise to all cell types of the body. Recent findings indicate that a particularly open chromatin state contributes to maintenance of pluripotency. Two emerging principles are that: specific factors maintain a globally open chromatin state that is accessible for transcriptional activation; and other chromatin regulators contribute locally to the silencing of lineage-specific genes until differentiation is triggered. These same principles may apply during reacquisition of an open chromatin state upon reprogramming to pluripotency, and during de-differentiation in cancer. PMID:21179060

  17. Sendai Virus-Based Reprogramming of Mesenchymal Stromal/Stem Cells from Umbilical Cord Wharton's Jelly into Induced Pluripotent Stem Cells.

    PubMed

    Miere, Cristian; Devito, Liani; Ilic, Dusko

    2016-01-01

    In an attempt to bring pluripotent stem cell biology closer to reaching its full potential, many groups have focused on improving reprogramming protocols over the past several years. The episomal modified Sendai virus-based vector has emerged as one of the most practical ones. Here we describe reprogramming of mesenchymal stromal/stem cells (MSC) derived from umbilical cord Wharton's Jelly into induced pluripotent stem cells (iPSC) using genome non-integrating Sendai virus-based vectors. The detailed protocols of iPSC colony cryopreservation (vitrification) and adaption to feeder-free culture conditions are also included. PMID:26246353

  18. Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes Under Defined Conditions.

    PubMed

    van den Berg, Cathelijne W; Elliott, David A; Braam, Stefan R; Mummery, Christine L; Davis, Richard P

    2016-01-01

    Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can differentiate to cardiomyocytes in vitro, offering unique opportunities to investigate cardiac development and disease as well as providing a platform to perform drug and toxicity tests. Initial cardiac differentiation methods were based on either inductive co-culture or aggregation as embryoid bodies, often in the presence of fetal calf serum. More recently, monolayer differentiation protocols have evolved as feasible alternatives and are often performed in completely defined culture medium and substrates. Thus, our ability to efficiently and reproducibly generate cardiomyocytes from multiple different hESC and hiPSC lines has improved significantly.We have developed a directed differentiation monolayer protocol that can be used to generate cultures comprising ~50% cardiomyocytes, in which both the culture of the undifferentiated human pluripotent stem cells (hPSCs) and the differentiation procedure itself are defined and serum-free. The differentiation method is also effective for hPSCs maintained in other culture systems. In this chapter, we outline the differentiation protocol and describe methods to assess cardiac differentiation efficiency as well as to identify and quantify the yield of cardiomyocytes. PMID:25626427

  19. Muse Cells, a New Type of Pluripotent Stem Cell Derived from Human Fibroblasts.

    PubMed

    Liu, Qi; Zhang, Ru-zhi; Li, Di; Cheng, Sai; Yang, Yu-hua; Tian, Ting; Pan, Xiao-ru

    2016-04-01

    A new type of mesenchymal stem cells (MSCs) that expresses stage-specific embryonic antigen 3 (SSEA-3) and the mesenchymal cell marker CD105 are known as multilineage-differentiating stress-enduring (Muse) cells. Studies have shown that stem cells in suspension cultures are more likely to generate embryoid body-like stem cell spheres and maintain an undifferentiated phenotype and pluripotency. We separated Muse cells derived from human dermal fibroblasts by long-term trypsin incubation (LTT) through suspension cultures in methylcellulose. The Muse cells obtained expressed several pluripotency markers, including Nanog, Oct4, Sox2, and SSEA-3, and could differentiate in vitro into cells of the three germ layers, such as hepatocytes (endodermal), neural cells (ectodermal) and adipocytes, and osteocytes (mesodermal cells). These cells showed a low level of DNA methylation and a high nucleo-cytoplasmic ratio. Our study provides an innovative and exciting platform for exploring the potential cell-based therapy of various human diseases using Muse cells as well as their great possibility for regenerative medicine. PMID:27055628

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

    PubMed

    Saito, Hidehito; Okita, Keisuke; Fusaki, Noemi; Sabel, Michael S; Chang, Alfred E; Ito, Fumito

    2016-01-01

    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. PMID:27057178

  1. Modeling Alzheimer's Disease with Induced Pluripotent Stem Cells: Current Challenges and Future Concerns

    PubMed Central

    Zhang, Weiwei; Jiao, Bin; Zhou, Miaojin; Zhou, Tao; Shen, Lu

    2016-01-01

    Alzheimer's disease (AD) is the most prevalent type of dementia and its pathology is characterized by deposition of extracellular β-amyloid plaques, intracellular neurofibrillary tangles, and extensive neuron loss. While only a few familial AD cases are due to mutations in three causative genes (APP, PSEN1, and PSEN2), the ultimate cause behind the rest of the cases, called sporadic AD, remains unknown. Current animal and cellular models of human AD, which are based on the Aβ and tau hypotheses only, partially resemble the familial AD. As a result, there is a pressing need for the development of new models providing insights into the pathological mechanisms of AD and for the discovery of ways to treat or delay the onset of the disease. Recent preclinical research suggests that stem cells can be used to model AD. Indeed, human induced pluripotent stem cells can be differentiated into disease-relevant cell types that recapitulate the unique genome of a sporadic AD patient or family member. In this review, we will first summarize the current research findings on the genetic and pathological mechanisms of AD. We will then highlight the existing induced pluripotent stem cell models of AD and, lastly, discuss the potential clinical applications in this field. PMID:27313629

  2. Impaired Neural Differentiation of Induced Pluripotent Stem Cells Generated from a Mouse Model of Sandhoff Disease

    PubMed Central

    Ogawa, Yasuhiro; Tanaka, Makoto; Tanabe, Miho; Suzuki, Toshihiro; Togawa, Tadayasu; Fukushige, Tomoko; Kanekura, Takuro; Sakuraba, Hitoshi; Oishi, Kazuhiko

    2013-01-01

    Sandhoff disease (SD) is a glycosphingolipid storage disease that arises from mutations in the Hexb gene and the resultant deficiency in β-hexosaminidase activity. This deficiency results in aberrant lysosomal accumulation of the ganglioside GM2 and related glycolipids, and progressive deterioration of the central nervous system. Dysfunctional glycolipid storage causes severe neurodegeneration through a poorly understood pathogenic mechanism. Induced pluripotent stem cell (iPSC) technology offers new opportunities for both elucidation of the pathogenesis of diseases and the development of stem cell-based therapies. Here, we report the generation of disease-specific iPSCs from a mouse model of SD. These mouse model-derived iPSCs (SD-iPSCs) exhibited pluripotent stem cell properties and significant accumulation of GM2 ganglioside. In lineage-directed differentiation studies using the stromal cell-derived inducing activity method, SD-iPSCs showed an impaired ability to differentiate into early stage neural precursors. Moreover, fewer neurons differentiated from neural precursors in SD-iPSCs than in the case of the wild type. Recovery of the Hexb gene in SD-iPSCs improved this impairment of neuronal differentiation. These results provide new insights as to understanding the complex pathogenic mechanisms of SD. PMID:23383290

  3. Generation of polyhormonal and multipotent pancreatic progenitor lineages from human pluripotent stem cells.

    PubMed

    Korytnikov, Roman; Nostro, Maria Cristina

    2016-05-15

    Generation of pancreatic β-cells from human pluripotent stem cells (hPSCs) has enormous importance in type 1 diabetes (T1D), as it is fundamental to a treatment strategy based on cellular therapeutics. Being able to generate β-cells, as well as other mature pancreatic cells, from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) will also enable the development of platforms that can be used for disease modeling and drug testing for a variety of pancreas-associated diseases, including cystic fibrosis. For this to occur, it is crucial to develop differentiation strategies that are robust and reproducible across cell lines and laboratories. In this article we describe two serum-free differentiation protocols designed to generate specific pancreatic lineages from hPSCs. Our approach employs a variety of cytokines and small molecules to mimic developmental pathways active during pancreatic organogenesis and allows for the in vitro generation of distinct pancreatic populations. The first protocol is designed to give rise to polyhormonal cells that have the potential to differentiate into glucagon-producing cells. The second protocol is geared to generate multipotent pancreatic progenitor cells, which harbor the potential to generate all pancreatic lineages including: monohormonal endocrine cells, acinar, and ductal cells. PMID:26515645

  4. Putative Immunogenicity Expression Profiling Using Human Pluripotent Stem Cells and Derivatives

    PubMed Central

    Awe, Jason P.; Gschweng, Eric H.; Vega-Crespo, Agustin; Voutila, Jon; Williamson, Mary H.; Truong, Brian; Kohn, Donald B.; Kasahara, Noriyuki

    2015-01-01

    Autologous human induced pluripotent stem cells (hiPSCs) should allow cellular therapeutics without an associated immune response. This concept has been controversial since the original report that syngeneic mouse iPSCs elicited an immune response after transplantation. However, an investigative analysis of any potential acute immune responses in hiPSCs and their derivatives has yet to be conducted. In the present study, we used correlative gene expression analysis of two putative mouse “immunogenicity” genes, ZG16 and HORMAD1, to assay their human homologous expression levels in human pluripotent stem cells and their derivatives. We found that ZG16 expression is heterogeneous across multiple human embryonic stem cell and hiPSC-derived cell types. Additionally, ectopic expression of ZG16 in antigen-presenting cells is insufficient to trigger a detectable response in a peripheral blood mononuclear cell coculture assay. Neither of the previous immunogenicity-associated genes in the mouse currently appears to be relevant in a human context. PMID:25575527

  5. Establishment of a primed pluripotent epiblast stem cell in FGF4-based conditions.

    PubMed

    Joo, Jin Young; Choi, Hyun Woo; Kim, Min Jung; Zaehres, Holm; Tapia, Natalia; Stehling, Martin; Jung, Koo Sung; Do, Jeong Tae; Schöler, Hans R

    2014-01-01

    Several mouse pluripotent stem cell types have been established either from mouse blastocysts and epiblasts. Among these, embryonic stem cells (ESCs) are considered to represent a "naïve", epiblast stem cells (EpiSCs) a "primed" pluripotent state. Although EpiSCs form derivatives of all three germ layers during in vitro differentiation, they rarely incorporate into the inner cell mass of blastocysts and rarely contribute to chimera formation following blastocyst injection. Here we successfully established homogeneous population of EpiSC lines with efficient chimera-forming capability using a medium containing fibroblast growth factor (FGF)-4. The expression levels of Rex1 and Nanog was very low although Oct4 level is comparable to ESCs. EpiSCs also expressed higher levels of epiblast markers, such as Cer1, Eomes, Fgf5, Sox17, and T, and further showed complete DNA methylation of Stella and Dppa5 promoters. However, the EpiSCs were clustered separately from E3 and T9 EpiSC lines and showed a completely different global gene expression pattern to ESCs. Furthermore, the EpiSCs were able to differentiate into all three germ layers in vitro and efficiently formed teratomas and chimeric embryos (21.4%) without germ-line contribution. PMID:25515008

  6. Single transcription factor reprogramming of hair follicle dermal papilla cells to induced pluripotent stem cells.

    PubMed

    Tsai, Su-Yi; Bouwman, Britta Am; Ang, Yen-Sin; Kim, Soo Jeong; Lee, Dung-Fang; Lemischka, Ihor R; Rendl, Michael

    2011-06-01

    Reprogramming patient-specific somatic cells into induced pluripotent stem (iPS) cells has great potential to develop feasible regenerative therapies. However, several issues need to be resolved such as ease, efficiency, and safety of generation of iPS cells. Many different cell types have been reprogrammed, most conveniently even peripheral blood mononuclear cells. However, they typically require the enforced expression of several transcription factors, posing mutagenesis risks as exogenous genetic material. To reduce this risk, iPS cells were previously generated with Oct4 alone from rather inaccessible neural stem cells that endogenously express the remaining reprogramming factors and very recently from fibroblasts with Oct4 alone in combination with additional small molecules. Here, we exploit that dermal papilla (DP) cells from hair follicles in the skin express all but one reprogramming factors to show that these accessible cells can be reprogrammed into iPS cells with the single transcription factor Oct4 and without further manipulation. Reprogramming was already achieved after 3 weeks and with efficiencies similar to other cell types reprogrammed with four factors. Dermal papilla-derived iPS cells are comparable to embryonic stem cells with respect to morphology, gene expression, and pluripotency. We conclude that DP cells may represent a preferred cell type for reprogramming accessible cells with less manipulation and for ultimately establishing safe conditions in the future by replacing Oct4 with small molecules. PMID:21563278

  7. Development of Gonadotropin-Releasing Hormone-Secreting Neurons from Human Pluripotent Stem Cells.

    PubMed

    Lund, Carina; Pulli, Kristiina; Yellapragada, Venkatram; Giacobini, Paolo; Lundin, Karolina; Vuoristo, Sanna; Tuuri, Timo; Noisa, Parinya; Raivio, Taneli

    2016-08-01

    Gonadotropin-releasing hormone (GnRH) neurons regulate human puberty and reproduction. Modeling their development and function in vitro would be of interest for both basic research and clinical translation. Here, we report a three-step protocol to differentiate human pluripotent stem cells (hPSCs) into GnRH-secreting neurons. Firstly, hPSCs were differentiated to FOXG1, EMX2, and PAX6 expressing anterior neural progenitor cells (NPCs) by dual SMAD inhibition. Secondly, NPCs were treated for 10 days with FGF8, which is a key ligand implicated in GnRH neuron ontogeny, and finally, the cells were matured with Notch inhibitor to bipolar TUJ1-positive neurons that robustly expressed GNRH1 and secreted GnRH decapeptide into the culture medium. The protocol was reproducible both in human embryonic stem cells and induced pluripotent stem cells, and thus provides a translational tool for investigating the mechanisms of human puberty and its disorders. PMID:27426041

  8. H1foo Has a Pivotal Role in Qualifying Induced Pluripotent Stem Cells.

    PubMed

    Kunitomi, Akira; Yuasa, Shinsuke; Sugiyama, Fumihiro; Saito, Yuki; Seki, Tomohisa; Kusumoto, Dai; Kashimura, Shin; Takei, Makoto; Tohyama, Shugo; Hashimoto, Hisayuki; Egashira, Toru; Tanimoto, Yoko; Mizuno, Saori; Tanaka, Shoma; Okuno, Hironobu; Yamazawa, Kazuki; Watanabe, Hideo; Oda, Mayumi; Kaneda, Ruri; Matsuzaki, Yumi; Nagai, Toshihiro; Okano, Hideyuki; Yagami, Ken-Ichi; Tanaka, Mamoru; Fukuda, Keiichi

    2016-06-14

    Embryonic stem cells (ESCs) are a hallmark of ideal pluripotent stem cells. Epigenetic reprogramming of induced pluripotent stem cells (iPSCs) has not been fully accomplished. iPSC generation is similar to somatic cell nuclear transfer (SCNT) in oocytes, and this procedure can be used to generate ESCs (SCNT-ESCs), which suggests the contribution of oocyte-specific constituents. Here, we show that the mammalian oocyte-specific linker histone H1foo has beneficial effects on iPSC generation. Induction of H1foo with Oct4, Sox2, and Klf4 significantly enhanced the efficiency of iPSC generation. H1foo promoted in vitro differentiation characteristics with low heterogeneity in iPSCs. H1foo enhanced the generation of germline-competent chimeric mice from iPSCs in a manner similar to that for ESCs. These findings indicate that H1foo contributes to the generation of higher-quality iPSCs. PMID:27237376

  9. Aneuploidy is permissive for hepatocyte-like cell differentiation from human induced pluripotent stem cells

    PubMed Central

    2014-01-01

    Background The characterization of induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) routinely includes analyses of chromosomal integrity. The belief is that pluripotent stem cells best suited to the generation of differentiated derivatives should display a euploid karyotype; although, this does not appear to have been formally tested. While aneuploidy is commonly associated with cell transformation, several types of somatic cells, including hepatocytes, are frequently aneuploid and variation in chromosomal content does not contribute to a transformed phenotype. This insight has led to the proposal that dynamic changes in the chromosomal environment may be important to establish genetic diversity within the hepatocyte population and such diversity may facilitate an adaptive response by the liver to various insults. Such a positive contribution of aneuploidy to liver function raises the possibility that, in contrast to existing dogma, aneuploid iPSCs may be capable of generating hepatocyte-like cells that display hepatic activities. Results We examined whether a human iPSC line that had multiple chromosomal aberrations was competent to differentiate into hepatocytes and found that loss of normal chromosomal content had little impact on the production of hepatocyte-like cells from iPSCs. Conclusions iPSCs that harbor an abnormal chromosomal content retain the capacity to generate hepatocyte–like cells with high efficiency. PMID:25002137

  10. Modeling Alzheimer's Disease with Induced Pluripotent Stem Cells: Current Challenges and Future Concerns.

    PubMed

    Zhang, Weiwei; Jiao, Bin; Zhou, Miaojin; Zhou, Tao; Shen, Lu

    2016-01-01

    Alzheimer's disease (AD) is the most prevalent type of dementia and its pathology is characterized by deposition of extracellular β-amyloid plaques, intracellular neurofibrillary tangles, and extensive neuron loss. While only a few familial AD cases are due to mutations in three causative genes (APP, PSEN1, and PSEN2), the ultimate cause behind the rest of the cases, called sporadic AD, remains unknown. Current animal and cellular models of human AD, which are based on the Aβ and tau hypotheses only, partially resemble the familial AD. As a result, there is a pressing need for the development of new models providing insights into the pathological mechanisms of AD and for the discovery of ways to treat or delay the onset of the disease. Recent preclinical research suggests that stem cells can be used to model AD. Indeed, human induced pluripotent stem cells can be differentiated into disease-relevant cell types that recapitulate the unique genome of a sporadic AD patient or family member. In this review, we will first summarize the current research findings on the genetic and pathological mechanisms of AD. We will then highlight the existing induced pluripotent stem cell models of AD and, lastly, discuss the potential clinical applications in this field. PMID:27313629

  11. Efficient CRISPR/Cas9-Based Genome Engineering in Human Pluripotent Stem Cells.

    PubMed

    Kime, Cody; Mandegar, Mohammad A; Srivastava, Deepak; Yamanaka, Shinya; Conklin, Bruce R; Rand, Tim A

    2016-01-01

    Human pluripotent stem cells (hPS cells) are rapidly emerging as a powerful tool for biomedical discovery. The advent of human induced pluripotent stem cells (hiPS cells) with human embryonic stem (hES)-cell-like properties has led to hPS cells with disease-specific genetic backgrounds for in vitro disease modeling and drug discovery as well as mechanistic and developmental studies. To fully realize this potential, it will be necessary to modify the genome of hPS cells with precision and flexibility. Pioneering experiments utilizing site-specific double-strand break (DSB)-mediated genome engineering tools, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have paved the way to genome engineering in previously recalcitrant systems such as hPS cells. However, these methods are technically cumbersome and require significant expertise, which has limited adoption. A major recent advance involving the clustered regularly interspaced short palindromic repeats (CRISPR) endonuclease has dramatically simplified the effort required for genome engineering and will likely be adopted widely as the most rapid and flexible system for genome editing in hPS cells. In this unit, we describe commonly practiced methods for CRISPR endonuclease genomic editing of hPS cells into cell lines containing genomes altered by insertion/deletion (indel) mutagenesis or insertion of recombinant genomic DNA. PMID:26724721

  12. Direct induction of haematoendothelial programs in human pluripotent stem cells by transcriptional regulators.

    PubMed

    Elcheva, Irina; Brok-Volchanskaya, Vera; Kumar, Akhilesh; Liu, Patricia; Lee, Jeong-Hee; Tong, Lilian; Vodyanik, Maxim; Swanson, Scott; Stewart, Ron; Kyba, Michael; Yakubov, Eduard; Cooke, John; Thomson, James A; Slukvin, Igor

    2014-01-01

    Advancing pluripotent stem cell technologies for modelling haematopoietic stem cell development and blood therapies requires identifying key regulators of haematopoietic commitment from human pluripotent stem cells (hPSCs). Here, by screening the effect of 27 candidate factors, we reveal two groups of transcriptional regulators capable of inducing distinct haematopoietic programs from hPSCs: pan-myeloid (ETV2 and GATA2) and erythro-megakaryocytic (GATA2 and TAL1). In both cases, these transcription factors directly convert hPSCs to endothelium, which subsequently transform into blood cells with pan-myeloid or erythro-megakaryocytic potential. These data demonstrate that two distinct genetic programs regulate the haematopoietic development from hPSCs and that both of these programs specify hPSCs directly to haemogenic endothelial cells. In addition, this study provides a novel method for the efficient induction of blood and endothelial cells from hPSCs via the overexpression of modified mRNA for the selected transcription factors. PMID:25019369

  13. Production of Human Pluripotent Stem Cell Therapeutics Under Defined Xeno-free Conditions: Progress and Challenges

    PubMed Central

    Fan, Yongjia; Wu, Jincheng; Ashok, Preeti; Hsiung, Michael; Tzanakakis, Emmanuel S.

    2014-01-01

    Recent advances on human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have brought us closer to the realization of their clinical potential. Nonetheless, tissue engineering and regenerative medicine applications will require the generation of hPSC products well beyond the laboratory scale. This also mandates the production of hPSC therapeutics in fully-defined, xeno-free systems and in a reproducible manner. Toward this goal, we summarize current developments in defined media free of animal-derived components for hPSC culture. Bioinspired and synthetic extracellular matrices for the attachment growth and differentiation of hPSCs are also reviewed. Given that most progress in xeno-free medium and substrate development has been demonstrated in two-dimensional rather than three dimensional culture systems, translation from the former to the latter poses unique difficulties. These challenges are discussed in the context of cultivation platforms of hPSCs as aggregates, on microcarriers or after encapsulation in biocompatible scaffolds. PMID:25077810

  14. Induced Pluripotent Stem Cells Generated from P0-Cre;Z/EG Transgenic Mice

    PubMed Central

    Ogawa, Yasuhiro; Eto, Akira; Miyake, Chisato; Tsuchida, Nana; Miyake, Haruka; Takaku, Yasuhiro; Hagiwara, Hiroaki; Oishi, Kazuhiko

    2015-01-01

    Neural crest (NC) cells are a migratory, multipotent cell population that arises at the neural plate border, and migrate from the dorsal neural tube to their target tissues, where they differentiate into various cell types. Abnormal development of NC cells can result in severe congenital birth defects. Because only a limited number of cells can be obtained from an embryo, mechanistic studies are difficult to perform with directly isolated NC cells. Protein zero (P0) is expressed by migrating NC cells during the early embryonic period. In the P0-Cre;Z/EG transgenic mouse, transient activation of the P0 promoter induces Cre-mediated recombination, indelibly tagging NC-derived cells with enhanced green fluorescent protein (EGFP). Induced pluripotent stem cell (iPSC) technology offers new opportunities for both mechanistic studies and development of stem cell-based therapies. Here, we report the generation of iPSCs from the P0-Cre;Z/EG mouse. P0-Cre;Z/EG mouse-derived iPSCs (P/G-iPSCs) exhibited pluripotent stem cell properties. In lineage-directed differentiation studies, P/G-iPSCs were efficiently differentiated along the neural lineage while expressing EGFP. These results suggest that P/G-iPSCs are useful to study NC development and NC-associated diseases. PMID:26382630

  15. Potential and Limitation of HLA-Based Banking of Human Pluripotent Stem Cells for Cell Therapy

    PubMed Central

    Villard, Jean

    2014-01-01

    Great hopes have been placed on human pluripotent stem (hPS) cells for therapy. Tissues or organs derived from hPS cells could be the best solution to cure many different human diseases, especially those who do not respond to standard medication or drugs, such as neurodege