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  • Variability in the generation of induced pluripotent stem cells: importance for disease modeling. 23197870

    In the field of disease modeling, induced pluripotent stem cells (iPSCs) have become an appealing choice, especially for diseases that do not have an animal model. They can be generated from patients with known clinical features and compared with cells from healthy controls to identify the biological bases of disease. This study was undertaken to determine the variability in iPSC lines derived from different individuals, with the aim of determining criteria for selecting iPSC lines for disease models. We generated and characterized 18 iPSC lines from eight donors and considered variability at three levels: (a) variability in the criteria that define iPSC lines as pluripotent cells, (b) variability in cell lines from different donors, and (c) variability in cell lines from the same donor. We found that variability in transgene expression and pluripotency marker levels did not prevent iPSCs from fulfilling all other criteria for pluripotency, including teratoma formation. We found low interindividual and interclonal variability in iPSCs that fulfilled the most stringent criteria for pluripotency, with very high correlation in their gene expression profiles. Interestingly, some cell lines exhibited reprogramming instability, spontaneously regressing from a fully to a partially reprogrammed state. This was associated with a low percentage of cells expressing the pluripotency marker stage-specific embryonic antigen-4. Our study shows that it is possible to define a similar "ground state" for each cell line as the basis for making patient versus control comparisons, an essential step in order to identify disease-associated variability above individual and cell line variability.
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  • Characterization of a novel embryonic stem cell line from an ICSI-derived blastocyst in the African green monkey. 19955206

    Several cell types from the African green monkey (Cercopithecus aethiops), such as red blood cells, primary culture cells from kidney, and the Vero cell line, are valuable sources for biomedical research and testing. Embryonic stem (ES) cells that are established from blastocysts have pluripotency to differentiate into these and other types of cells. We examined an in vitro culture system of zygotes produced by ICSI in African green monkeys and attempted to establish ES cells. Culturing with and without a mouse embryonic fibroblast (MEF) cell monolayer resulted in the development of ICSI-derived zygotes to the blastocyst stage, while culturing with a buffalo rat liver cell monolayer yielded no development (3/14, 21.4% and 6/31, 19.4% vs 0/23, 0% respectively; P0.05). One of the nine blastocysts, which had been one of the zygotes co-cultured with MEF cells, formed flat colonies consisting of cells with large nuclei, similar to other primate ES cell lines. The African green monkey ES (AgMES) cells expressed pluripotency markers, formed teratomas consisting of three embryonic germ layer tissues, and had a normal chromosome number. Furthermore, expression of the germ cell markers CD9 and DPPA3 (STELLA) was detected in the embryoid bodies, suggesting that AgMES cells might have the potential ability to differentiate into germ cells. The results suggested that MEF cells greatly affected the quality of the inner cell mass of the blastocysts. In addition, AgMES cells would be a precious resource for biomedical research such as other primate ES cell lines.
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  • Bovine induced pluripotent stem cells are more resistant to apoptosis than testicular cells in response to mono-(2-ethylhexyl) phthalate. 24658443

    Although the androgen receptor (AR) has been implicated in the promotion of apoptosis in testicular cells (TSCs), the molecular pathway underlying AR-mediated apoptosis and its sensitivity to environmental hormones in TSCs and induced pluripotent stem cells (iPSCs) remain unclear. We generated the iPSCs from bovine TSCs via the electroporation of OCT4. The established iPSCs were supplemented with leukemia inhibitory factor and bone morphogenetic protein 4 to maintain and stabilize the expression of stemness genes and their pluripotency. Apoptosis signaling was assessed after exposure to mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of di-(2-ethylhexyl) phthalate. Here, we report that iPSCs were more resistant to MEHP-induced apoptosis than were original TSCs. MEHP also repressed the expression of AR and inactivated WNT signaling, and then led to the commitment of cells to apoptosis via the cyclin dependent kinase inhibitor p21CIP1. The loss of the frizzed receptor 7 and the gain of p21CIP were responsible for the stimulatory effect of MEHP on AR-mediated apoptosis. Our results suggest that testicular iPSCs can be used to study the signaling pathways involved in the response to environmental disruptors, and to assess the toxicity of environmental endocrine disruptors in terms of the maintenance of stemness and pluripotency.
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  • Expression of pluripotent stem cell markers in the human fetal ovary. 18203707

    Human primordial germ cells (PGCs) can give rise to pluripotent stem cells such as embryonal carcinoma cells (ECCs) and embryonic germ cells (EGCs).In order to determine whether PGCs express markers associated with pluripotency in EGCs and ECCs, the following study cross examines the expression patterns of multiple pluripotent markers in the human fetal ovary, 5.5-15 weeks post-fertilizaton (pF) and relates this expression with the ability to derive pluripotent EGCs in vitro.Specific subpopulations were identified which included OCT4(+)/Nanog(+)/cKIT(+)/VASA(+) PGCs and oogonia. Interestingly, these cells also expressed SSEA1 and alkaline phosphatase (AP) and SSEA4 expression occurred throughout the entire gonad. Isolation of SSEA1(+) cells from the gonad resulted in AP(+) EGC colony formation. The number of OCT4(+) or Nanog(+) expressing cells peaked by week 8 and then diminished after week 9 pF, as oogonia enter meiosis. In addition, the efficiency of EGC derivation was associated with the number of OCT4(+) cells. TRA-1-60 and TRA-1-81 were only detected in the lining of the mesonephric ducts and occasionally in the gonad.These results demonstrate that PGCs, a unipotent cell, express most, but not all, of the markers associated with pluripotent cells in the human fetal ovary.
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  • A novel approach for the derivation of putative primordial germ cells and sertoli cells from human embryonic stem cells. 18845765

    Using human embryonic stem cells (hESCs), we describe a novel method for the rapid derivation and enrichment of cells that are comparable to primordial germ cells (PGCs) and Sertoli cells. The methodology described is based on modest changes to the growth conditions commonly used to expand hESCs and does not require genetic manipulation or complex three-dimensional culture. Remarkably, we have determined that simply reducing the size of cultured ESC colonies and manipulating the number of feeding cycles, results in the rapid emergence of cells that are comparable to migratory PGCs. Importantly, these cells can be monitored and purified on the basis of the expression of the chemokine receptor CXCR4. Under more stringent differentiating conditions these cells mature and upregulate the expression of specific germ cell markers. Importantly, this process is accompanied by the development of Sertoli-like support cells. Such cells normally provide trophic support and immunoprotection to developing germ cells and may have significant clinical utility in the prevention of graft rejection. The putative Sertoli-germ cell cocultures generated in this study may ultimately be developed to study and manipulate interactions and processes involved in human gametogenesis.
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  • Methionine sulfoxide reductase A (MsrA) protects cultured mouse embryonic stem cells from H2O2-mediated oxidative stress. 20506347

    Methionine sulfoxide reductase A (MsrA), a member of the Msr gene family, can reduce methionine sulfoxide residues in proteins formed by oxidation of methionine by reactive oxygen species (ROS). Msr is an important protein repair system which can also function to scavenge ROS. Our studies have confirmed the expression of MsrA in mouse embryonic stem cells (ESCs) in culture conditions. A cytosol-located and mitochondria-enriched expression pattern has been observed in these cells. To confirm the protective function of MsrA in ESCs against oxidative stress, a siRNA approach has been used to knockdown MsrA expression in ES cells which showed less resistance than control cells to hydrogen peroxide treatment. Overexpression of MsrA gene products in ES cells showed improved survivability of these cells to hydrogen peroxide treatment. Our results indicate that MsrA plays an important role in cellular defenses against oxidative stress in ESCs. Msr genes may provide a new target in stem cells to increase their survivability during the therapeutic applications.
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  • Primed pluripotent cell lines derived from various embryonic origins and somatic cells in pig. 23326334

    Since pluripotent embryonic stem cell (ESC) lines were first derived from the mouse, tremendous efforts have been made to establish ESC lines in several domestic species including the pig; however, authentic porcine ESCs have not yet been established. It has proven difficult to maintain an ESC-like state in pluripotent porcine cell lines due to the frequent occurrence of spontaneous differentiation into an epiblast stem cell (EpiSC)-like state during culture. We have been able to derive EpiSC-like porcine ESC (pESC) lines from blastocyst stage porcine embryos of various origins, including in vitro fertilized (IVF), in vivo derived, IVF aggregated, and parthenogenetic embryos. In addition, we have generated induced pluripotent stem cells (piPSCs) via plasmid transfection of reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) into porcine fibroblast cells. In this study, we analyzed characteristics such as marker expression, pluripotency and the X chromosome inactivation status in female of our EpiSC-like pESC lines along with our piPSC line. Our results show that these cell lines demonstrate the expression of genes associated with the Activin/Nodal and FGF2 pathways along with the expression of pluripotent markers Oct4, Sox2, Nanog, SSEA4, TRA 1-60 and TRA 1-81. Furthermore all of these cell lines showed in vitro differentiation potential, the X chromosome inactivation in female and a normal karyotype. Here we suggest that the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines.
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  • Adaptation to robust monolayer expansion produces human pluripotent stem cells with improved viability. 23486835

    The generation of human pluripotent stem cells (hPSCs) of sufficient quantity and quality remains a major challenge for biomedical application. Here we present an efficient feeder-free, high-density monolayer system in which hPSCs become SSEA-3-high and gradually more viable than their feeder-dependent counterparts without changes attributed to culture adaptation. As a consequence, monolayer hPSCs possess advantages over their counterparts in embryoid body development, teratoma formation, freezing as a single-cell suspension, and colony-forming efficiency. Importantly, this monolayer culture system is reversible, preserving the competence of hPSCs to gradually reacquire features of colony growth, if necessary. Therefore, the monolayer culture system is highly suitable for long-term, large-scale propagation of hPSCs, which is necessary in drug development and pluripotent stem cell-based therapies.
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  • Reprogramming factors involved in hybrids and cybrids of human embryonic stem cells fused with hepatocytes. 20936904

    Embryonic stem cells (ESCs) have the potential to reprogram somatic cells into ESC-like cells through cell fusion. In the present study, the potential of human (h)ESC cytoplasts and karyoplasts to reprogram human hepatocytes was evaluated. Green fluorescent protein (GFP) transfected hESCs (ENVY cells) were fused with SNARF-1 (CellTracker)-labeled human hepatocytes using polyethylene glycol (PEG) and fluorescence-activated cell sorting (FACS) to produce hESC-hepatocyte hybrids. Immunocytochemical analysis of ESC markers showed that the hybrids expressed OCT4, TRA-1-60, TRA-1-81, SSEA-4, and GCTM-2. However, SSEA-1, which is typically low or absent on hESCs, was detected on hESC–hepatocyte hybrids. Moreover, reverse transcriptase polymerase chain reaction (RT-PCR) showed that alpha-fetoprotein, which is highly expressed in hepatocytes, was erased in the hybrids. These results indicated that hESCs have the potential to reprogram hepatocyte phenotype to a relatively undifferentiated state, but such hybrid cells are not identical to hESCs. Although hESC–hepatocyte hybrids were aneuploid, they were able to differentiate into embryoid bodies and some types of somatic cells. Furthermore, cybrids of enucleated hESCs and hepatocytes were produced by cell fusion, but the cybrids were unable to self-renew in the same way as hESCs. Presumably, the reprogramming factors are associated with the karyoplast and not the cytoplast of hESCs.
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  • Efficient, high-throughput transfection of human embryonic stem cells. 20659329

    Genetic manipulation of human embryonic stem cells (hESC) has been limited by their general resistance to common methods used to introduce exogenous DNA or RNA. Efficient and high throughput transfection of nucleic acids into hESC would be a valuable experimental tool to manipulate these cells for research and clinical applications.We investigated the ability of two commercially available electroporation systems, the Nucleofection® 96-well Shuttle® System from Lonza and the Neon™ Transfection System from Invitrogen to efficiently transfect hESC. Transfection efficiency was measured by flow cytometry for the expression of the green fluorescent protein and the viability of the transfected cells was determined by an ATP catalyzed luciferase reaction. The transfected cells were also analyzed by flow cytometry for common markers of pluripotency.Both systems are capable of transfecting hESC at high efficiencies with little loss of cell viability. However, the reproducibility and the ease of scaling for high throughput applications led us to perform more comprehensive tests on the Nucleofection® 96-well Shuttle® System. We demonstrate that this method yields a large fraction of transiently transfected cells with minimal loss of cell viability and pluripotency, producing protein expression from plasmid vectors in several different hESC lines. The method scales to a 96-well plate with similar transfection efficiencies at the start and end of the plate. We also investigated the efficiency with which stable transfectants can be generated and recovered under antibiotic selection. Finally, we found that this method is effective in the delivery of short synthetic RNA oligonucleotides (siRNA) into hESC for knockdown of translation activity via RNA interference.Our results indicate that these electroporation methods provide a reliable, efficient, and high-throughput approach to the genetic manipulation of hESC.
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