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PubMed2004

Neural differentiation of mouse embryonic stem cells in vitro and after transplantation into eyes of mutant mice with rapid retinal degeneration.

Embryonic stem (ES) cells can differentiate into many specialized cell types, including those of the nervous system. We evaluated the differentiation of enhanced green fluorescent protein (EGFP)-expressing B5 mouse ES cells in vitro and in vivo after transplantation into the eyes of mice with hereditary retinal degeneration. After neural induction with retinoic acid, the majority of cells in embryoid bodies expressed markers for neural progenitors as well as for immature and mature neurons and glial cells. When induced ES cells were plated in vitro, further differentiation was observed and the majority of cells expressed beta-III Tubulin, a marker for immature neurons. In addition, many plated cells expressed markers for mature neurons or glial cells. Four days after intravitreal transplantation into the eyes of rd1 mice (a model of rapid retinal degeneration), donor cells appeared attached to the vitreal surface of the retina. After 6 weeks in vivo, most transplanted cells remained adherent to the inner retinal surface, and some donor cells had integrated into the retina. Transplanted cells exhibited some properties typical of neurons, including extensive process outgrowth with numerous varicosities and expression of neuronal and synaptic markers. Therefore, after induction B5 ES cells can acquire the morphologies of neural cells and display markers for neuronal and glial cells in vitro and in vivo. Furthermore, when placed in the proper microenvironment ES-derived neural precursors can associate closely with or migrate into nervous tissue where differentiation appears to be determined by cues provided by the local environment, in this case the degenerating neural retina.

Animals↗

PubMed2006

Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b.

DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b cooperatively regulate cytosine methylation in CpG dinucleotides in mammalian genomes, providing an epigenetic basis for gene silencing and maintenance of genome integrity. Proper CpG methylation is required for the normal growth of various somatic cell types, indicating its essential role in the basic cellular function of mammalian cells. Previous studies using Dnmt1(-/-) or Dnmt3a(-/-)Dnmt3b(-/-) ES cells, however, have shown that undifferentiated embryonic stem (ES) cells can tolerate hypomethylation for their proliferation. In an attempt to investigate the effects of the complete loss of CpG DNA methyltransferase function, we established mouse ES cells lacking all three of these enzymes by gene targeting. Despite the absence of CpG methylation, as demonstrated by genome-wide methylation analysis, these triple knockout (TKO) ES cells grew robustly and maintained their undifferentiated characteristics. TKO ES cells retained pericentromeric heterochromatin domains marked with methylation at Lys9 of histone H3 and heterochromatin protein-1, and maintained their normal chromosome numbers. Our results indicate that ES cells can maintain stem cell properties and chromosomal stability in the absence of CpG methylation and CpG DNA methyltransferases.

Amino Acid Sequence↗

PubMed2005

Electrophysiological properties of pluripotent human and mouse embryonic stem cells.

Pluripotent embryonic stem cells (ESCs) possess promising potential for cell-based therapies, but their electrophysiological properties have not been characterized. Here we describe the presence of ionic currents in mouse (m) and human (h) ESCs and their physiological function. In mESCs, tetraethylammonium (TEA)-sensitive depolarization-activated delayed rectifier K+ currents (IK(DR)) (8.6 +/- 0.9 pA/pF at +40 mV; IC50 = 1.2 +/- 0.3 mM), which contained components sensitive to 4-aminopyridine (4-AP) (IC50 = 0.5 +/- 0.1 mM) and 100 nM Ca2+-activated K+ current (IK(Ca)) blocker iberiotoxin (IBTX),were detected in 52.3% of undifferentiated cells.IK(DR) was similarly present in hESCs (approximately 100%) but with an approximately sixfold higher current density (47.5 +/- 7.9 pA/pF at +40 mV). When assayed by bromodeoxyurindine incorporation, application of TEA, 4-AP, or IBTX significantly reduced the proliferation of mESCs and hESCs in a dose-dependent manner (p < .05). A hyperpolarization-activated inward current (I(h)) (-2.2 +/- 0.4 pA/pF at -120 mV) was detected in 23% of mESCs but not hESCs. Neither Na(v) nor Ca(v) currents were detected in mESCs and hESCs. Microarray and reverse transcription-polymerase chain reaction analyses identified several candidate genes for the ionic currents discovered. Collectively, our results indicate that pluripotent ESCs functionally express several specialized ion channels and further highlight similarities and differences between the two species. Practical considerations for the therapeutic use of ESCs are discussed.

4-Aminopyridine↗

PubMed2006

Efficient establishment of mouse embryonic stem cell lines from single blastomeres and polar bodies.

Recently, ES cell lines were established from single blastomeres taken from eight-cell embryos in mice and humans with success rates of 4% and 2%, respectively, which suggests that the method could be used in regenerative medicine to reduce ethical concerns over harm to embryos. However, those studies used other ES cells as supporting cells. Here, we report a simple and highly efficient method of establishing mouse ES cell lines from single blastomeres, in which single blastomeres are simply plated onto a feeder layer of mouse embryonic fibroblasts with modified ES cell medium. A total of 112 ES cell lines were established from two-cell (establishment rate, 50%-69%), early four-cell (28%-40%), late four-cell (22%), and eight-cell (14%-16%) stage embryos. We also successfully established 18 parthenogenetic ES cell lines from first (36%-40%) and second polar bodies (33%), the nuclei of which were reconstructed to embryos by nuclear transfer. Most cell lines examined maintained normal karyotypes and expressed markers of pluripotency, including germline transmission in chimeric mice. Our results suggest that the single cells of all early-stage embryos or polar bodies have the potential to be converted into ES cells without any special treatment.

Animals↗

PubMed2005

Branching ducts similar to mesonephric ducts or ureteric buds in teratomas originating from mouse embryonic stem cells.

Ureteric bud epithelial cells and metanephric mesenchymal cells that comprise the metanephric kidney primordium are capable of producing nephrons and collecting ducts through reciprocal inductive interaction. Once these cells are induced from pluripotent embryonic stem (ES) cells, they have the potential to become powerful tools in the regeneration of kidney tissues. In this study, we investigated these renal primordial cells and structures in mouse ES cell outgrowths and their transplants. Gene expression essential for early kidney development was examined by RT-PCR in embryoid body (EB) outgrowths and their transplants in adult mice. Histochemical detection of kidney primordial structures and gene expression analysis coupled with laser microdissection were performed in transplant tissues. RT-PCR analysis detected gene expression of Pax-2, Lim-1, c-Ret, Emx2, Sall1, WT-1, Eya-1, GDNF, and Wnt-4 in the EB outgrowths from days 6-9 of expansion onward, and also in the teratoma tissues 14 and 28 days after transplantation. Histochemical analysis 14 days after transplantation showed that some ducts were positive for Pax-2, endo A cytokeratin, kidney-specific cadherin, and Dolichos biflorus agglutinin and that dichotomous branching of these ducts had occurred. These staining patterns and morphological features are intrinsic for mesonephric ducts and ureteric buds. In long-term survival of 28 days, Pax-2-immunoreactivity disappeared in some renal primordia-like structures, indicating their differentiation. Some ducts were accompanied by mesonephric nephron-like convoluted tubules. RT-PCR analysis of those structures collected by microdissection confirmed that they expressed kidney development-related genes. In conclusion, these data suggest the potential of ES cells to produce renal primordial duct structures and provides an insight into the regeneration of kidney tissues.

Animals↗

PubMed2001

Deriving and propagating mouse embryonic stem cell lines for studying genomic imprinting.

Embryonic stem (ES) cells are a cell culture derivative of the blastocyst inner cell mass (ICM), the latter giving rise to the embryo, the amnion, the yolk sac, and the chrorioallantoic portion of the placenta. Blastocyst injection chimera experiments show that ES cells are similar to early-stage ICM cells in that they contribute to the primitive ectoderm and endoderm derivatives (1). However, it is probably not possible to equate these two cell types, as ES cells appear to be produced by the cell culture environment and have no exact counterpart in the blastocyst. Instead, ES cells could be thought of as being ICM cells that, instead of undergoing rapid differentiation as they would in vivo, are abnormally locked into continuing cycles of division in the undifferentiated state by virtue of the action of exogenous factors. Leukemia inhibitory factor, LIF, is one such factor (2,3) and is indispensable for the propagation of mouse ES cells at least when primary embryo fibroblasts (PEFs) are used as feeder layers (4).

Animals↗

PubMed2001

Proteomic characterization of early-stage differentiation of mouse embryonic stem cells into neural cells induced by all-trans retinoic acid in vitro.

Embryonic stem (ES) cells are totipotent stem cells, which can differentiate into various kinds of cell types, including neurons. They are widely used as a model system for investigating mechanisms of differentiation events during early mouse development. In this study, proteomic techniques were used to approach the protein profile associated with the early-stage differentiation of ES cells into neuronal cells induced by all-trans retinoic acid (ATRA) in vitro. In comparison of the protein profile of parent ES cells with that of ES-derived neural-committed cells, which was induced by ATRA for four days, 24 differentially displayed protein spots were selected from two-dimensional electrophoresis (2-DE) gels for further protein identification by pepide mass fingerprinting (PMF). Nine proteins were known to being involved in the process of neural differentiation and/or neural survival. Of those, alpha-3/alpha-7 tubulin and vimentin were down-regulated, while cytokeratin 8, cytokeratin 18, G1/S-special cyclin D2, follistatin-related protein, NEL protein, platelet-activating factor acetylhydrolase IB alpha-subunit, and thioredoxin peroxidase 2 were upregulated during differentiation of ES cells to neural cells. Additionally, other 12 protein (five upregulated and seven downregulated) spots associated with ES cell differentiation into neuronal cells were not matched to known proteins so far, implicating that they might be novel proteins. The results above indicated that the molecular mechanisms of differentiation of ES cells to neural cells in vitro might be similar to those of other neural systems in vitro and identified that proteomic analysis is an effective strategy to comprehensively unravel the regulatory network of differentiation.

Animals↗

PubMed2005

Insulin stimulates Ca2+ uptake via PKC, cAMP, and p38 MAPK in mouse embryonic stem cells.

Embryonic stem (ES) cells are provided as a powerful tool for developmental biology and have been shown to respond to insulin. However, little is known about the effect of insulin on [Ca2+]i regulation in the ES cells, although many cellular functions are tightly regulated by [Ca2+]i. Therefore, we examined the effect of insulin on Ca2+ uptake and its related signal pathways in the mouse ES cells. Mouse ES cells expressed alkaline phosphatase (AP), transcription factor Oct-4, and stage-specific embryonic antigen-1 (SSEA-1). Insulin increased the Ca2+ uptake in a time- and dose-dependent manner and the effect was blocked by L-type Ca2+ channel blockers, nifedifine and methoxyverapamil. Genistein or herbimycin A (tyrosine kinase inhibitors), wortmannin (PI-3K inhibitor), and staurosporine or bisindolylmaleimide I (PKC inhibitors) completely prevented insulin-induced increase of Ca2+ uptake. Wortmannin blocked insulin-induced PKC activation, but SQ 22536 (adenylate cyclase inhibitor) did not. Insulin also rapidly increased formation of inositol phosphates (IPs). We examined the involvement of MAPKs in mediating the effect of insulin on Ca2+ uptake. SB 203580 (p38 MAPK inhibitor) but not PD 98059 (p44/42 MAPKs inhibitor) blocked insulin-induced increase of Ca2+ uptake. Insulin significantly increased the phosphorylation of p38 MAPK but not p44/42 MAPKs. In addition, genistein, PKI, and bisindolylmaleimide I blocked the phosphorylation of p38 MAPK by insulin, suggesting a causal relationship. In conclusion, insulin partially stimulated Ca2+ uptake via PKC, cAMP, and p38 MAPK signaling pathways in mouse ES cells.

Analysis of Variance↗

PubMed2005

DNA methylation is required for silencing of ant4, an adenine nucleotide translocase selectively expressed in mouse embryonic stem cells and germ cells.

The capacity for cellular differentiation is governed not only by the repertoire of available transcription factors but by the accessibility of cis-regulatory elements. Studying changes in epigenetic modifications during stem cell differentiation will help us understand how cells maintain or lose differentiation potential. We investigated changes in DNA methylation during the transition of pluripotent embryonic stem cells (ESCs) into differentiated cell types. Using a methylation-sensitive restriction fingerprinting method, we identified a novel adenine nucleotide (ADP/ATP) translocase gene, Ant4, that was selectively hypomethylated and expressed in undifferentiated mouse ESCs. In contrast to other pluripotent stem cell-specific genes such as Oct-4 and Nanog, the Ant4 gene was readily derepressed in differentiated cells after 5-aza-2'-deoxycytidine treatment. Moreover, expression of de novo DNA methyltransferases Dnmt3a and Dnmt3b was essential for repression and DNA methylation of the Ant4 gene during ESC differentiation. Although the deduced amino acid sequence of Ant4 is highly homologous to the previously identified Ant isoforms, the expression of Ant4 was uniquely restricted to developing gametes in adult mice, and its promoter hypomethylation was observed only in testis. Additionally, Ant4 was expressed in primordial germ cells. These data indicate that Ant4 is a pluripotent stem cell- and germ cell-specific isoform of adenine nucleotide translocase in mouse and that DNA methylation plays a primary role in its transcriptional silencing in somatic cells.

Animals↗

PubMed2004

Messenger RNA electroporation is highly efficient in mouse embryonic stem cells: successful FLPe- and Cre-mediated recombination.

Development of efficient short-term gene transfer technologies for embryonic stem (ES) cells is urgently needed for various existing and new ES cell-based research strategies. In this study, we present a highly efficient, nonviral non-DNA technology for genetic loading of mouse ES cells based on electroporation of defined mRNA. Here, we show that mouse ES cells can be efficiently loaded with mRNA encoding a green fluorescent reporter protein, resulting in a level of at least 90% of transgene expression without loss of cell viability and phenotype. To show that transgenes, introduced by mRNA electroporation, exert a specific cellular function in transfected cells, we electroporated stably transfected ES cell lines with mRNA encoding FLPe or Cre recombinase proteins in order to excise an FRT- or LoxP-flanked reporter gene. The results, as determined by the disappearance and/or appearance of a fluorescent reporter gene expression, show that FLPe and Cre recombinase proteins, introduced by mRNA electroporation, efficiently exert their function without influence on further culture of undifferentiated ES cell populations and their ability to differentiate towards a specific lineage.

Animals↗

PubMed2007

Expansion of mouse embryonic stem cells on microcarriers.

Embryonic stem (ES) cells have been shown to differentiate in vitro into a wide variety of cell types having significant potential for tissue regeneration. Therefore, the operational conditions for the ex vivo expansion and differentiation should be optimized for large-scale cultures. The expansion of mouse ES cells has been evaluated in static culture. However, in this system, culture parameters are difficult to monitor and scaling-up becomes time consuming. The use of stirred bioreactors facilitates the expansion of cells under controlled conditions but, for anchorage-dependent cells, a proper support is necessary. Cytodex-3, a microporous microcarrier made up of a dextran matrix with a collagen layer at the surface, was tested for its ability to support the expansion of the mouse S25 ES cell line in spinner flasks. The effect of inocula and microcarrier concentration on cell growth and metabolism were analyzed. Typically, after seeding, the cells exhibited a growth curve consisting of a short death or lag phase followed by an exponential phase leading to the maximum cell density of 2.5-3.9 x 10(6) cells/mL. Improved expansion was achieved using an inoculum of 5 x 10(4) cells/mL and a microcarrier concentration of 0.5 mg/mL. Medium replacement allowed the supply of the nutrients and the removal of waste products inhibiting cell growth, leading to the maintenance of the cultures in steady state for several days. These conditions favored the preservation of the S25 cells pluripotent state, as assessed by quantitative real-time PCR and immunostaining analysis.

Animals↗

PubMed2004

Mouse embryonic stem cells efficiently lipofected with nuclear localization peptide result in a high yield of chimeric mice and retain germline transmission potency.

Embryonic stem (ES) cells are an important tool in developmental biology, genomics, and transgenic methods, as well as in potential clinical applications such as gene therapy or tissue engineering. Electroporation is the standard transfection method for mouse ES (mES) cells because lipofection is quite inefficient. It is also unclear if mES cells treated with cationic lipids maintain pluripotency. We have developed a simple lipofection method for high efficiency transfection and stable transgene expression by employing the nonclassical nuclear localization signal M9 derived from the heterogeneous nuclear ribonucleoprotein A1. In contrast to using 20 microg DNA for 10 x 10(6) cells via electroporation which resulted in 10-20 positive cells/mm2, M9-assisted lipofection of 2 x 10(5) cells with 2 microg DNA resulted in > 150 positive cells/mm2. Electroporation produced only 0.16% EGFP positive cells with fluorescence intensity (FI) > 1000 by FACS assay, while M9-lipofection produced 36-fold more highly EGFP positive cells (5.75%) with FI > 1000. Using 2.5 x 10(6) ES cells and 6 microg linearized DNA followed by selection with G418, electroporation yielded 17 EGFP expressing colonies, while M9-assisted lipofection yielded 72 EGFP expressing colonies. The mES cells that stably expressed EGFP following M9-assisted lipofection yielded > 66% chimeric mice (8 of 12) and contributed efficiently to the germline. In an example of gene targeting, a knock-in mouse was produced from an ES clone screened from 200 G418-resistant colonies generated via M9-assisted lipofection. To our knowledge, this is the first report of generation of transgenic or knock-in mice obtained from lipofected mES cells and this method may facilitate large scale genomic studies of ES developmental biology or large scale generation of mouse models of human disease.

Animals↗

PubMed2002

In vitro differentiation of mouse embryonic stem cells after activation by retinoic acid.

Embryonic stem (ES) cells are pluripotent cells isolated from the inner cell mass of blastocysts. ES cells are able to differentiate into the three primitive layers (endoderm, mesoderm, and ectoderm) of the organism, including the germline. 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. In this study, we investigated the induction of mouse ES cell differentiation, using culture of embryoid bodies (EBs) into the diverse tissues. EBs were formed by culturing ES cells (129/SV strain) in DMEM supplemented with 10% FBS, in the absence of feeder cells and leukemia inhibitory factor (LF). EBs were induced to differentiate by treatment with retinoic acid (RA). In control medium (non-RA medium) beating muscles, blood vessels, hemocytes, and cartilages were frequently observed in EBs. Moreover, when EBs were cultured in medium including RA (5 x 10(-8) M, and 5 x 10(-9) M), differentiation of the optic vesicle, lens, retina, and neural groove was observed. In this study we demonstrated that an efficient system for inducing the differentiation of ES cells using EBs.

Animals↗

PubMed2005

Neural differentiation of mouse embryonic stem cells in chemically defined medium.

Directed differentiation of embryonic stem (ES) cells has enormous potential to derive a wide variety of defined cell populations of therapeutic value. To achieve this, it is necessary to use protocols that promote cell differentiation under defined culture conditions. Furthermore, understanding the mechanisms of cell differentiation in vitro will allow the development of rationale approaches to systematically manipulate cell fates. Here we have analysed the differentiation of mouse ES cells to the neural lineage under serum and feeder cell-free conditions, using a previously described chemically defined medium (CDM). In CDM, ES cell differentiation is highly neurogenic. Cell differentiation was monitored by analysis of a gene expression array (Clontech-Atlas) and by semi-quantitative RT-PCR for a panel of genes involved in cell lineage specification and patterning of the epiblast. In addition to expression of neural markers, data identified a transient expression of several genes associated with the organising activities of the embryonic node and visceral endoderm, including regulators of WNT, BMP, Hedgehog and FGF signaling pathways. Neural differentiation in CDM does not occur by a simple default mechanism, but was dependent on endogenous FGF signaling, and could be blocked by adding BMP4, and LiCl to simulate WNT activation. Neural differentiation was also inhibited by antagonising endogenous hedgehog activity. Taken together the profile of gene expression changes seen in CDM cultures recapitulates those seen in the early embryo, and is suggestive of common developmental mechanisms.

Animals↗

PubMed2005

Efficient gene transfer into mouse embryonic stem cells with adenovirus vectors.

Efficient and transient gene transfer into embryonic stem (ES) cells is expected to be of use for basic studies in developmental biology and for applications in regenerative medicine. Here, we report the development of an adenovirus (Ad) vector that efficiently expresses foreign genes in mouse ES (mES) cells. We prepared four LacZ-expressing Ad vectors, each of which contained one of the following: Rous sarcoma virus (RSV), cytomegalovirus (CMV), beta-actin promoter/CMV enhancer (CA), or EF-1alpha promoter. While the RSV and CMV promoters were inactive in mES cells, the CA and EF-1alpha promoters strongly drove LacZ expression in more than 90% of the mES cells. The EF-1alpha promoter was found to be slightly more efficient than the CA promoter. mES cells were found to express the Ad primary receptor, coxsackievirus and adenovirus receptor, suggesting that while Ad vectors could introduce the exogenous gene into mES cells, the choice of a suitable promoter was critical for efficient gene expression. Fiber-mutant Ad vectors containing RGD or polylysine peptide on the fiber knob mediated efficient LacZ expression, not only in mES cells, but also in feeder cells. Exogenous expression of Oct-3/4 or the dominant-negative mutant of STAT3 (STAT3F) by conventional Ad vectors containing the EF-1alpha promoter promoted the differentiation of mES cells into the cells of three germ layers, and STAT3F-mediated differentiation was rescued by the coexpression of Nanog. These results suggest that Ad vectors can be used for basic research using ES cells and that they may be of great utility for therapeutic applications in gene-modified regenerative medicine based on ES cells.

Actins↗

PubMed1998

Mouse embryonic stem cells with aberrant transforming growth factor beta signalling exhibit impaired differentiation in vitro and in vivo.

Embryonic stem (ES) cells are resistant to transforming growth factor beta (TGF beta). We have shown previously that they lack type-II binding receptors (T beta RII) and in this respect resemble the inner cell mass and ectoderm cells of mouse embryos 4.5-7.5 days post coitum (dpc); they do however express type-I (alk-5) signalling receptors. Here we show that in contrast to several tumour cell lines, stable transfection of wtT beta RII is not sufficient for ES cells to become biologically sensitive to TGF beta. We analysed the expression of several down-stream molecules known to be involved in TGF beta signalling (Smads) and TGF beta-mediated cell cycle regulation (cyclins D) during the differentiation of control and wtT beta RII-expressing ES cells and showed that upregulation of these molecules correlated with (i) an increase in plasminogen activator inhibitor-1 (PAI-1) synthesis and (ii) growth inhibition, following addition of TGF beta 1. These TGF beta responses were reduced in an ES cell line expressing a dominant negative (truncated) T beta RII (delta T beta RII). The differentiation pattern of control and wtT beta RII-expressing ES cells was indistinguishable in monolayer culture and as embryoid bodies, but in delta T beta RII ES cells, the capacity to form mesodermal derivatives in monolayer cultures in response to the addition of retinoic acid (RA) and removal of leukemia inhibitory factor (LIF) was lost, and only endoderm-like cells formed. The T beta RII and delta T beta RII ES cells were, however, both distinguishable from control ES cells when allowed to differentiate in chimaeric embryos following aggregation with morula-stage hosts. Conceptuses containing mutant cells, recovered from pseudopregnant females at the equivalent of 9.5 dpc, exhibited highly defective yolk sac development; most strikingly, no blood vessels were present and in addition the yolk sacs with derivatives of ES cells containing wtT beta RII were blistered and lacked haematopoietic cells. The implications for understanding TGF beta signalling in early mouse development are discussed.

Animals↗

PubMed2003

Generation of hepatocytes from cultured mouse embryonic stem cells.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of fertilized blastocysts in vitro. ES cells can be induced to undergo differentiation into potentially all cell types. The aim of this study is to examine the differentiating potential of mouse ES cells into hepatocytes in the presence of retinoic acid (RA), hepatocyte growth factor (HGF), and beta-nerve growth factor (beta-NGF). RA, HGF, and beta-NGF were added to the cell culture. Hepatocyte induction was confirmed morphologically, as well as biochemically, through immunohistochemical assays of alpha1-antitrypsin (alpha1-AT) and alfafetaprotein (AFP) expression and reverse-transcriptase polymerase chain reaction tests for the presence of albumin, transthyretin, glucose 6 phosphates, hepatic nuclear factor 4, and SAPK/ERK kinase-1 (SEK1) messenger RNA, produced only by functioning hepatocytes. Fifteen days after the addition of HGF and beta-NGF to the cell culture, many epithelioid cells were noticed. alpha1-AT, AFP, albumin, transthyretin, glucose 6 phosphates, hepatic nuclear factor 4, and SEK1 messenger RNA expression also was detected, indicating successful ES cell differentiation into functioning hepatocytes. However, in the presence of RA alone, only transthyretin messenger RNA was positive, whereas no other expression pertaining to functioning hepatocytes could be detected. In the presence of HGF and beta-NGF, mouse ES cells can differentiate into functioning hepatocytes, whereas RA function is limited.

Animals↗

PubMed2001

[The specific labelling of mouse embryonic stem cells].

Despite the wide application of ES cell technology, little is known about the pluripotent nature of ES cells. This is partly due to the heterogeneity of ES cell population in culture. This report described the specific labelling of undifferentiated cells in ES cell lines. oct-4 gene is specifically expressed in all the totipotent cells in mouse embryos and undifferentiated ES cells. A constructed pG18NG was obtained by inserting the reporter gene beta geo into the oct-4 transcription elements. ES cell lines MESPU22 and MESPU13 were transfected with this construct and stable integrated cell clones were selected out. With the experiments of in vitro cultivation, differentiation and chimeras production, it was confirmed that we have successfully labelled the undifferentiated cells in ES cell lines, and this label was both valid in vitro and in vivo.

Animals↗