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PubMed1997

Developmental fate of single embryonic stem cells microinjected into 8-cell-stage mouse embryos.

Embryonic stem (ES) cells are pluripotent and capable of differentiating into somatic as well as germ cell lineages when conjoined with blastomeres of early mouse embryos. However, the developmental potential of single ES cells has not been fully investigated. We injected single murine ES cells (A3-1 cell line) of 129 origin into 8-cell mouse embryos (B6xBDF1) and examined the patterns of distribution of ES-cell-derived cells in the blastocysts as well as in the fully grown chimeric mice. The ES cells underwent 1-2 cycles of mitosis between the 8-cell and the blastocyst stage when they were introduced as single cells, whereas those introduced as groups of 2-5 cells did not proliferate during the same period of development. The ES cells and their daughter cells were predominantly incorporated into the ICM. From the 63 8-cell embryos which received single ES cells microinjected into the perivitelline space, 24 newborns were obtained, and 4 (2 fertile males, 1 sterile female and 1 hermaphrodite) of them (16.6%) were chimeric. The test breeding studies revealed that all the progeny of the two chimeric males were derived from spermatozoa of 129 genotype. The relative contribution of the host-derived and the ES-cell-derived cells in different tissues of the chimeric mice was assessed by PCR analyses of the microsatellite polymorphism of genomic DNA extracted from the tissues. In two male germ line chimeras, the testes, the kidneys and the dorsal skeletal muscles exhibited exceptionally high 129 contents. Our results demonstrated that single ES cells which maintain totipotency or pluripotency of high degree are present in a colony of ES cells, and that single ES cells conjoined with the blastomeres of 8-cell-stage embryos may colonize, if the circumstances allow, almost exclusively the germ cells and concomitantly the urogenital cell lineages. Possible correlation between the allocation of the germ line and the urogenital lineages is discussed.

Animals↗

PubMed1992

At least two distinct epigenetic mechanisms are correlated with high-frequency "switching" for APRT phenotypic expression in mouse embryonal carcinoma stem cells.

A series of clones displaying high frequency "switching" phenotypes for expression of the adenine phosphoribosyltransferase (aprt) gene were previously isolated from the P19 mouse embryonal carcinoma stem cell line. Most clones contained only one aprt allele. We report here the characterization of each of these clones with regards to enzymatic activity, mRNA steady state levels, DNA methylation, and chromatin conformation. When clones were selected for resistance to the purine analog 2,6-diaminopurine, which requires markedly reduced levels of APRT enzymatic activity, two distinct classes were observed. The first class was associated with reduced or undetectable levels of aprt mRNA, hypermethylation of the 5' CpG island, and a closed chromatin conformation within this region. When clones of this class were selected for reacquisition of APRT enzymatic activity they were found to have increased mRNA levels, a hypomethylated CpG island, and an open chromatin conformation. In contrast, the second class of clones displayed wild-type levels of mRNA, CpG island hypomethylation, and an open chromatin conformation regardless of whether they were selected for the presence or absence of APRT enzymatic activity. The implications of these results for general mechanisms of epigenetic change in somatic cells and the possibility that expression of the mouse aprt gene may be developmentally regulated are discussed.

Adenine Phosphoribosyltransferase↗

PubMed2005

Survival and engraftment of mouse embryonic stem cell-derived implants in the guinea pig brain.

alpha-Mannosidosis is a lysosomal storage disease resulting from a deficiency of the enzyme alpha-D-mannosidase. A major feature of alpha-mannosidosis is progressive neurological decline, for which there is no safe and effective treatment available. We have a guinea pig model of alpha-mannosidosis that models the human condition. This study investigates the feasibility of implanting differentiated mouse embryonic stem cells in the neonatal guinea pig brain in order to provide a source of alpha-mannosidase to the affected central nervous system. Cells implanted at a low dose (1.5 x 10(3)cells per hemisphere) at 1 week of age were found to survive in very low numbers in some immunosuppressed animals out to 8 weeks. Four weeks post-implantation, cells implanted in high numbers (10(5) cells per hemisphere) formed teratomas in the majority of the animals implanted. Although implanted cells were found to migrate extensively within the brain and differentiate into mature cells of neural (and other) lineages, the safety issue related to uncontrolled cell proliferation precluded the use of this cell type for longer-term implantation studies. We conclude that the pluripotent cell type used in this study is unsuitable for achieving safe engraftment in the guinea pig brain.

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PubMed1992

Gene targeting using a mouse HPRT minigene/HPRT-deficient embryonic stem cell system: inactivation of the mouse ERCC-1 gene.

A convenient system for gene targeting that uses hypoxanthine phosphoribosyltransferase (HPRT) minigenes as the selectable marker in HPRT-deficient mouse embryonic stem (ES) cells is described. Improvements to the expression of HPRT minigenes in ES cells were achieved by promoter substitution and the provision of a strong translational initiation signal. The use of minigenes in the positive-negative selection strategy for gene targeting was evaluated and the smaller minigenes were found to be as effective as a more conventional marker--the herpes simplex virus thymidine kinase gene. Minigenes were used to target the DNA repair gene ERCC-1 in ES cells. A new HPRT-deficient ES cell line was developed that contributes with high frequency to the germ line of chimeric animals. The ability to select for and against HPRT minigene expression in the new HPRT-deficient ES cell line will make this system useful for a range of gene-targeting applications.

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PubMed2002

Noggin and chordin have distinct activities in promoting lineage commitment of mouse embryonic stem (ES) cells.

To examine the role of secreted signaling molecules and neurogenic genes in early development, we have developed a culture system for the controlled differentiation of mouse embryonic stem (ES) cells. In the current investigation, two of the earliest identified BMP antagonists/neural-inducing factors, noggin and chordin, were expressed in pluripotent mouse ES cells. Neurons were present as early as 24 h following transfection of ES cells with a pCS2/noggin expression plasmid, with differentiation peaking at 72 h. With neuronal differentiation, stem cell marker genes were down-regulated and neural determination genes expressed. Coculture experiments and exposure to noggin-conditioned medium produced similar neuronal differentiation of control ES cells, while addition of BMP-4 to noggin expressants strikingly inhibited neuronal differentiation. Transfection of ES cells with a pCS2/chordin expression vector or exposure to chordin-conditioned medium produced a more complex pattern of differentiation; ES cells formed neurons, mesenchymal cells as well as N-CAM-positive, nestin-positive neuroepithelial progenitors. These data suggest that, consistent with their different expression fields, noggin and chordin may play distinct roles in patterning the early mouse embryo.

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PubMed2003

Molecular signature of human embryonic stem cells and its comparison with the mouse.

The molecular mechanism underlying pluripotency is largely unknown. Here, we provide the first global transcriptional profile of the state of "stemness" in human embryonic stem cells (HESCs). We have identified a set of 918 genes enriched in undifferentiated HESCs compared with their differentiated counterparts. These include ligand/receptor pairs and secreted inhibitors of the FGF, TGFbeta/BMP, and Wnt pathways, highlighting a prevalent role for these pathways in HESCs. Importantly, a significant number of HESCs-enriched genes, including several signaling components, are found to be intersected with published mouse embryonic stem cell data, indicating that a "core molecular program" is shared between the two pluripotent stem cells.

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PubMedDate not supplied

Use of embryonic stem cell-derived endothelial cells as a cell source to generate vessel structures in vitro.

Embryonic stem (ES) cells could potentially serve as an excellent cell source for various applications in regenerative medicine and tissue engineering. Our laboratory is particularly interested in generating a reproducible endothelial cell source for the development of prevascularized materials for tissue/organ reconstruction. After developing methods to isolate highly purified (>96%) proliferating populations of endothelial cells from mouse embryonic stem cells, we tested their ability to form three-dimensional (3-D) vascular structures in vitro. The ES cell-derived endothelial cells were embedded in 3-D collagen gel constructs with rat tail collagen type I (2 mg/mL) at a concentration of 10(6) cells/mL of gel. The gels were observed daily with a phase-contrast microscope to analyze the time course for endothelial cell assembly. The first vessels were observed between days 3 and 5 after gel construct formation. The number and complexity of structures steadily increased, reaching a maximum before beginning to regress. By 2 weeks, all vessel-like structures had regressed back to single cells. Histology and fluorescent images of the vessel-like structures verified that tube structures were multicellular and could develop patent lumens. We have shown that endothelial cells derived, purified and expanded in vitro from ES cells sustain an important endothelial cell function, the ability to undergo vasculogenesis in collagen gels, indicating that endothelial products derived in vitro from stem cells could be useful in regenerative medicine applications.

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PubMed2003

Generation of neural crest-derived peripheral neurons and floor plate cells from mouse and primate embryonic stem cells.

To understand the range of competence of embryonic stem (ES) cell-derived neural precursors, we have examined in vitro differentiation of mouse and primate ES cells into the dorsal- (neural crest) and ventralmost (floor plate) cells of the neural axis. Stromal cell-derived inducing activity (SDIA; accumulated on PA6 stromal cells) induces cocultured ES cells to differentiate into rostral CNS tissues containing both ventral and dorsal cells. Although early exposure of SDIA-treated ES cells to bone morphogenetic protein (BMP)4 suppresses neural differentiation and promotes epidermogenesis, late BMP4 exposure after the fourth day of coculture causes differentiation of neural crest cells and dorsalmost CNS cells, with autonomic system and sensory lineages induced preferentially by high and low BMP4 concentrations, respectively. In contrast, Sonic hedgehog (Shh) suppresses differentiation of neural crest lineages and promotes that of ventral CNS tissues such as motor neurons. Notably, high concentrations of Shh efficiently promote differentiation of HNF3beta(+) floor plate cells with axonal guidance activities. Thus, SDIA-treated ES cells generate naive precursors that have the competence of differentiating into the "full" dorsal-ventral range of neuroectodermal derivatives in response to patterning signals.

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PubMed2006

Germ cells from mouse and human embryonic stem cells.

Mammalian gametes are derived from a founder population of primordial germ cells (PGCs) that are determined early in embryogenesis and set aside for unique development. Understanding the mechanisms of PGC determination and differentiation is important for elucidating causes of infertility and how endocrine disrupting chemicals may potentially increase susceptibility to congenital reproductive abnormalities and conditions such as testicular cancer in adulthood (testicular dysgenesis syndrome). Primordial germ cells are closely related to embryonic stem cells (ESCs) and embryonic germ (EG) cells and comparisons between these cell types are providing new information about pluripotency and epigenetic processes. Murine ESCs can differentiate to PGCs, gametes and even blastocysts - recently live mouse pups were born from sperm generated from mESCs. Although investigations are still preliminary, human embryonic stem cells (hESCs) apparently display a similar developmental capacity to generate PGCs and immature gametes. Exactly how such gamete-like cells are generated during stem cell culture remains unclear especially as in vitro conditions are ill-defined. The findings are discussed in relation to the mechanisms of human PGC and gamete development and the biotechnology of hESCs and hEG cells.

Animals↗

PubMed1998

Efficient poly A trap approach allows the capture of genes specifically active in differentiated embryonic stem cells and in mouse embryos.

Special vectors have been constructed that allow the trapping of genes in mouse embryonic stem (ES) cells. These vectors generally contain the neomycin phosphotransferase (neo) gene for selection and the beta-galactosidase (beta-gal) gene as a marker. Promoterless vectors can be used to identify genes that are active in undifferentiated ES cells. To also have access to genes that are inactive in totipotent ES cells, we constructed a polyadenylation (poly A) trap vector in which the expression of a poly A less neo gene is driven by a constitutive promoter, whereas the expression of beta-gal depends on the trapped sequences. We demonstrate here that this vector integrates with a high frequency into transcription units and that it traps genes with very different expression patterns in vitro and in vivo. The vector integrates efficiently into transcription units that are inactive in undifferentiated ES cells and which can be activated through in vitro differentiation. Furthermore, in vivo expression patterns demonstrate that this vector integrates into genes that exhibit a highly specific temporal and spatial expression pattern during embryogenesis.

Animals↗

PubMed2003

Survival and function of mouse embryonic stem cell-derived cardiomyocytes in ectopic transplants.

OBJECTIVE: Embryonic stem cell-derived cardiomyocytes are a useful source for cell transplantation into the heart, as well as for tissue engineering of the extracardiac vascular system. The present study was designed to investigate the survival and contractile function of embryonic stem cell-derived cardiomyocytes around large blood vessels to assess the feasibility of their ectopic use for future engineering of cardiovascular tissues. METHODS: The mouse embryonic stem cell-derived cardiomyocytes were transplanted into the retroperitoneum of the adult nude mice, and the myocardial tissues that developed were characterized by electrophysiological and histological techniques. RESULTS: Macroscopic and electrophysiological analyses showed spontaneously contracting transplants in the host retroperitoneum 7 and 30 days after transplantation. Immunohistochemistry detected developing cardiomyocytes in the transplants on Day 7, which formed the myocardial tissues. They were positive for cardiac troponin I, cadherin, connexin 43, and proliferating cell nuclear antigen, but negative for alpha-smooth muscle actin. Vascular formation was discernible in the transplant tissues. By Day 30, more mature myocardial tissues had been established in the transplants. Electron microscopic study emphasized that the transplant tissues comprised cardiomyocytes, in which myofibrils with organized sarcomeres were observed. Desmosomes, fasciae adherens and gap junctions were evident in the cellular junctions. CONCLUSIONS: The cardiomyocytes derived from the mouse ES cells were demonstrated to be viable and function in the ectopic site of the host retroperitoneum up to Day 30, following a process of proliferation and differentiation. Vascularization and host perfusion beneficial for the survival of the cardiomyocytes occurred in the transplants.

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PubMed2006

Guiding embryonic stem cells towards differentiation: lessons from molecular embryology.

Embryonic stem cells are uniquely endowed with the capacity of self-renewal and the potential to give rise to all possible cell types, including germ cells. These qualities have made mouse embryonic stem cells a valuable resource for genetic manipulation of the mouse genome. In addition, they present a powerful system for the in vitro dissection of mammalian embryonic development. The recent isolation of human embryonic stem cells has raised a lot of interest for the potential of transposing our knowledge of lineage-specific differentiation of embryonic stem cells to cell-based therapy of human disease. Recent reports have provided insights into the specific differentiation of embryonic stem cells to different cell types of the embryo. However, progress in this direction seems to depend on the knowledge of the mechanisms controlling lineage decisions during embryogenesis.

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PubMed2005

Subtype switching of T-type Ca 2+ channels from Cav3.2 to Cav3.1 during differentiation of embryonic stem cells to cardiac cell lineage.

BACKGROUND: The developmental changes of Ni(2+)-sensitivity to automaticity of Nkx2.5-positive cells derived from mouse embryonic stem cell have been identified, suggesting developmental regulation of expressing Ni(2+)-sensitive T-type Ca(2+) channel, although the mechanism of the change has not been fully studied. METHODS AND RESULTS: Transcripts of Cav3.2, Cav3.1 and Cav1.2 genes of beating Nkx2.5-positive cells, which encode the Ni(2+)-sensitive T-type Ca(2+) channel, Ni(2+)-insensitive T-type Ca(2+) channel, and L-type Ca(2+) channel, respectively, were investigated by real-time reverse-transcriptase-polymerase chain reaction, and the current density of each channel was measured by patch-clamp techniques at the early and late stages of differentiation. The expression of the Cav3.2 transcript predominated in the early stage whereas those of Cav3.1 and Cav1.2 transcripts were upregulated in the late stage, which was consistent with the change in each current density, suggesting the expression of channel proteins is largely determined at the transcriptional level. CONCLUSION: The results indicate that the mechanism of change of Ni(2+)-sensitivity is partly, if not completely, the subtype switch of T-type Ca(2+) channel from Cav3.2 to Cav3.1 at the transcriptional level, and that the expression of the L-type Ca(2+) channel might have an attenuating effect on Ni(2+)-sensitivity to automaticity in the late stage of differentiation.

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PubMed1993

Culturing the epiblast cells of the pig blastocyst.

Pig epiblast cells that had been separated from other early embryonic cells were cultured in vitro. A three-step dissection protocol was used to isolate the epiblast from trophectoderm and primitive endoderm before culturing. Blastocysts collected at 7 to 8 days postestrus were immunodissected to obtain the inner cell mass (ICM) and destroy trophectodermal cells. The ICM was cultured for 2 to 3 days on STO feeder cells. The epiblast was then physically dissected free of associated primitive endoderm. Epiblast-derived cells, grown on STO feeders, produced colonies of small cells resembling mouse embryonic stem cells. This primary cell morphology changed as the colonies grew and evolved into three distinct colony types (endodermlike, neural rosette, or complex). Cell cultures derived from these three colony types spontaneously differentiated into numerous specialized cell types in STO co-culture. These included fibroblasts, endodermlike cells, neuronlike cells, pigmented cells, adipogenic cells, contracting muscle cells, dome-forming epithelium, ciliated epithelium, tubule-forming epithelium, and a round amoeboid cell type resembling a plasmacyte after Wright staining. The neuronlike cells, contracting muscle cells, and tubule-forming epithelium had normal karyotypes and displayed finite or undefined life spans upon long-term STO co-culture. The dome-forming epithelium had an indefinite life span in STO co-culture and also retained a normal karyotype. These results demonstrate the in vitro pluripotency of pig epiblast cells and indicate the epiblast can be a source for deriving various specialized cell cultures or cell lines.

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PubMed1997

Gene transfer into mouse embryonic stem cell-derived cardiac myocytes mediated by recombinant adenovirus.

The main purpose of this study was to examine, for the first time, the ability of recombinant adenovirus to mediate gene transfer into cardiac myocytes derived from mouse embryonic stem (ES) cells differentiating in vitro. In addition, observations were made on the effect of adenovirus infection on cardiac myocyte differentiation and contractility in this in vitro system of cardiogenesis. ES cell cultures were infected at various times of differentiation with a recombinant adenovirus vector (AdCMVlacZ) containing the bacterial lacZ gene under the control of the cytomegalovirus (CMV) promoter. Expression of the lacZ reporter gene was determined by histochemical staining for beta-galactosidase activity. LacZ expression was not detected in undifferentiated ES cells infected with AdCMVlacZ. In contrast, infection of differentiating ES cell cultures showed increasing transgene expression with continued time in culture. Expression in ES-cell-derived cardiac myocytes was demonstrated by codetection of beta-galactosidase activity and troponin T with indirect immunofluorescence. At 24 h postinfection, approximately 27% of the cardiac myocytes were beta-galactosidase positive, and lacZ gene expression appeared to be stable for up to 21 d postinfection. Adenovirus infection had no apparent effect on the onset, extent, or duration of spontaneously contracting ES-cell-derived cardiomyocytes, indicating that cardiac differentiation and contractile function were not significantly altered in the infected cultures. The demonstration of adenovirus-mediated gene transfer into ES-cell-derived cardiac myocytes will aid studies of gene expression with this in vitro model of cardiogenesis and may facilitate future studies involving the use of these myocytes for grafting experiments in vivo.

Adenoviridae↗

PubMed2003

The antimalaria agent artemisinin exerts antiangiogenic effects in mouse embryonic stem cell-derived embryoid bodies.

Artemisinin is widely used as an agent to treat malaria; the possible antiangiogenic effects of this compound are unknown. In the present study, the antiangiogenic effects of artemisinin were investigated in mouse embryonic stem cell-derived embryoid bodies, which are a model system for early postimplantation embryos and which efficiently differentiate capillaries. Artemisinin dose dependently inhibited angiogenesis in embryoid bodies and raised the level of intracellular reactive oxygen species. Furthermore impaired organization of the extracellular matrix component laminin and altered expression patterns of matrix metalloproteinases 1, 2, and 9 were observed during the time course of embryoid body differentiation. Consequently accelerated penetration kinetics of the fluorescent anthracycline doxorubicin occurred within the tissue, indicating increased tissue permeability. Artemisinin down-regulated hypoxia-inducible factor-1alpha and vascular endothelial growth factor (VEGF) expression, which control endothelial cell growth. The antiangiogenic effects and the inhibition of hypoxia-inducible factor-1alpha and VEGF were reversed upon cotreatment with the free radical scavengers mannitol and vitamin E, indicating that artemisinin may act via reactive oxygen species generation. Furthermore, capillary formation was restored upon coadministration of exogenous VEGF. The data of the present study suggest that the antiangiogenic activity of artemisinin and the increase in tissue permeability for cytostatics may be exploited for anticancer treatment.

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PubMed2006

Embryonic stem cell-derived endothelial cells may lack complete functional maturation in vitro.

Stem cell therapies will only become clinically relevant if the stem cells differentiated in vitro function as their in vivo counterparts. Here, we employed our previously developed techniques for deriving endothelial cells (>96% purity) from mouse embryonic stem cells (ESC) and compared these with mouse aortic endothelial cells (MAEC) obtained from thoracic aortas. Immunocytochemical analysis of ESC-derived endothelial cells (EC) demonstrates that both cell types are positive for the EC markers endothelial nitric oxide synthase (eNOS), Flk-1, Flt-1, vascular endothelial cadherin (VEcad), platelet-endothelial cell adhesion molecule-1 (PECAM-1), and CD34. However, ESC-derived EC express slightly lower levels of PECAM-1 and VE-cadherin, and significantly lower levels of acetylated low-density lipoprotein (LDL) uptake and von Willebrand factor. Although ESC-derived EC do express VE-cadherin, the VE-cadherin in the ESC-derived EC did not localize as well at the cell-cell junctions as in the MAEC. Interestingly, ESC-derived EC express much greater levels of the endothelial and hematopoietic stem cell marker CD34 and vasculogenic and angiogenic sprouting than MAEC. These results indicate that ESC-derived EC share some key characteristics of 'mature' EC, while retaining markers of alternate phenotypes including immature endothelium.

Animals↗

PubMed2006

[Progress in the research of germ cell from human embryonic stem cells].

As the development of spontaneous differentiation of germ cells and gametogenesis from mouse embryonic stem cells (mES) in vitro, hES (human embryonic stem cells) also have potential to differentiated into germ cells in theory. This review focuses on the stem cells niches and genes regulating the hES differentiation toward germ cells, as well as the recent advance and application on the reproductive medicine and therapy of infertility.

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