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Results for “Embryonic stem cells”

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PubMed2006

[PI3-kinase inhibitors LY294002 and wortmannin have different effects on proliferation of murine embryonic stem cells].

Murine embryonic stem (mES) cells can proliferate independently of the presence of growth factors in the medium. It is yet unknown what intrinsic activity triggers cell cycle events in mES cells. Here we investigated the contribution of the PI3-kinase cascade to autonomous proliferation of mES cell using PI3-kinase inhibitors wortmannin and LY294002. Wortmannin displays a weaker inhibitory effect on phosphorylation of PI3-kinase pathway target PKB as compared with LY294002, and does not downregulate mES cells proliferation, while LY294002 causes a strong decrease in the share of cells in S-phase and accumulation of cells in G1 phase. Both inhibitors cause significant decrease in cyclin D1 amount. The treatment with LY294002, rather than with wortmannin results in a decrease of cyclin E amount and cyclin E-assossiated kinase activity. In mES cells, inactivation of PI3-kinase-dependent pathway and G1 arrest are not accompanied by induction of p27kip 1 transcription and accumulation of this inhibitor of cyclin-cdk complexes at the protein level, implying that these events accomplished by some p27kip 1-independent mechanism. Both LY294002 and wortmannin cause apoptotic death of mES cells and downregulate the growth of population. Thus, inactivation of PI3-kinase in mES cells may lead to apoptosis rather than to cell cycle arrest.

Androstadienes↗

PubMed2003

Differentiation of radial glia-like cells from embryonic stem cells.

Radial glial cells play important roles in neural development. They provide support and guidance for neuronal migration and give rise to neurons and glia. In vitro, neurons, astrocytes, and oligodendrocytes can be generated from neural and embryonic stem cells, but the generation of radial glial cells from these stem cells has not yet been reported. Since the differentiation of radial glial cells is indispensable during brain development, we hypothesize that stem cells also generate radial glial cells during in vitro neural differentiation. To test this hypothesis, we utilized five different clones of mouse embryonic (ES) and embryonal carcinoma (EC) stem cell lines to investigate the differentiation of radial glial cells during in vitro neural differentiation. Here, we demonstrate that radial glia-like cells can be generated from ES/EC cell lines. These ES/EC cell-derived radial glia-like cells are similar in morphology to radial glial cells in vivo, i.e., they are bipolar with an unbranched long process and a short process. They also express several cytoskeletal markers, such as nestin, RC2, and/or GFAP, that are characteristics of radial glial cells in vivo. The processes of these in vitro generated radial glia-like cells are organized into parallel arrays that resemble the radial glial scaffolds in neocortical development. Since radial glia-like cells were observed in all five clones of ES/EC cells tested, we suggest that the differentiation of radial glial cells may be a common pathway during in vitro neural differentiation of ES cells. This novel in vitro model system should facilitate the investigation of regulation of radial glial cell differentiation and its biological function.

Animals↗

PubMed2004

Transforming growth factor-beta1 signaling contributes to development of smooth muscle cells from embryonic stem cells.

Knockout of transforming growth factor (TGF)-beta1 or components of its signaling pathway leads to embryonic death in mice due to impaired yolk sac vascular development before significant smooth muscle cell (SMC) maturation occurs. Thus the role of TGF-beta1 in SMC development remains unclear. Embryonic stem cell (ESC)-derived embryoid bodies (EBs) recapitulate many of the events of early embryonic development and represent a more physiological context in which to study SMC development than most other in vitro systems. The present studies showed induction of the SMC-selective genes smooth muscle alpha-actin (SMalphaA), SM22alpha, myocardin, smoothelin-B, and smooth muscle myosin heavy chain (SMMHC) within a mouse ESC-EB model system. Significantly, SM2, the SMMHC isoform associated with fully differentiated SMCs, was expressed. Importantly, the results showed that aggregates of SMMHC-expressing cells exhibited visible contractile activity, suggesting that all regulatory pathways essential for development of contractile SMCs were functional in this in vitro model system. Inhibition of endogenous TGF-beta with an adenovirus expressing a soluble truncated TGF-beta type II receptor attenuated the increase in SMC-selective gene expression in the ESC-EBs, as did an antibody specific for TGF-beta1. Of interest, the results of small interfering (si)RNA experiments provided evidence for differential TGF-beta-Smad signaling for an early vs. late SMC marker gene in that SMalphaA promoter activity was dependent on both Smad2 and Smad3 whereas SMMHC activity was Smad2 dependent. These results are the first to provide direct evidence that TGF-beta1 signaling through Smad2 and Smad3 plays an important role in the development of SMCs from totipotential ESCs.

Actins↗

PubMed2002

Characterization of the expression of MHC proteins in human embryonic stem cells.

Human embryonic stem (ES) cells are pluripotent cells that may be used in transplantation medicine. These cells can be induced to differentiate into cells from the three embryonic germ layers both in vivo and in vitro. To determine whether human ES cells might be rejected after transplantation, we examined cell surface expression of the MHC proteins in these cells. Our results show very low expression levels of MHC class I (MHC-I) proteins on the surface of human ES cells that moderately increase on in vitro or in vivo differentiation. A dramatic induction of MHC-I proteins was observed when the cells were treated with IFN-gamma but not with IFN-alpha or -beta. However, all three IFNs induced expression of MHC-I proteins in differentiated human ES cells. MHC-II proteins and HLA-G were not expressed on the surface of undifferentiated or differentiated cells. Ligands for natural killer cell receptors were either absent or expressed in very low levels in human ES cells and in their differentiated derivatives. In accordance, natural killer cytotoxic assays demonstrated only limited lysis of both undifferentiated and differentiated cells. To initiate a histocompatibility databank of human ES cells, we have isotyped several of the published ES cell lines for their human leukocyte antigens. In conclusion, our results demonstrate that human ES cells can express high levels of MHC-I proteins and thus may be rejected on transplantation.

Cell Differentiation↗

PubMed2005

Hypothesis: cell plasticity, linking embryonal stem cells to adult stem cell reservoirs and metastatic cancer cells?

Embryonal stem (ES) cells are the earliest ontogenetically identifiable stem cells of the embryo proper for all subsequent mesenchymal stem cells and for highly specialized differentiated cells. This review characterizes, in a working hypothesis, the role of reversible EMT/MET (epithelialmesenchymal transition) as a manifestation of cell plasticity 1) in the development of ES cells to adult stem cells (hematopoietic stem cells) and 2) in metastasizing cancer cells. Animal studies support the concept that EMT/MET is a key manifestation of cell plasticity in the development of ES cells to adult stem cells, and in conversion of localized to metastasizing cancer cells. In fact, ES cells may persist to postnatal life, in cytologically verifiable form and/or within the frame of EMT/MET, as ultimate reservoir for adult stem cells. Furthermore, EMT could possibly serve as a conceptional link between physiologic and pathologic signaling pathways. Clonal confirmation in humans is necessary.

Adult↗

PubMed2006

Nuclear reprogramming of somatic cells by embryonic stem cells is affected by cell cycle stage.

Hybrid embryonic stem (ES)-like clones were generated by fusion of murine ES cells with somatic cells that carried a neo resistance gene under the transcriptional control of the Oct-4 promoter. The Oct-4 promoter was reactivated in hybrid ES cells formed by fusion with fetal fibroblasts, and all hybrid colonies were of ES rather than fibroblast phenotype, suggesting efficient reprogramming of fibroblast chromosomes. Like normal diploid murine ES cells, hybrid lines expressed alkaline phosphatase activity and formed differentiated cells derived from the three embryonic germ layers both in vitro and in vivo. Treatments thought to affect nuclear transfer efficiency (ES cell confluence and serum starvation of primary embryonic fibroblasts) were investigated to determine whether they had an effect on reprogramming in cell hybrids. Serum starvation of primary embryonic fibroblasts increased hybrid colony number 50-fold. ES cells were most effective at reprogramming when they contained a high proportion of cells in the S and G2/M phases of the cell cycle. These data suggest that nuclear reprogramming requires an initial round of somatic DNA replication of quiescent chromatin in the presence of ES-derived factors produced during S and G2/M phases.

Alkaline Phosphatase↗

PubMed2004

Available human feeder cells for the maintenance of human embryonic stem cells.

Mouse embryonic fibroblasts (MEFs) have been previously used as feeder cells to support the growth of human embryonic stem cells (hESCs). In this study, human adult uterine endometrial cells (hUECs), human adult breast parenchymal cells (hBPCs) and embryonic fibroblasts (hEFs) were tested as feeder cells for supporting the growth of hESCs to prevent the possibility of contamination from animal feeder cells. Cultured hUECs, hBPCs and hEFs were mitotically inactivated and then plated. hESCs (Miz-hES1, NIH registered) initially established on mouse feeder layers were transferred onto each human feeder layer and split every 5 days. The morphology, expression of specific markers and differentiation capacity of hESCs adapted on each human feeder layer were examined. On hUEC, hBPC and hEF feeder layers, hESCs proliferated for more than 90, 50 and 80 passages respectively. Human feeder-based hESCs were positive for stage-specific embryonic antigen (SSEA)-3 and -4, and Apase; they also showed similar differentiation capacity to MEF-based hESCs, as assessed by the formation of teratomas and expression of tissue-specific markers. However, hESCs cultured on hUEC and hEF feeders were slightly thinner and flatter than MEF- or hBPC-based hESCs. Our results suggest that, like MEF feeder layers, human feeder layers can support the proliferation of hESCs without differentiation. Human feeder cells have the advantage of supporting more passages than when MEFs are used as feeder cells, because hESCs can be uniformly maintained in the undifferentiated stage until they pass through senescence. hESCs established and/or maintained under stable xeno-free culture conditions will be helpful to cell-based therapy.

Adult↗

PubMed1989

Expression of c-myb in embryonal carcinoma cells and embryonal stem cells.

Mouse c-myb has been implicated in the regulation of differentiation and proliferation of haematopoietic cells. Analysis of the chromatin structure of the promoter region of c-myb in embryonal carcinoma (EC) cells and embryonal stem (ES) cells reveals a DNAse I-hypersensitive site coincident with a site found in c-myb-expressing haematopoietic cells, but absent in murine fibroblasts (which do not express c-myb). EC and ES cells were found to express c-myb mRNA, albeit at a level lower than found in haematopoietic cells. Differentiation of ES cells into embryoid bodies resulted in an elevated level of c-myb expression.

Animals↗

PubMed2006

Definitive-like erythroid cells derived from human embryonic stem cells coexpress high levels of embryonic and fetal globins with little or no adult globin.

Human embryonic stem cells are a promising tool to study events associated with the earliest ontogenetic stages of hematopoiesis. We describe the generation of erythroid cells from hES (H1) by subsequent processing of cells present at early and late stages of embryoid body (EB) differentiation. Kinetics of hematopoietic marker emergence suggest that CD45+ hematopoiesis peaks at late D14EB differentiation stages, although low-level CD45- erythroid differentiation can be seen before that stage. By morphologic criteria, hES-derived erythroid cells were of definitive type, but these cells both at mRNA and protein levels coexpressed high levels of embryonic (epsilon) and fetal (gamma) globins, with little or no adult globin (beta). This globin expression pattern was not altered by the presence or absence of fetal bovine serum, vascular endothelial growth factor, Flt3-L, or coculture with OP-9 during erythroid differentiation and was not culture time dependent. The coexpression of both embryonic and fetal globins by definitive-type erythroid cells does not faithfully mimic either yolk sac embryonic or their fetal liver counterparts. Nevertheless, the high frequency of erythroid cells coexpressing embryonic and fetal globin generated from embryonic stem cells can serve as an invaluable tool to further explore molecular mechanisms.

Cell Differentiation↗

PubMed2002

Enhanced transgene expression in primitive hematopoietic progenitor cells and embryonic stem cells efficiently transduced by optimized retroviral hybrid vectors.

Oncoretroviral vectors have been successfully used in gene therapy trials, yet low transduction rates and loss of transgene expression are still major obstacles for their application. To overcome these problems we modified the widely used Moloney murine leukemia virus-derived retroviral vector pMX by replacing the 3'LTR with the spleen focus-forming virus LTR and inserting the woodchuck hepatitis B virus post-translational regulatory element. To compare requirements crucial for efficient transgene expression, we generated the hybrid retroviral vectors pMOWS and pOWS that harbor the complete murine embryonic stem cell virus (MESV)-leader sequence or a shortened MESV-leader not comprising primer binding site (PBS) and splice donor (SD). Applying these retroviral vectors significantly augmented transgene expression in hematopoietic cell lines and progenitor cells. For transduction of murine embryonic stem (ES) cells the retroviral vector pMOWS that harbors the MESV-PBS and -SD was superior resulting in 65% green fluorescent protein (GFP) expressing ES cells. Surprisingly, in murine and human primitive hematopoietic progenitor cells (HPC), the highest efficiency of up to 66% GFP expressing cells was achieved with pOWS, a retroviral vector that retains the negative regulatory element coinciding with the MoMuLV-PBS. In summary our hybrid retroviral vectors facilitate significantly improved transgene expression in multipotent cells and thus possess great potential for reconstituting genes in primary cells of disease models, as well as for gene therapy.

Animals↗

PubMed1998

Primate embryonic stem cells.

Primate embryonic stem (ES) cells are derived from preimplantation embryos, have a normal karyotype, and are capable of indefinite, undifferentiated proliferation. Even after culture for more than a year, primate ES cells maintain the potential to differentiate to trophoblast and derivatives of embryonic endoderm, mesoderm, and ectoderm. In this review, we compare the characteristics of ES cell lines from two primate species, the rhesus monkey (Macaca mulatta) and the common marmoset (Callithrix jacchus), with the characteristics of mouse ES cells and human embryonal carcinoma cells. We also discuss the implications of using primate ES cells to understand early human development and discuss the practical and ethical implications for the understanding and treatment of human disease.

Animals↗

PubMed1996

Synapse formation and establishment of neuronal polarity by P19 embryonic carcinoma cells and embryonic stem cells.

A number of different cell lines that exhibit a partial neuronal phenotype have been identified, but in many cases the full extent of their neuronal differentiation has not been directly addressed by functional studies. We have used electrophysiology and immunofluorescence to examine the formation of synapses and the development of neuronal polarity by murine embryonic stem (ES) cells and the mouse P19 embryonic carcinoma cell line. Within 2-3 weeks after induction by retinoic acid, subsets of P19 and ES cells formed excitatory synapses, mediated by glutamate receptors, or inhibitory synapses, mediated by receptors for GABA or glycine. In ES-cell cultures, both NMDA and non-NMDA receptors contributed to the excitatory postsynaptic response. Staining with antibodies to growth-associated protein-43 and microtubule-associated protein-2 revealed segregation of immunoreactivity into separate axonal and somato-dendritic compartments, respectively. Consistent with our physiological evidence for synapse formation, intense punctate staining was observed with antibodies to the synaptic vesicle proteins synapsin, SV2, and synaptophysin. These results demonstrate the in vitro acquisition by pluri-potent cell lines of neuronal polarity and functional synaptic transmission that is characteristic of CNS neurons.

Action Potentials↗

PubMed2006

Complement-dependent control of teratoma formation by embryonic stem cells.

The fetus has pluripotent stem cells that when transferred to mature individuals can generate tumors. However, for reasons yet unknown, tumors form rarely in the fetus and/or the mother during normal gestation. We questioned whether the complement system might protect against tumor formation by pluripotent stem cells. Murine embryonic stem cells were notably more susceptible than cardiomyocytes differentiated from those cells to lysis by complement in heterologous and homologous sera. Treatment of embryonic stem cells with heterologous serum averted tumor formation after residual cells were transplanted into mice. Confirming the importance of homologous complement in preventing formation of tumors, untreated embryonic stem cells formed tumors more quickly in C3-deficient than in wild-type mice. Susceptibility of embryonic stem cells to complement required an intact alternative pathway and was owed at least in part to a relative deficiency of sialic acid on cell surfaces compared with differentiated cells. Susceptibility to complement and resistance to tumors was inversely related to the number of cells transferred. These findings show that formation of tumors from embryonic stem cells is controlled in part by the alternative pathway of complement and suggest that susceptibility to complement might represent a general property of pluripotent stem cells that can be exploited to prevent tumor formation.

Animals↗

PubMed2003

Characterization and culture of human embryonic stem cells.

Human embryonic stem (ES) cells are cultured cell lines derived from the inner cell mass of the blastocyst that can be grown indefinitely in their undifferentiated state, yet also are capable of differentiating into all cells of the adult body as well as extraembryonic tissue. Detailed investigation of the properties of embryonal carcinoma cells of both the mouse and human as well as mouse and primate ES cells led to the initial isolation and subsequent culture of human ES cells. The methodologies that were developed to culture and characterize these cell lines have provided a template for the development of human ES cells. The existing data illustrate a number of important differences and similarities between human ES cells and the other cell lines. This review aims to provide a brief historic account of the development of the mammalian pluripotent stem cell field; describe how this led to the isolation, culture, and characterization of human ES cells; and discuss the potential implications of recent advances.

Animals↗

PubMed2005

Derivation of adipocytes from human embryonic stem cells.

Human embryonic stem (hES) cells are undifferentiated and pluripotent cells that hold great therapeutic potential, but are hampered by our limited knowledge to promote specific cell differentiation. Here we provide the first report of the directed differentiation of hES cells into adipocytes. Embryoid bodies (EBs) derived from hES cells are shown to respond to factors that promote adipogenesis. Differentiated cells were observed that displayed the key features of adipocytes, i.e., expression of specific molecular markers, such as peroxisome proliferator-activated receptor gamma2 (PPARgamma2), adipocyte fatty acid binding protein (aP2) and adiponectin, the secretion of leptin, and the accumulation of lipid droplets in cytoplasm. Taken together, our results demonstrate that adipocytes derived from hES cells in vitro can provide a novel model system to study human adipogenesis and obesity.

Adipocytes↗

PubMed2006

Enrichment of cardiomyocytes derived from mouse embryonic stem cells.

BACKGROUND: Embryonic stem (ES) cell-derived cardiomyocytes transplantation and tissue engineering together represent a promising approach for the treatment of myocardial infarction, despite the limited supply of cardiac myocytes. This study examines whether functional cardiomyocytes can be efficiently enriched from mouse embryonic stem (mES) cells. METHODS: mES cells were induced by ascorbic acid to differentiate into cardiomyocytes. Beating cells were observed after 1 week and increased in number with time while under differentiation conditions. Furthermore, the differentiated cultures could be dissociated and enriched by Percoll gradient density centrifugation. RESULTS: The beating cells expressed markers characteristic of cardiomyocytes, such as cardiac troponin T (cTnT). The enriched population contained 88.7% cardiomyocytes and showed expression of cardiomyocyte markers of troponin T and cardiac genes, including alpha-MHC, beta-MHC, ANF and Nkx2.5. However, Oct-4, a marker of early-stage ES cells, was not expressed in the mES cell-derived cardiac cell clusters. Moreover, the mES cell-derived and Percoll-enriched cardiomyocytes responded appropriately to cardioactive drugs, as did normal neonatal rat cardiomyocytes. CONCLUSIONS: mES cell-derived functional cardiomyocytes can be enriched by the method of discontinuous Percoll gradient centrifugation. The ability to differentiate and enrich for functional mouse cardiomyocytes makes it possible for further development of these cells as a model of myocardial repair through cell transplantation or tissue engineering.

Animals↗

PubMed2002

Proteome analysis of conditioned medium from mouse embryonic fibroblast feeder layers which support the growth of human embryonic stem cells.

Human embryonic stem (ES) cells are pluripotent cells with the potential to differentiate into a variety of cell types, which could be used for cell transplantation therapies as well as drug discovery studies. However, the large-scale culture of undifferentiated human ES cells is currently limited by their dependency on mouse embryonic fibroblast feeder layers. The proteomics approach was employed to characterize the environment that supports the growth of undifferentiated human ES cells and to identify factors critical for their independent growth. Conditioned medium from mouse embryonic fibroblast feeder layers, STO cell line, was concentrated and subjected to analyses by two-dimensional electrophoresis mass spectrometry. In total, 136 unique protein species were identified which included some that are known to participate in cell growth and differentiation, extracellular matrix formation and remodeling, in addition to the unexpected but interesting finding of many nominally intracellular proteins. This approach has thus revealed the complexity of the environment provided by the feeder cells and provides a useful starting point for future studies. Moreover, candidates from the initial list of identified proteins can be further investigated for their effects on the growth and differentiation of human ES cells in a defined culture environment.

Animals↗

PubMed2000

In vivo immunological function of mast cells derived from embryonic stem cells: an approach for the rapid analysis of even embryonic lethal mutations in adult mice in vivo.

An important goal of tissue engineering is to achieve reconstitution of specific functionally active cell types by transplantation of differentiated cell populations derived from normal or genetically altered embryonic stem cells in vitro. We find that mast cells derived in vitro from wild-type or genetically manipulated embryonic stem cells can survive and orchestrate immunologically specific IgE-dependent reactions after transplantation into mast cell-deficient Kit(W)/Kit(W-v) mice. These findings define a unique approach for analyzing the effects of mutations of any genes that are expressed in mast cells, including embryonic lethal mutations, in vitro or in vivo.

Anaphylaxis↗