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Results for “Mouse Embryonic Stem Cells”

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PubMed2006

Inducible and reversible suppression of Npm1 gene expression using stably integrated small interfering RNA vector in mouse embryonic stem cells.

The tetracycline (Tc)-inducible small interference RNA (siRNA) is a powerful tool for studying gene function in mammalian cells. However, the system is infrequently utilized in embryonic stem (ES) cells. Here, we present the first application of the Tc-inducible, stably integrated plasmid-based siRNA system in mouse ES cells to down-regulate expression of Npm1, an essential gene for embryonic development. The physiological role of Npm1 in ES cells has not been defined. Our data show that the knock-down of Npm1 expression by this siRNA system was not only highly efficient, but also Tc- dose- and induction time-dependent. Particularly, the down-regulation of Npm1 expression was reversible. Importantly, suppression of Npm1 expression in ES cells resulted in reduced cell proliferation. Taken together, this system allows for studying gene function in a highly controlled manner, otherwise difficult to achieve in ES cells. Moreover, our results demonstrate that Npm1 is essential for ES cell proliferation.

Animals↗

PubMed2006

ATP stimulates mouse embryonic stem cell proliferation via protein kinase C, phosphatidylinositol 3-kinase/Akt, and mitogen-activated protein kinase signaling pathways.

This study investigated the effect of ATP and its related signal cascades on the proliferation of mouse ESCs. ATP increased the level of [(3)H]thymidine/5-bromo-2'-deoxyuridine incorporation and the number of cells in both a time- and dose-dependent manner. AMP-CPP (a P2X(1) and P2X(3) agonist), ATP-gammaS (a P2Y agonist), and 2-methylthio-ATP (a P2X and P2Y agonist) stimulated [(3)H]thymidine incorporation. P2 purinoceptor antagonists (suramin, reactive blue 2) inhibited the ATP-induced increase in [(3)H]thymidine incorporation. Reverse transcription-polymerase chain reaction analysis revealed P2X(3), P2X(4), P2Y(1), and P2Y(2) expression in mouse ESCs. Adenylate cyclase inhibitor (SQ 22536), phospholipase C inhibitors (neomycin or U 73122), and protein kinase C (PKC) inhibitors (bisindolylmaleimide I or staurosporine) inhibited the ATP-induced increase in [(3)H]thymidine incorporation. ATP increased the level of intracellular cAMP and inositol phosphates. ATP translocated PKC alpha, delta, and zeta from the cytosol to the membrane compartment. ATP and its agonists increased [Ca(2+)](i). In addition, the ATP-induced increase in [(3)H]thymidine incorporation was completely inhibited by a combination of EGTA (extracellular Ca(2+) chelator) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM (intracellular Ca(2+) chelator). ATP phosphorylated Akt and p44/42 mitogen-activated protein kinases (MAPKs) in a time-dependent manner, and either suramin or reactive blue 2 (RB2) blocked the ATP-induced phosphorylation of Akt. Suramin, RB2, the phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin), or the Akt inhibitor inhibited the phosphorylation of p44/42 MAPKs. The ATP-induced increase in [(3)H]thymidine incorporation was inhibited by wortmannin, the Akt inhibitor, and the MAPK kinase inhibitor (PD 98059). Suramin, RB2, PD 98059, and wortmannin blocked the ATP-induced increase in the cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK4 levels. In conclusion, ATP stimulates mouse ESC proliferation through PKC, PI3K/Akt, and MAPKs via the P2 purinoceptors.

Adenosine Monophosphate↗

PubMed2005

Overexpression of Nodal promotes differentiation of mouse embryonic stem cells into mesoderm and endoderm at the expense of neuroectoderm formation.

Understanding how to direct the fate of embryonic stem (ES) cells upon differentiation is critical to their eventual use in therapeutic applications. Clues for controlling ES cell differentiation may be found in the early embryo because mouse ES cells form derivatives of all three embryonic germ layers upon injection into blastocysts. One promising candidate for influencing the differentiation of ES cells into the embryonic germ layers is the transforming growth factor-beta (TGF-beta) growth factor, Nodal. Nodal null mouse mutants lack mesoderm, and injection of Nodal mRNA into nonmammalian embryos induces mesodermal and endodermal tissues. We find that overexpression of Nodal in mouse ES cells leads not only to up-regulation of mesodermal and endodermal cell markers but also to downregulation of neuroectodermal markers. These findings demonstrate the importance of Nodal's influence on the differentiation of pluripotent cells to all three of the primary germ layers. Accordingly, altering expression of factors responsible for cell differentiation in the intact embryo provides an approach for directing ES cell fates in vitro toward therapeutically useful cell types.

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PubMed2004

X-chromosome inactivation in differentiating mouse embryonic stem cells carrying X-linked GFP and lacZ transgenes.

Three new female ES cell lines (GLM1, GLP1 and GLP2) were established from mouse embryos carrying GFP (green fluorescent protein) and HMG-lacZ transgenes on one of two X chromosomes in cis. Using these cell lines, we studied the temporal relationships among three events relevant to X-chromosome inactivation: replication asynchrony of the X chromosome, and quenching of GFP fluorescence and beta-galactosidase (beta-gal) activity, during cell differentiation induced by embryoid body (EB) formation and retinoic acid (RA) treatment. In embryoid bodies adhering to the bottom of culture dishes, GFP-negative cells appeared first in the peripheral outgrowths 4 days after the initiation of EB formation, followed about 24 hours later by the appearance of cells negative for beta-gal and those having a single allocyclic X chromosome. Although the frequency of cells with an allocyclic X chromosome could reach 80% in adherent embryoid bodies, it tended to remain low and variable in embryoid bodies maintained in suspension. In spite of apparently parallel extinction of GFP and lacZ in embryoid bodies, their concurrent occurrence did not always characterize RA-induced differentiation. Moreover, an allocyclic X chromosome was identified in not more than 20 percent of informative metaphase cells up to 10 days after initiation of RA treatment. These findings suggest that RA-induced differentiation of female ES cells does not always accompany X-inactivation.

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PubMed2005

Enrichment of hepatocytes differentiated from mouse embryonic stem cells as a transplantable source.

BACKGROUND: We previously reported that hepatocytes can be differentiated from embryonic stem (ES) cells by way of embryoid body (EB) formation and are transplantable into the mouse liver. However, the transplantation of EB-derived cells frequently resulted in teratoma formation in the recipient liver. In the present study, we eliminated the tumorigenic cells from EB outgrowths and examined the effects of enriched ES-cell-derived hepatocyte transplantation into an injured liver. METHODS: On day 15 in culture, the EBs were partially disaggregated and subcultured. Hepatocytes in the subcultured cells were examined by the expression of hepatocyte markers. Undifferentiated cells contaminating in the EB-derived cells were eliminated by Percoll discontinuous gradient centrifugation. Furthermore, undifferentiated cells, endothelial cells, and macrophages were eliminated by magnetic cell sorting using platelet/endothelial cell adhesion molecule (PECAM)-1 and Mac-1 antibodies. These enriched ES-cell-derived hepatocytes were then transplanted into the injured mouse liver. RESULTS: Percoll centrifugation and PECAM-1 antibodies eliminated the undifferentiated cells expressing Oct-3/4 from the EB-derived cells. ES-cell-derived hepatocytes showed expression of liver-related genes, synthesis of urea and glycogen, and structural characteristics during subculture. A transplantation study showed that the enriched ES-cell-derived hepatocytes integrated into the injured mouse liver and produced no teratomas. When the ES-cell-derived hepatocytes were transplanted into a CCl4-injured liver, the liver function was subsequently improved. CONCLUSIONS: Functional hepatocytes can be differentiated from mouse ES cells by way of EB formation. The elimination of undifferentiated cells from the EBs provides transplantable cells for liver failure without tumorigenicity.

Animals↗

PubMed1996

Maintenance of pluripotency in mouse embryonic stem cells persistently infected with murine coronavirus.

A persistently coronavirus-infected embryonic stem (ES) cell line A3/MHV was established by infecting an ES cell line, A3-1, with mouse hepatitis virus type-2. Although almost all A3/MHV cells were found infected, both A3/MHV and A3-1 cells expressed comparable levels of cell surface differentiation markers. In addition, A3/MHV cells retained the ability to form embryoid bodies. These results suggest that persistent coronavirus infection does not affect the differentiation of ES cells.

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PubMed2004

Induction of epithelial progenitors in vitro from mouse embryonic stem cells and application for reconstruction of damaged cornea in mice.

PURPOSE: Severe ocular surface diseases and injuries cause loss of the corneal limbal epithelium, leading to re-epithelialization by bulbar conjunctival cells, resulting in vascularization of the cornea, conjunctival scarring, and loss of visual acuity. In this study, the optimal culture condition for induction of differentiation of epithelial progenitor cells from embryonic stem (ES) cells was determined for use in transplantation to damaged cornea in mice. METHODS: Mouse ES cells were cultured on Petri dishes coated with several extracellular matrix proteins, and the markers for epithelial cells were analyzed with RT-PCR and Western blot analysis. The optimal condition for induction of epithelial progenitor cells was determined, and the progenitors were transplanted onto mouse eyes with corneal epithelia that had been damaged by exposure to n-heptanol. RESULTS: Epithelial progenitors were successfully induced by culturing mouse ES cells on type IV collagen for 8 days. These progenitors expressed keratin (K)12, which is specific to corneal epithelial cells, and cell surface CD44 and E-cadherin, both of which are essential in corneal epithelial wound healing. Complete re-epithelialization of the corneal surface occurred within 24 hours after transplantation. The resultant corneal epithelial cells expressed markers of the grafted cells, and no teratomata were observed during the follow-up period. CONCLUSIONS: Epithelial progenitors were successfully induced in vitro from ES cells and were applicable as grafts for treating corneal epithelial injury. ES cells may become an unlimited donor source of corneal epithelial cells for corneal transplantation and may restore useful vision in patients with a deficiency of limbal epithelial cells. This is an important first trial toward assessing the use of ES cells to reconstruct corneal epithelial cells.

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PubMed2002

In vivo differentiation of mouse embryonic stem cells into hepatocytes.

Embryonic stem (ES) cells have been regarded as a powerful resource for cell replacement therapy. In recent reports mouse ES cells have been successfully applied in the treatment of spinal cord injury, hereditary myelin disorder of the central nervous system, and diabetes mellitus. Another type of disease that could benefit from the availability of stem cell therapy is liver disease. However, for this potential to be realized, it is necessary to demonstrate the differentiation of ES cells into hepatocytes. To demonstrate the in vivo differentiation potential of mouse ES cells, we injected ES cells into the spleen of immunosuppressed nude mice. Histological analysis of teratomas derived from injected ES cells revealed that some areas contained typical hepatocytes arranged in a sinusoidal structure. The hepatic nature of these cells was further confirmed by showing that transcripts of liver-specific genes were present in the differentiated teratoma using reverse transcriptase-polymerase chain reaction and immunohistochemistry using several liver-specific antibodies including HEP-PAR, phenylalanine hydroxylase, and mouse N-system aminotransferase to identify the respective proteins in the differentiated hepatocytes. This is the first demonstration that mouse ES cells can differentiate in vivo into a mixed population of hepatocytes of varying maturity. This finding extends the potential use of ES cells in the cell replacement therapy by including its possible application for treating liver diseases.

Animals↗

PubMed2000

Apoptosis of mouse embryonic stem cells induced by single cell suspension.

Embryonic stem cells (ES cells) are pluripotential, and are therefore used to construct gene knock-out mice. We found that the apoptosis of mouse ES cells was induced when the cells were dispersed as single cells, whereas this process was suppressed when they proliferated in aggregates. The apoptosis of ES cells was repressed when the cells were cultured on feeders prepared from STO cells, a cell line established from embryonic fibroblasts. Culture supernatants from STO cells did not block the apoptosis of ES cells, which suggests that a direct interaction between ES cells and STO cells is required for the suppression of apoptosis. The viability of ES cells examined by the trypan blue exclusion test or by the MTT ((3-4,5-dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay decreased dramatically when the cells were dispersed in phosphate-buffered saline PBS. Cellular activity was restored by the addition of culture medium for ES cells. Glucose in the medium was found to be a major factor responsible for the restoration. Amino acids also restored the decrease in reduction of MTT. Suspension of the ES cells in PBS(-) caused leakage of the nucleosome into cytoplasm. Results indicate that the single cell suspension of ES cells leads to leakage of substrates for oxidative phosphorylation from the mitochondria, and that these cells finally become committed to apoptosis.

Amino Acids↗

PubMed2005

Wnt4-transformed mouse embryonic stem cells differentiate into renal tubular cells.

Embryonic stem (ES) cells have the potential to differentiate into various progenitor cells. Here we investigated the capacity of mouse ES cells to differentiate into renal tubular cells both in vitro and in vivo. After stably transfecting Wnt4 cDNA to mouse ES cells (Wnt4-ES cells), undifferentiated ES cells were incubated by the hanging drop culture method to induce differentiation to embryoid bodies (EBs). During culturing of the EBs derived from the Wnt4-ES cells, aquaporin-2 (AQP2) mRNA and protein were expressed within 15-20 days. The expression of AQP2 in Wnt4-EBs was enhanced in the presence of hepatocyte growth factor (HGF) and activin A. We next performed in vivo experiments by transplanting the Wnt4-EBs into the mouse renal cortex. Four weeks after transplantation, some portions of the EB-derived cells expressing AQP2 in the kidney assembled into tubular-like formations. In conclusion, our in vitro and in vivo experiments revealed two new findings: first, that cultured Wnt4-EBs have an ability to differentiate into renal tubular cells; and second, that Wnt4, HGF, and activin A may promote the differentiation of ES cells to renal tubular cells.

Activins↗

PubMed2005

In vitro differentiation and maturation of mouse embryonic stem cells into hepatocytes.

It is difficult to induce the maturation of embryonic stem (ES) cells into hepatocytes in vitro. We previously reported that Thy1-positive mesenchymal cells derived from the mouse fetal liver promote the maturation of hepatic progenitor cells. Here, we isolated alpha-fetoprotein (AFP)-producing cells from mouse ES cells for subsequent differentiation into hepatocytes in vitro by coculture with Thy1-positive cells. ES cells expressing green fluorescent protein (GFP) under the control of an AFP promoter were cultured under serum- and feeder layer-free culture conditions. The proportion of GFP-positive cells plateaued at 41.6 +/- 12.2% (means +/- SD) by day 7. GFP-positive cells, isolated by flow cytometry, were cultured in the presence or absence of Thy1-positive cells as a feeder layer. Isolated GFP-positive cells were stained for AFP, Foxa2, and albumin. The expression of mRNAs encoding tyrosine amino transferase, tryptophan 2,3-dioxygenase, and glucose-6-phosphatase were only detected following coculture with Thy1-positive cells. Following coculture with Thy1-positive cells, the isolated cells produced and stored glycogen. Ammonia clearance activity was also enhanced following coculture. Electron microscopic analysis indicated that the cocultured cells exhibited the morphologic features of mature hepatocytes. In conclusion, coculture with Thy1-positive cells in vitro induced the maturation of AFP-producing cells isolated from ES cell cultures into hepatocytes.

Animals↗

PubMed2004

In vitro organogenesis of gut-like structures from mouse embryonic stem cells.

Embryonic stem (ES) cells have pluripotency and give rise to many cell types and tissues, including representatives of all three germ layers in the embryo. We have reported previously that mouse ES cells formed contracting gut-like organs from embryoid bodies (EBs). These gut-like structures contracted spontaneously, and had large lumens surrounded by three layers, i.e. epithelium, lamina propria and muscularis. Ganglia were scattered along the periphery, and interstitial cells of Cajal (ICC) were distributed among the smooth muscle cells. In the present study, to determine whether they can be a model of gut organogenesis, we investigated the formation process of the gut-like structures in comparison with embryonic gut development. As a result, we found that the fundamental process of formation in vitro was similar to embryonic gut development in vivo. The result indicates that the gut-like structure is a useful tool not only for developmental study to determine the factors that induce gut organogenesis, but also for studies of enteric neurone and ICC development.

Animals↗

PubMed2006

The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells.

Oct4 and Nanog are transcription factors required to maintain the pluripotency and self-renewal of embryonic stem (ES) cells. Using the chromatin immunoprecipitation paired-end ditags method, we mapped the binding sites of these factors in the mouse ES cell genome. We identified 1,083 and 3,006 high-confidence binding sites for Oct4 and Nanog, respectively. Comparative location analyses indicated that Oct4 and Nanog overlap substantially in their targets, and they are bound to genes in different configurations. Using de novo motif discovery algorithms, we defined the cis-acting elements mediating their respective binding to genomic sites. By integrating RNA interference-mediated depletion of Oct4 and Nanog with microarray expression profiling, we demonstrated that these factors can activate or suppress transcription. We further showed that common core downstream targets are important to keep ES cells from differentiating. The emerging picture is one in which Oct4 and Nanog control a cascade of pathways that are intricately connected to govern pluripotency, self-renewal, genome surveillance and cell fate determination.

Animals↗

PubMed2004

Functional properties of motoneurons derived from mouse embryonic stem cells.

The capacity of embryonic stem (ES) cells to form functional motoneurons (MNs) and appropriate connections with muscle was investigated in vitro. ES cells were obtained from a transgenic mouse line in which the gene for enhanced green fluorescent protein (eGFP) is expressed under the control of the promotor of the MN specific homeobox gene Hb9. ES cells were exposed to retinoic acid (RA) and sonic hedgehog agonist (Hh-Ag1.3) to stimulate differentiation into MNs marked by expression of eGFP and the cholinergic transmitter synthetic enzyme choline acetyltransferase. Whole-cell patch-clamp recordings were made from eGFP-labeled cells to investigate the development of functional characteristics of MNs. In voltage-clamp mode, currents, including EPSCs, were recorded in response to exogenous applications of GABA, glycine, and glutamate. EGFP-labeled neurons also express voltage-activated ion channels including fast-inactivating Na(+) channels, delayed rectifier and I(A)-type K(+) channels, and Ca(2+) channels. Current-clamp recordings demonstrated that eGFP-positive neurons generate repetitive trains of action potentials and that l-type Ca(2+) channels mediate sustained depolarizations. When cocultured with a muscle cell line, clustering of acetylcholine receptors on muscle fibers adjacent to developing axons was seen. Intracellular recordings of muscle fibers adjacent to eGFP-positive axons revealed endplate potentials that increased in amplitude and frequency after glutamate application and were sensitive to TTX and curare. In summary, our findings demonstrate that MNs derived from ES cells develop appropriate transmitter receptors, intrinsic properties necessary for appropriate patterns of action potential firing and functional synapses with muscle fibers.

Acetylcholine↗

PubMed2005

Redox control of angiogenic factors and CD31-positive vessel-like structures in mouse embryonic stem cells after direct current electrical field stimulation.

The molecular mechanisms driving angiogenesis in tissues derived from embryonic stem (ES) cells are currently unknown. Herein we investigated the effects of direct current (DC) electrical field treatment on endothelial cell differentiation and angiogenesis of mouse ES cells. Treatment of ES cell-derived embryoid bodies with field strengths ranging from 250 V/m to 750 V/m, applied for 60 s, dose-dependently increased the capillary area staining positive for the endothelial-specific marker platelet endothelial cell adhesion molecule-1 (PECAM-1), indicating stimulation of endothelial cell differentiation and angiogenesis. Consequently, increased expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) within 24 h was observed. Electric field treatment raised reactive oxygen species (ROS) generation for at least 48 h, which was blunted by NADPH-oxidase inhibitors diphenylen iodonium chloride (DPI) as well as 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), and increased the expression of NADPH-oxidase subunits p22-phox, p47-phox, p67-phox, and gp91-phox within 24 h. Electrical field treatment resulted in activation of extracellular regulated kinase 1,2 (ERK1,2), p38, as well as c-Jun NH2-terminal kinase (JNK). Pretreatment with the JNK inhibitor SP600125 resulted in a significant decrease in capillary areas under control conditions as well as under conditions of electrical field treatment, whereas the p38 inhibitor SB203580 was without effects. By contrast, the ERK1,2 antagonist UO126 inhibited electrical field-induced angiogenesis, whereas angiogenesis under control conditions was unimpaired. The increase in capillary areas and VEGF expression as well as activation of JNK and ERK1,2 was significantly inhibited in the presence of the free radical scavenger vitamin E underscoring the role of ROS in electrical field-induced angiogenesis of ES cells.

Angiogenesis Inducing Agents↗

PubMed2006

15-Deoxy-delta12,14-prostaglandin J2 regulates leukemia inhibitory factor signaling through JAK-STAT pathway in mouse embryonic stem cells.

Embryonic stem (ES) cells are genetically normal, pluripotent cells, capable of self-renewal and differentiation into all cell lineages. While leukemia inhibitory factor (LIF) maintains pluripotency in mouse ES cells, retinoic acid and other nuclear hormones induce neuro-glial differentiation in mouse and human ES cells in culture. Peroxisome-proliferator-activated receptors (PPARs) are ligand-dependent nuclear receptor transcription factors that regulate cell growth and differentiation in many cell types. However, the role of PPARs in the regulation of ES cell growth and differentiation is not known. In this study, we show that LIF induces proliferation and self-renewal of mouse D3-ES cells in culture. However, treatment with 15-Deoxy-Delta(12,14)-Prostaglandin J(2) (15d-PGJ2), a natural ligand for PPARgamma, or all-trans retinoic acid (ATRA) results in a dose-dependent decrease in proliferation and self-renewal in D3-ES cells. Immunoprecipitation and Western blot analyses showed that LIF induces tyrosine phosphorylation of JAK1, TYK2 and STAT3 in 30 min and treatment with 15d-PGJ2 or ATRA results in a dose-dependent decrease in LIF-induced phosphorylation of JAK1 and STAT3 in D3-ES cells. However, treatment of D3-ES cells with Ciglitazone or 15d-PGJ2 for 48 h in culture resulted in a dose-dependent increase in PPARgamma protein expression. These results suggest that PPARgamma agonists regulate LIF signaling through JAK-STAT pathway leading to growth and self-renewal of ES cells.

Animals↗

PubMed2004

Nitric oxide facilitates cardiomyogenesis in mouse embryonic stem cells.

Stem cell therapy holds great promise for the replacement of damaged or dysfunctional myocardium. Nitric oxide (NO) has been shown to promote embryonic stem (ES) cell differentiation in other systems. We hypothesized that NO, through NO synthase gene transfer or exogenous NO exposure, would promote the differentiation of mouse ES cells into cardiomyocytes (CM). In our study, NO treatment increased both the number and the size of beating foci in embryoid body (EB) outgrowths. Within 2 weeks, 69% of the inducible NO synthase-transduced EB displayed spontaneously beating foci, as did 45% of the NO donor-treated EB, compared with only approximately 15% in controls. Cardiac-specific genes and protein expression were significantly increased in NO-treated ES. Electron microscopy and immunocytochemistry revealed that these NO-induced contracting cells exhibited characteristics consistent with CM. At day 7 in culture, troponin T was expressed in 45.6 +/- 20.6% of the NO-treated ES cells but in only 9.25 +/- 1.77% of control cells. Interestingly, 50.4 +/- 18.4% of NO-treated ES cells were troponin T-negative and annexin V-positive. This apoptotic phenotype was seen in <1% of the control ES cells. These data strongly support our hypothesis that mouse ES cells can be accelerated to differentiate into CM by NO treatment. NO may influence cardiac differentiation by both inducing a switch toward a cardiac phenotype and inducing apoptosis in cells not committed to cardiac differentiation.

Animals↗

PubMed2006

Factors derived from mouse embryonic stem cells promote self-renewal of goat embryonic stem-like cells.

Goat embryonic stem (ES)-like cells could be isolated from primary materials-inner cell masses (ICMs) and remain undifferentiated for eight passages in a new culture system containing mouse ES cell conditioned medium (ESCCM) and on a feeder layer of mouse embryo fibroblasts (MEFs). However, when cultured in medium without mouse ESCCM, goat ES-like cells could not survive for more than three passages. In addition, no ES-like cells could be obtained when ICMs were cultured on goat embryo fibroblasts or the primary materials-whole goat blastocysts were cultured on MEFs. Goat ES-like cells isolated from ICMs had a normal karyotype and highly expressed alkaline phosphatase. Multiple differentiation potency of the ES-like cells was confirmed by differentiation into neural cells and fibroblast-like cells in vitro. These results suggest that mouse ES cells might secrete factors playing important roles in promoting goat ES-like cells' self-renewal, moreover, the feeder layers and primary materials could also influence the successful isolation of goat ES-like cells.

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