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PubMed2002

Generation of cardiomyocytes from embryonic stem cells experimental studies.

BACKGROUND: Cardiomyopathy is characterized by the loss of functional cardiomyocytes resulting in heart failure. More recently, there is increasing evidence from animal studies that transplantation of cardiomyocytes may represent a valuable approach for the treatment of severe heart failure. DEVELOPMENT OF CARDIAC CELLS: Treatment of cardiovascular diseases using cardiomyocytes derived from embryonic stem cells prerequisites establishment of pure lineages of early embryonic cardiomyocytes from human embryonic stem cells. The development of cardiac cells from embryonic stem cells is regulated by several growth factors such as TGF-beta, IGF FGF and erythropoietin. After binding to their receptor, growth factors induce expression of a number of cardiac specific transcription factors such as the zinc finger GATA proteins and Nkx-2.5, a coactivator of the GATA-4 protein. The GATA-4 transcription factor and Nkx-2.5 are essential for heart development. In parallel to adult cardiomyocytes, embryonic stem cell-derived cardiomyocytes developmentally express cardiac specific proteins and ion channels. GENERATION FROM EMBRYONIC STEM CELLS: Recently, it has been shown that pure cardiomyocytes can be generated from genetically manipulated embryonic stem cells. In order to achieve the selective cardiac differentiation of embryonic stem cells different culture conditions are currently tested to examine in the future the influence of different growth factors. However, although significant progress has been made in generating pure cardiomyocytes, further efforts are required to avoid possible immunological rejection of the engrafted cardiomyocytes. Thus, a main challenge in the future will be the establishment of pure lineages of cardiomyocytes derived from human embryonic stem cells.

Adult↗

PubMed2003

Derivation of embryonic germ cells and male gametes from embryonic stem cells.

Egg and sperm cells (gametes) of the mouse are derived from a founder population of primordial germ cells that are set aside early in embryogenesis. Primordial germ cells arise from the proximal epiblast, a region of the early mouse embryo that also contributes to the first blood lineages of the embryonic yolk sac. Embryonic stem cells differentiate in vitro into cystic structures called embryoid bodies consisting of tissue lineages typical of the early mouse embryo. Because embryoid bodies sustain blood development, we reasoned that they might also support primordial germ cell formation. Here we isolate primordial germ cells from embryoid bodies, and derive continuously growing lines of embryonic germ cells. Embryonic germ cells show erasure of the methylation markers (imprints) of the Igf2r and H19 genes, a property characteristic of the germ lineage. We show that embryoid bodies support maturation of the primordial germ cells into haploid male gametes, which when injected into oocytes restore the somatic diploid chromosome complement and develop into blastocysts. Our ability to derive germ cells from embryonic stem cells provides an accessible in vitro model system for studies of germline epigenetic modification and mammalian gametogenesis.

Animals↗

PubMed2006

A molecular scheme for improved characterization of human embryonic stem cell lines.

BACKGROUND: Human embryonic stem cells (hESC) offer a renewable source of a wide range of cell types for use in research and cell-based therapies to treat disease. Inspection of protein markers provides important information about the current state of the cells and data for subsequent manipulations. However, hESC must be routinely analyzed at the genomic level to guard against deleterious changes during extensive propagation, expansion, and manipulation in vitro. RESULTS: We found that short tandem repeat (STR) analysis, human leukocyte antigen (HLA) typing, single nucleotide polymorphism (SNP) genomic analysis, mitochondrial DNA sequencing, and gene expression analysis by microarray can be used to fully describe any hESC culture in terms of its identity, stability, and undifferentiated state. CONCLUSION: Here we describe, using molecular biology alone, a comprehensive characterization of 17 different hESC lines. The use of amplified nucleic acids means that for the first time full characterization of hESC lines can be performed with little time investment and a minimum of material. The information thus gained will facilitate comparison of lines and replication of results between laboratories.

Cell Differentiation↗

PubMed2006

Derivation and characterization of alveolar epithelial cells from murine embryonic stem cells in vitro.

We present a protocol that has been developed for induction of the differentiation of murine embryonic stem (ES) cells to alveolar type II cells. With this protocol, undifferentiated murine ES cells are induced to form embryoid bodies (EBs). The 10-d-old EBs are transferred to adherent culture conditions and are fed with high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% v/v fetal bovine serum, 2 mM L-glutamine, and 0.1 mM 2-mercaptoethanol for 20 d without splitting. Then, the cells are fed with a medium designed for the maintenance and growth of mature distal airway epithelial cells (small airway growth medium, SAGM) for 3 d. Characterization of the alveolar type II cells was done using real-time reverse transcriptase polymerase chain reaction detection of surfactant protein C mRNA and immunocytochemical detection of prosurfactant protein C. Real-time reverse transcriptase polymerase chain reaction revealed that SAGM increases the mRNA expression level of SPC by a factor of 8 when compared to that of cells grown in supplemented high-glucose DMEM (p < 0.05, Student t-test). Immunocytochemistry revealed that proSPC-expressing cells comprised 2.8 +/- 0.23% of the total cell population in SAGM-treated samples and 0.5 +/- 0.1% in samples treated with supplemented high-glucose DMEM (p < 0.05, chi2 test).

Animals↗

PubMed2004

Endothelial and hematopoietic cell fate of human embryonic stem cells originates from primitive endothelium with hemangioblastic properties.

The cellular organization and relationships among precursors that initiate embryonic angiogenesis and hematopoiesis in the human have yet to be characterized. Here, we identify a subpopulation of primitive endothelial-like cells derived from human embryonic stem cells (hESCs) that express PECAM-1, Flk-1, and VE-cadherin, but not CD45 (CD45negPFV cells), and that are uniquely responsible for endothelial and hematopoietic development. Molecular profiling of CD45negPFV cells is consistent with endothelial and hematopoietic competency. Clonal isolation demonstrates that the CD45negPFV population includes bipotent cells with endothelial and hematopoietic capacity. We suggest that human hematopoiesis and endothelial maturation originate exclusively from a subset of embryonic endothelium that possesses hemangioblastic properties and offers a model system to study these lineage relationships in the human.

Antigens, CD↗

PubMed1997

Development of erythroid cells from mouse embryonic stem cells in culture: potential use for erythroid transcription factor study.

We developed an efficient differentiation induction system from mouse embryonic stem (ES) cells into blood cells by coculture on a novel stromal cell line named OP9, in order to analyze molecular mechanisms of hematopoietic cell development and differentiation. ES cells could give rise to adult type definitive erythrocytes, myeloid and B lineage cells via multipotential hematopoietic precursor cells, when the cells were simply cocultured with the OP9 stromal cells. The temporal pattern of the appearance of erythroid lineage cells during the differentiation induction was very similar to that detected in mouse ontogeny. This differentiation induction method should facilitate to dissect the function of erythroid transcription factors during erythroid lineage cell development.

Animals↗

PubMed2005

SDF-1/CXCL12 enhances survival and chemotaxis of murine embryonic stem cells and production of primitive and definitive hematopoietic progenitor cells.

Understanding embryonic stem cell (ESC) regulation is important for realizing how best to control their growth and differentiation ex vivo for potential therapeutic benefit. Stromal cell-derived factor-1 (SDF-1/CXCL12) and its receptor, CXCR4, have been implicated as important regulators of a number of fetal and adult cell functions, including survival/antiapoptosis and migration/homing of hematopoietic stem and progenitor cells. We hypothesized that the SDF-1/CXCL12-CXCR4 axis would also be important for regulation of murine ESC functions. ESCs secreted low levels of SDF-1/CXCL12 and expressed low levels of CXCR4; however, both increased with differentiation of ESCs. Endogenously produced/released SDF-1/CXCL12 enhanced survival/antiapoptosis of ESCs in the presence of leukemia inhibitory factor but absence of serum, and survival/antiapoptosis was further enhanced by exogenous administration of SDF-1/CXCL12. Furthermore, SDF-1/CXCL12 induced chemotaxis of ESCs, and chemotaxis could be enhanced by diprotin A inhibition of CD26/dipeptidylpeptidase IV. Endogenous and exogenous SDF-1/CXCL12 enhanced embryoid body production of primitive and definitive erythroid, granulocyte-macrophage, and multipotential progenitors. SDF-1/CXCL12 did not noticeably affect production of hemangioblasts. These results demonstrate functional activities of SDF-1/CXCL12 on survival, chemotaxis, and hematopoietic differentiation of murine ESCs that may be relevant for their ex vivo manipulation.

Animals↗

PubMed2003

Organogenesis of heart-vascular system derived from mouse 2 cell stage embryos and from early embryonic stem cells in vitro.

Regenerative medical treatment with embryonic stem cells (an ES cell) is a goal for organ transplantation. Structures that are tubular in nature (i.e. blood capillaries) were induced from early embryonic stem (EES) cells in vitro using embryotrophic factor (ETFs). In addition, cardiac muscle cells could be identified as well. However, differentiation of EES cells into a complete cardiovascular system was difficult because 3 germ layer primordial organs are directed embryologically in various ways and it is not possible to guide only cardiovascular organs. Thus, we introduced ETFs after the formation of an embryoid body and were successful in cloning cell clusters that beat, thus deriving only cardiovascular organs. The application of this to the treatment of various cardiovascular diseases is promising.

Animals↗

PubMed2005

Stage- and site-specific DNA demethylation during neural cell development from embryonic stem cells.

Activation of the transcription factor STAT3 is important for astrocyte differentiation during neural development. Demethylation of the methyl-CpG dinucleotide in the STAT3 binding site in the promoter of the glial fibrillary acidic protein (GFAP) gene, a marker for astrocytes, was previously shown to be a crucial cue for neural progenitors to express this gene in response to astrogenic signals during brain development. In this study, we analyzed the methylation status of the STAT3 binding site in the GFAP gene promoter during neural cell development from mouse embryonic stem (ES) cells in vitro. The CpG dinucleotide in the STAT3 binding site in the GFAP gene promoter exhibited a high incidence of cytidine-methylation in undifferentiated pluripotent ES cells. The high incidence of methylation of this particular cytidine was maintained in ES cell-derived neuroectoderm-like cells, but it underwent demethylation when the neural lineage cells became competent to express GFAP in response to a STAT3 activation signal. In contrast, hypermethylation of the CpG site was maintained in non-neural cells generated from the same ES cells. Progressive demethylation of the STAT3 binding site in the GFAP gene promoter was also observed in primary embryonic neuroepithelial cells during in vitro culture, whereas non-neural cells maintained hypermethylation of this site even after culture. Taken together, these results demonstrate that the astrocyte gene-specific cytidine-demethylation is programmed when neural progenitors from pluripotent cells are committed to a neural lineage that is capable of producing astrocytes.

Animals↗

PubMed1998

UTF1, a novel transcriptional coactivator expressed in pluripotent embryonic stem cells and extra-embryonic cells.

We have obtained a novel transcriptional cofactor, termed undifferentiated embryonic cell transcription factor 1 (UTF1), from F9 embryonic carcinoma (EC) cells. This protein is expressed in EC and embryonic stem cells, as well as in germ line tissues, but could not be detected in any of the other adult mouse tissues tested. Furthermore, when EC cells are induced to differentiate, UTF1 expression is rapidly extinguished. In normal mouse embryos, UTF1 mRNA is present in the inner cell mass, the primitive ectoderm and the extra-embryonic tissues. During the primitive streak stage, the induction of mesodermal cells is accompanied by the down-regulation of UTF1 in the primitive ectoderm. However, its expression is maintained for up to 13.5 days post-coitum in the extra-embryonic tissue. Functionally, UTF1 boosts the level of transcription of the adenovirus E2A promoter. However, unlike the pluripotent cell-specific E1A-like activity, which requires the E2F sites of the E2A promoter for increased transcriptional activation, UTF1-mediated activation is dependent on the upstream ATF site of this promoter. This result indicates that UTF1 is not a major component of the E1A-like activity present in pluripotent embryonic cells. Further analyses revealed that UTF1 interacts not only with the activation domain of ATF-2, but also with the TFIID complex in vivo. Thus, UTF1 displays many of the hallmark characteristics expected for a tissue-specific transcriptional coactivator that works in early embryogenesis.

Activating Transcription Factor 2↗

PubMed2005

Three-dimensional nanofibrillar surfaces promote self-renewal in mouse embryonic stem cells.

The regulation of mouse embryonic stem cell (mESC) fate is controlled by the interplay of signaling networks that either promote self-renewal or induce differentiation. Leukemia inhibitory factor (LIF) is a cytokine that is required for stem cell renewal in mouse but not in human embryonic stem cells. However, feeder layers of embryonic fibroblasts are capable of inducing stem cell renewal in both cell types, suggesting that the self-renewal signaling pathways may also be promoted by other triggers, such as alternative cytokines and/or chemical or physical properties of the extracellular matrix (ECM) secreted by feeder fibroblasts. We have recently used a synthetic polyamide matrix (Ultra-Web) whose three-dimensional (3D) nanofibrillar organization resembles the ECM/basement membrane. Growth of mESCs on this nanofibrillar surface greatly enhanced proliferation and self-renewal in comparison with growth on tissue culture surfaces without nanofibers, despite the presence of LIF in both systems. Enhanced proliferation and self-renewal of the stem cells on nanofibrillar surfaces were correlated with the activation of the small GTPase Rac, the activation of phosphoinositide 3-kinase (PI3K) pathway, and the enhanced expression of Nanog, a homeoprotein required for maintenance of pluripotency. Inhibitors of PI3K reduced the expression level of Nanog in mESCs cultured on 3D nanofibrillar surfaces. These results provide support for the view that the three-dimensionality of the culture surface may function as a cue for the activation of Rac and PI3K signaling pathways, resulting in stem cell proliferation and self-renewal.

Alkaline Phosphatase↗

PubMed2005

Cell adhesion molecule l1-transfected embryonic stem cells with enhanced survival support regrowth of corticospinal tract axons in mice after spinal cord injury.

Previous studies have indicated that the cell adhesion molecule L1 enhances neuronal survival and neurite outgrowth. L1-mediated promotion of neurite outgrowth has been shown to occur also in an inhibitory environment not only in vitro, but also in vivo. To further investigate the effects of L1 in spinal cord injury, we transfected embryonic stem cells with a plasmid encoding the full-length mouse L1 molecule under the control of PGK promoter. An embryonic stem cell line derived from C57BL/6J transgenic mice that express green fluorescent protein under control of the beta-actin promoter was transfected with L1 and injected into the lesion site of 3-month-old C57BL/6J female mice 7 days after compression injury. Non-transfected embryonic stem cells were detectable at the lesion site 3 days after transplantation, but lost their cellular integrity 7 days after transplantation and were barely detectable 1 month after transplantation. In contrast, L1-transfected embryonic stem cells were detected 1 month after transplantation in numbers comparable to those of the injected cells and demonstrated extended processes. Further, in contrast to the few detectable nontransfected stem cells that remained at the injection site 1 month post-transplantation, the L1-transfected embryonic stem cells had migrated rostrally and caudally from the lesion. Anterogradely labeled corticospinal tract axons showed interdigitation with L1-transfected embryonic stem cells and, in contrast to non-transfected stem cells, extended into the lesion site 1 month after transplantation and, in some cases, extended beyond it. Our observations encourage the use of L1-transfected embryonic stem cells that express L1 not only at the cell surface, but also as a soluble and secreted form. Their use could condition the inhibitory environment for homophilic L1-enhanced axon regrowth not only in spinal cord regeneration, but also in other lesion paradigms.

Animals↗

PubMed2006

Alginate-PLL microencapsulation: effect on the differentiation of embryonic stem cells into hepatocytes.

The emergence of hepatocyte based clinical and pharmaceutical technologies, has been limited by the absence of a stable hepatocyte cell source. Embryonic stem cells may represent a potential solution to this cell source limitation problem since they are highly proliferative, renewable, and pluripotent. Although many investigators have described techniques to effectively differentiate stem cells into a variety of mature cell lineages, their practicality is limited by: (1) low yields of fully differentiated cells, (2) absence of large scale processing considerations, and (3) ineffective downstream enrichment protocols. Thus, a differentiation platform that may be modified to induce and sustain differentiated cell function and scaled to increase differentiated cell yield would improve current stem cell differentiation strategies. Microencapsulation provides a vehicle for the discrete control of key cell culture parameters such as the diffusion of growth factors, metabolites, and wastes. In addition, both cell seeding density and bead composition may be manipulated. In order to assess the feasibility of directing stem cell differentiation via microenvironment regulation, we have developed a murine embryonic stem cell (ES) alginate poly-l-lysine microencapsulation hepatocyte differentiation system. Our results indicate that the alginate microenvironment maintains cell viability, is conducive to ES cell differentiation, and maintains differentiated cellular function. This system may ultimately assist in developing scalable stem cell differentiation strategies.

Albumins↗

PubMed2006

More than 40,000 transcripts, including novel and noncoding transcripts, in mouse embryonic stem cells.

To study the transcriptome of embryonic stem cells, we used a new gene expression profiling method that can measure the expression levels of unknown and rarely expressed transcripts precisely. We detected a total of 33,136 signal peaks representing transcripts in mouse embryonic stem cells, E14. Subsequent random cloning of the peaks suggests that mouse embryonic stem cells express at least 40,000 transcripts, of which about 2,000 are still unknown. In addition, we identified 1,022 noncoding transcripts, several of which change depending on differentiation in gene expression. Our database provides a high-resolution expression profile of E14 cells and is applicable to other mouse embryonic stem cell analyses. It includes most transcription regulation factor-encoding genes and a significant number of unknown and noncoding transcripts.

Animals↗

PubMed1996

A limited temporal window for the derivation of multilineage repopulating hematopoietic progenitors during embryonal stem cell differentiation in vitro.

Embryonal stem cells have been shown to differentiate in vitro into all hematopoietic lineages. This has been used successfully as one approach to the study of genetic events occurring during haematopoiesis. However, studies on the commitment of mesodermal precursors to the hematopoietic lineage have been limited due to the inability to define a system in which embryonal stem (ES) cells will give rise to primitive hematopoietic stem cells in vitro. Using a colony forming assay (CFU-A), we determined that the earliest time point at which primitive multilineage hematopoietic precursors can be detected during ES cell differentiation in vitro in the absence of exogenous conditioned medium or stromal cell culture is 4 days. Lethally irradiated adult recipient mice that received differentiated ES cells from this time point survived for more than 3 weeks; and in two out three experiments, peripheral blood from these animals contained ES-derived progeny. Fluorescence activated cell sorting (FACS) found ES-derived CD45+ hematopoietic cells in both lymphoid and myeloid compartments at 12 weeks posttransplantation, suggesting that the population of day 4 differentiated ES cells contains primitive hematopoietic precursors. A preliminary RT-PCR analysis of gene expression around this time point suggests that there are very few hematopoietic cells present. This approach should prove useful in studies of genetic control of commitment to and maintenance of hematopoietic lineages in vitro and in vivo.

Amino Acid Sequence↗

PubMed2004

In vitro and in vivo characterization of pigment epithelial cells differentiated from primate embryonic stem cells.

PURPOSE: To determine whether primate embryonic stem (ES) cell-derived pigment epithelial cells (ESPEs) have the properties and functions of retinal pigment epithelial (RPE) cells in vitro and in vivo. METHODS: Cynomolgus monkey ES cells were induced to differentiate into pigment epithelial cells by coculturing them with PA6 stromal cells in a differentiating medium. The expanded, single-layer ESPEs were examined by light and electron microscopy. The expression of standard RPE markers by the ESPEs was determined by RT-PCR, Western blot, and immunocytochemical analyses. The ESPEs were transplanted into the subretinal space of 4-week-old Royal College of Surgeons (RCS) rats, and the eyes were analyzed immunohistochemically at 8 weeks after grafting. The effect of the ESPE graft on the visual function of RCS rats was estimated by optokinetic reflex. RESULTS: The expanded ESPEs were hexagonal and contained significant amounts of pigment. The ESPEs expressed typical RPE markers: ZO-1, RPE65, CRALBP, and Mertk. They had extensive microvilli and were able to phagocytose latex beads. When transplanted into the subretinal space of RCS rats, the grafted ESPEs enhanced the survival of the host photoreceptors. The effects of the transplanted ESPEs were confirmed by histologic analyses and behavioral tests. CONCLUSIONS: The ESPEs had morphologic and physiological properties of normal RPE cells, and these findings suggest that these cells may provide an unlimited source of primate cells to be used for the study of pathogenesis, drug development, and cell-replacement therapy in eyes with retinal degenerative diseases due to primary RPE dysfunction.

Animals↗

PubMed2005

Therapeutic potential of embryonic stem cells.

Nearly 20 years after murine embryonic stem cells (mESC) were isolated, the first report of the derivation of human embryonic stem cells (hESC) in 1998 spawned the field of hESC research [Evans MJ, Kaufman MH, Establishment in culture of pluripotential cells from mouse embryos. Nature 1981; 292 (5819): 154-6; Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts. Science 1998; 282 (5391): 1145-7.]. Although this field is only in its infancy, hESC represent a theoretically inexhaustible source of precursor cells that could be differentiated into any cell type to treat degenerative, malignant, or genetic diseases, or injury due to inflammation, infection, and trauma. This pluripotent, endlessly dividing cell has been hailed as a possible means for treating diabetes, Parkinson's disease, Alzheimer's, spinal cord injury, heart failure, and bone marrow failure. But the regenerative medicine applications of embryonic stem cells are only one facet of hESC therapeutic potential. Human ESC are an invaluable research tool to study development, both normal and abnormal, and can serve as a platform to develop and test new therapies. In addition to discussing the therapeutic potential of hESC, this chapter will cover limitations to using hESC for replacement cell therapy, strategies to overcome these limitations, and alternative methods of deriving hESC.

Animals↗

PubMed2004

Immunogenicity of human embryonic stem cells: can we achieve tolerance?

Human embryonic stem cells are unique in their capacity to propagate without losing pluripotency, and at the same time may readily differentiate to various cell types of the three embryonic germ layers. It is widely accepted today that differentiated human embryonic stem cells may in the future enable repair of vital tissues of the body. Detailed differentiation protocols need to be developed and safety issues associated with cellular therapeutics must be examined. One of the greatest hurdles facing transplantation is the development of immune rejection processes towards non-autologous cells. Profiling of histocompatibility antigens expressed on the cells reveals that they might be subjected to immune response. Here we describe the routes of immune recognition that can identify these antigens and the proposed ways for overcoming the rejection of human embryonic stem cell derivatives.

Cell Differentiation↗