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PubMed2006

Myocardial regeneration by embryonic stem cell transplantation: present and future trends.

Embryonic stem cells are a promising source for myocardial regeneration due to their pluripotency and plasticity. In theory, embryonic stem cells are capable of self-renewal in an unlimited fashion, and can differentiate into any cell type required for cell-based therapy, including cardiac myocytes. In recent years, embryonic stem cells have been transplanted for cardiac regeneration in animal models, and the results are encouraging. However, there are still many hurdles to be overcome for the clinical application of embryonic stem cells.

Animals↗

PubMed2003

Transplantation of motoneuron-enriched neural cells derived from mouse embryonic stem cells improves motor function of hemiplegic mice.

Embryonic stem (ES) cells are expected to be a potential donor source for neural transplantation. We have obtained motoneuron-enriched neural progenitor cells by culturing mouse ES cells with retinoic acid (RA). The cells also expressed mRNA of a neurotrophic factor, neurotrophin-3 (NT-3). The left motor cortex area of mice was damaged by cryogenic brain injury, and the neural cells were transplanted underneath the injured motor cortex, neighboring to the paraventricular region. We found that the cells expressing neuronal phenotypes not only remained close to the implantation site, but also exhibited substantial migration penetrating into the damaged lesion, in a seemingly directed manner up to cortical region. We found that some of the neural cells differentiated into Islet1-positive motoneurons. It seems likely that the ability of the ES cell-derived neural progenitor cells to respond in vivo to guidance cues and signals that can direct their migration and differentiation may contribute to functional recovery of the recipient mice. We found that an "island of the mature neuronal cells" of recipient origin emerged in the damaged motor cortex. This may be associated with the neuroprotective effects of the ES cell-derived neural cells. The ES cells differentiated into CD31+ vasculoendothelial cells with the RA treatment in vitro. Furthermore, the grafted cells may provide sufficient neurotrophic factors such as NT-3 for neuroprotection and regeneration. The grafted neural cells that migrated into residual cortex and differentiated into neurons had purposefully elongated axons that were stained with anti-neurofilament middle chain (NFM) antibody. Our study suggests that motoneurons can be induced from ES cells, and ES cells become virtually an unlimited source of cells for experimental and clinical neural cell transplantation.

Animals↗

PubMed2003

Tissue engineering of blood vessels with endothelial cells differentiated from mouse embryonic stem cells.

Endothelial cells (TEC3 cells) derived from mouse embryonic stem (ES) cells were used as seed cells to construct blood vessels. Tissue engineered blood vessels were made by seeding 8 106 smooth muscle cells (SMCs) obtained from rabbit arteries onto a sheet of nonwoven polyglycolic acid (PGA) fibers, which was used as a biodegradable polymer scaffold. After being cultured in DMEM medium for 7 days in vitro, SMCs grew well on the PGA fibers, and the cell-PGA sheet was then wrapped around a silicon tube, and implanted subcutaneously into nude mice. After 6~8 weeks, the silicon tube was replaced with another silicon tube in smaller diameter, and then the TEC3 cells (endothelial cells differentiated from mouse ES cells) were injected inside the engineered vessel tube as the test group. In the control group only culture medium was injected. Five days later, the engineered vessels were harvested for gross observation, histological and immunohistochemical analysis. The preliminary results demonstrated that the SMC-PGA construct could form a tubular structure in 6~8 weeks and PGA fibers were completely degraded. Histological and immunohistochemical analysis of the newly formed tissue revealed a typical blood vessel structure, including a lining of endothelial cells (ECs) on the lumimal surface and the presence of SMC and collagen in the wall. No EC lining was found in the tubes of control group. Therefore, the ECs differentiated from mouse ES cells can serve as seed cells for endothelium lining in tissue engineered blood vessels.

Animals↗

PubMed2006

Concise review: epigenetic mechanisms contribute to pluripotency and cell lineage determination of embryonic stem cells.

Epigenetic mechanisms, such as histone modifications and DNA methylation, have been shown to play a key role in the regulation of gene transcription. Results of recent studies indicate that a novel "bivalent" chromatin structure marks key developmental genes in embryonic stem cells (ESCs), wherein a number of untranscribed lineage-control genes, such as Sox1, Nkx2-2, Msx1, Irx3, and Pax3, are epigenetically modified with a unique combination of activating and repressive histone modifications that prime them for potential activation (or repression) upon cell lineage induction and differentiation. However, results of these studies also showed that a subset of lineage-control genes, such as Myf5 and Mash1, were not marked by these histone modifications, suggesting that distinct epigenetic mechanisms might exist for lineage-control genes in ESCs. In this review article, we summarize evidence regarding possible mechanisms that control these unique histone modifications at lineage-control gene loci in ESCs and consider their possible contribution to ESC pluripotency. In addition, we propose a novel "histone modification pulsing" model wherein individual pluripotent stem cells within the inner cell mass of blastocysts undergo transient asynchronous histone modifications at these developmental gene loci, thereby conferring differential responsiveness to environmental cues and morphogenic gradients important for cell lineage determination. Finally, we consider how these rapid histone modification exchanges become progressively more stable as ESCs undergo differentiation and maturation into specialized cell lineages.

Animals↗

PubMed2006

A novel chemical-defined medium with bFGF and N2B27 supplements supports undifferentiated growth in human embryonic stem cells.

Traditionally, undifferentiated human embryonic stem cells (hESCs) are maintained on mouse embryonic fibroblast (MEF) cells or on matrigel with an MEF-conditioned medium (CM), which hampers the clinical applications of hESCs due to the contamination by animal pathogens. Here we report a novel chemical-defined medium using DMEM/F12 supplemented with N2, B27, and basic fibroblast growth factor (bFGF) [termed NBF]. This medium can support prolonged self-renewal of hESCs. hESCs cultured in NBF maintain an undifferentiated state and normal karyotype, are able to form embryoid bodies in vitro, and differentiate into three germ layers and extraembryonic cells. Furthermore, we find that hESCs cultured in NBF possess a low apoptosis rate and a high proliferation rate compared with those cultured in MEF-CM. Our findings provide a novel, simplified chemical-defined culture medium suitable for further therapeutic applications and developmental studies of hESCs.

Apoptosis↗

PubMed2004

Temporally induced Nurr1 can induce a non-neuronal dopaminergic cell type in embryonic stem cell differentiation.

The nuclear transcription factor Nurr1 is involved in the development and maintenance of the midbrain dopaminergic (DA) neuronal phenotype. We analysed the cellular and biological effects of Nurr1 during embryonic stem (ES) cell differentiation using the ROSA26-engineered Tet-inducible ES cell line J1-rtTA that does not express transgenes in mature neurons. Induction of Nurr1 at nestin-positive precursor and later stages of ES cell differentiation produced a non-neuronal DA cell type including functional DA transporters. In these cells, we found a clear correlation between Nurr1 and TH gene expression and specific midbrain DA cellular markers such as AADC, AHD2 and calbindin. Nurr1 did not alter gene expression of non-DA neuronal phenotypes and did not influence other midbrain developmental transcription factors, such as Otx1, Otx2, En-1, GBX2, Pitx3 and lmx1b. In addition, Nurr1 expression was required for maintenance of the DA phenotype and mediated up-regulation of the tyrosine kinase Ret and associated trophic factor GDNF-family receptors alpha 1, 2, and 4. This demonstrates that Nurr1 is sufficient to induce and maintain a midbrain-like DA biochemical and functional cellular phenotype independent of neurogenesis.

Animals↗

PubMed2003

Limb bud progenitor cells induce differentiation of pluripotent embryonic stem cells into chondrogenic lineage.

Pluripotent embryonic stem (ES) cells are the most versatile cells, with the potential to differentiate into all types of cells. However, the cellular and molecular mechanisms responsible for their differentiation into specific lineages have not been elucidated. Recent studies in human ES cells have challenged the scientific community to focus research on the basic mechanisms of stem cell differentiation for their potential applications in regenerative medicine and cell-based therapies. The majority of studies thus far have focused on cells that are already committed to specific lineages. The current studies were designed to establish a system for investigating the mechanisms of cell fate determination starting from undifferentiated ES cells, to gain insight into events during the normally inaccessible period of development. Here we demonstrate that pluripotent ES cells can be programmed to differentiate into chondrocytes, the cartilage-producing cells, by co-culture with progenitor cells from the limb buds of the developing embryo. Almost 60%-80% of the cells exhibited phenotypic characteristics of mature chondrocytes and expressed genes such as sox9, collagen type II, and proteoglycans, which was accompanied by a decrease in ES cell-specific transcription factor Oct-4. Collagen type II, which is expressed in two different forms during chondrogenic differentiation due to the alternate splicing of mRNA, was also properly regulated. The studies presented here suggest that the signals produced by progenitor cells from the developing embryo can induce lineage-specific differentiation. The system described here may serve as an in vitro model to study the mechanisms of cell fate determination by stem cells.

Animals↗

PubMed2003

Generation of hepatocyte-like cells from human embryonic stem cells.

Use of human hepatocytes for therapeutic and drug discovery applications is hampered by limited tissue source and the inability of hepatocytes to proliferate and maintain function long term in vitro. Human embryonic stem (hES) cells are immortal and pluripotent and may provide a cell source for functional human hepatocytes. We report here that hES cells can be induced to differentiate into hepatocyte-like cells. Treatment with sodium butyrate induced hepatic differentiation as well as significant cell death, resulting in approximately 10-15% yield of a homogeneous population of cells. The differentiated cells have morphological features similar to that of primary hepatocytes and 70-80% of the cells express liver-associated proteins (albumin, alpha-1-antitrypsin, cytokeratin 8 and 18), accumulate glycogen, have inducible cytochrome P450 activity, and do not express alpha-fetoprotein. Because of the inherent proliferative capacity of hES cells, these cells may provide a reliable source of normal human hepatocytes for research and transplantation.

Adult↗

PubMed2006

Staining embryonic stem cells using monoclonal antibodies to stage-specific embryonic antigens.

Stage-specific embryonic antigens (SSEAs) are cell-surface molecules that exhibit lineage-restricted patterns of expression during development. These antigens provide useful markers for identifying embryonic stem cells and their differentiated derivatives. SSEA-1 provides a surface marker for mouse and human primordial germ cells and mouse embryonic stem cells. SSEA-3 and SSEA-4 provide surface markers for human embryonic stem cells. Here, we describe methods for staining embryonic stem cells in culture and suspension using specific primary antibodies and fluorescent dye- or enzyme-conjugated secondary antibodies. Methods for alkaline phosphatase staining and immunohistochemical localization of antigens in paraffin sections are also described. Monoclonal antibodies directed to SSEAs are valuable research tools that can be used to: (1) determine the percentage of stem cells in mixed cultures; (2) examine the spatial distribution of stem cells and differentiated derivatives in monolayer cultures and embryoid bodies; and (3) isolate populations of differentiated and undifferentiated cells by immunomagnetic or fluorescence-activated cell sorting for cellular and molecular analyses.

Alkaline Phosphatase↗

PubMed2006

Derivation and characterization of lentoid bodies and retinal pigment epithelial cells from monkey embryonic stem cells in vitro.

For cell replacement therapies of retinal diseases, the most-needed cells are retinal pigment epithelial cells and photoreceptor cells; lens cells are needed for replacement of the cataract. Stromal cell-derived inducing activity induces the differentiation of mouse embryonic stem (ES) cells into neural cells, including midbrain tyrosine hydroxylase-positive dopaminergic neurons, and ocular cells; the method also works with primate ES cells. We describe the methods to induce retinal pigment epithelial and lens cells from monkey ES cells using stromal cell-derived inducing activity and show the characteristics of those cells in vitro and in vivo.

Animals↗

PubMed2006

A method for the selection of human embryonic stem cell sublines with high replating efficiency after single-cell dissociation.

Human embryonic stem cells (hESCs) exhibit pluripotency and indefinite proliferation and are a potential source of cells for transplantation therapies and drug discovery. These applications will require large amounts of hESCs. However, hESCs are difficult to culture and maintain at larger scales, in part because of their low resistance to dissociation during passaging. To circumvent this, we developed a simple and easy method for establishing hESC sublines tolerant of complete dissociation. These cells exhibit high replating efficiency and also high cloning efficiency, and they maintain their ability to differentiate into the three germ layers. Several sublines have no detectable abnormalities in their karyotypes, and they retained their characteristics under feeder-free culture conditions and after freeze-thawing. Thus, these hESC sublines would be valuable for hESC applications.

Biomarkers↗

PubMed2006

Embryonic stem cells from parthenotes.

While human embryonic stem cells (hESCs) hold tremendous therapeutic potential, they also create societal and ethical dilemmas. Adult and placental stem cells represent two alternatives to the hESC, but may have technical limitations. An additional alternative is the stem cell derived from parthenogenesis. Parthenogenesis is a reproductive mechanism that is common in lower organisms and produces a live birth from an oocyte activated in the absence of sperm. However, parthenogenetic embryos will develop to the blastocyst stage and so can serve as a source of embryonic stem cells. Parthenogenetic ESCs (pESCs) have been shown to have the properties of self-renewal and the capacity to generate cell derivatives from the three germ layers, confirmed by contributions to chimeric animals and/or teratoma formation when injected into SCID mice. Therefore, this mechanism for generating stem cells has the ethical advantage of not involving the destruction of viable embryos. Moreover, the cells do not involve the union of male and female and so genetic material will be derived exclusively from the female oocyte donor (with the attendant potential immunological advantages). This chapter describes the biology underlying parthenogenesis, as well as provides detailed technical considerations for the production of pESCs.

Animals↗

PubMed2006

Uniform adherent neural progenitor populations from rhesus embryonic stem cells.

Rhesus and human embryonic stem cells (ESCs) are similar, making rhesus ESCs an appropriate preclinical allograft model for refining stem cell therapies. Use of rhesus ESC-derived neural progenitors (NPs) in preclinical applications will be enhanced if the neural derivation process is scalable and free from contaminating ESCs or nonneural cells. In this study, we have quantified temporal gene expression changes of rhesus ESC differentiated to uniform NPs using simple feeder-free adherent cultures. NPs exhibited a significant up-regulation of neural-specific genes and a downregulation of pluripotency genes. Additionally, expression of Hu, MAP2, and Tuj1, shows that NPs can form post-mitotic neurons. This study represents a simple and scalable means of producing adherent primate NPs for preclinical testing of neural cell-based therapy.

Animals↗

PubMed2005

Human embryonic stem cells: biology and clinical implications.

Embryonic stem cells (ESCs) are derived from the inner cell mass of the preimplantation stage embryo and are capable of prolonged symmetrical self-renewal (both daughter cells remain escs) as well as differentiation into derivatives of all three embryonic germ layers. ESCs therefore have the potential to provide an unlimited supply of transplantable cells to replace or regenerate damaged or diseased tissues. However, several barriers must be overcome before successful clinical trials are possible: for example, pure populations of the desired cell type need to be selected and expanded in clinically relevant numbers, and a method for preventing immunological rejection of the transplanted cells without long-term immunosuppressive therapy is also required. In this review, we highlight recent developments in human ESC derivation and expansion, outline current understanding of the signalling pathways underlying stem cell renewal, and discuss challenging problems related to the selective differentiation and immune properties of human ESCs.

Animals↗

PubMed2006

Generation of new human embryonic stem cell lines with diploid and triploid karyotypes.

Human pluripotent embryonic stem cells (hESC) have great promise for research into human developmental biology and the development of cell therapies for the treatment of diseases. To meet the increased demand for characterized hESC lines, we present the derivation and characterization of five hESC lines on mouse embryonic fibroblast cells. Our stem cell lines are characterized by morphology, long-term expansion, and expression profiles of a number of specific markers, including TRA-1-60, TRA-1-81, alkaline phosphatase, connexin 43, OCT-4, NANOG, CXCR4, NODAL, LEFTY2, THY-1, TDGF1, PAX6, FOXD3, SOX2, EPHA2, FGF4, TAL1, AC133 and REX-1. The pluripotency of the cell line was confirmed by spontaneous differentiation under in vitro conditions. Whereas all of the cell lines expressed all the characteristics of undifferentiated pluripotent hESC, two of the cell lines carried a triploid karyotype.

Biomarkers↗

PubMed2006

Efficient generation of retinal progenitor cells from human embryonic stem cells.

The retina is subject to degenerative conditions, leading to blindness. Although retinal regeneration is robust in lower vertebrates, regeneration does not occur in the adult mammalian retina. Thus, we have developed efficient methods for deriving retinal neurons from human embryonic stem (hES) cells. Under appropriate culture conditions, up to 80% of the H1 line can be directed to the retinal progenitor fate, and express a gene expression profile similar to progenitors derived from human fetal retina. The hES cell-derived progenitors differentiate primarily into inner retinal neurons (ganglion and amacrine cells), with functional glutamate receptors. Upon coculture with retinas derived from a mouse model of retinal degeneration, the hES cell derived retinal progenitors integrate with the degenerated mouse retina and increase in their expression of photoreceptor-specific markers. These results demonstrate that human ES cells can be selectively directed to a neural retinal cell fate and thus may be useful in the treatment of retinal degenerations.

Adaptor Proteins, Signal Transducing↗

PubMed1991

Fidelity of targeted recombination in human fibroblasts and murine embryonic stem cells.

Targeted recombination in murine embryonic stem cells promises to be a powerful tool for introducing specific mutations into target genes to study development in mice and to create animal models of human disease. Gene targeting also holds potential for correcting genetic defects as an approach to human gene therapy. To precisely modify target genes, homologous recombination must proceed with high fidelity. However, several results have suggested that targeted recombination may be highly mutagenic. To test the accuracy of gene targeting we analyzed 44 independent targeted recombinants at the hypoxanthine phosphoribosyltransferase (HPRT) locus in a human fibroblast cell line and in mouse embryonic stem cells. We surveyed 80 kilobases around the sites of recombination by using chemical cleavage of mismatches. Only two mutations were found: a T----G transversion and a thymidine deletion. Thus, gene targeting in mammalian cells can be extremely accurate. These results demonstrate the feasibility of generating precise modifications of mammalian genomes by gene targeting.

Animals↗

PubMed2002

Efficient marking of neural stem cell-derived neurons with a modified murine embryonic stem cell virus, MESV2.

Treatments for nervous system disorders that involve transplanting genetically modified neural stem cells may ultimately be feasible. As a step towards this therapeutic approach, a novel murine embryonic stem cell gammaretroviral vector was developed with features designed to optimize transgene expression in neural stem cells and to increase vector safety. All potential start sites of translation in the 5' leader were removed. These sites may compete with an inserted transgene for translation initiation, and also produce potentially immunogenic peptides. Further, all of the gag gene sequences were replaced with a well-defined constitutive transport element from avian leukemia virus to promote nuclear export of viral RNA, and to eliminate any homology between the vector and a murine leukemia virus-derived gag-pol packaging plasmid. Two versions of the virus were made in which EGFP expression was driven either by the Rous sarcoma virus U3 enhancer or by a combination of sequences from the Syn1 and Pgk-1 promoters. Both of these viruses efficiently transduced neural stem cells isolated from embryonic rat hippocampus, and robust EGFP expression was observed in neurons derived from these cells following differentiation in vitro.

Animals↗