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PubMed2003

[Directional development and differentiation of mouse embryonic stem cells into hepatocytes in vitro].

OBJECTIVE: To investigate the mechanism and regulation of differentiation from embryonic stem (ES) cells to hepatocytes in vitro and to find a new source of cell types for hepatocytes replacement therapies in the treatment of hepatic failure. METHODS: ES cells of BALB/c mice were cultured and maintained undifferentiated in gelatin-coated dishes in Dubecco's modified Eagle's medium (DMEM) containing 1 000 U/L leukaemia inhibitory factor (LIF). Then, the ES cells were cultured in DMEM without LIF so as to develop into embryonic bodies (EBs). For directional differentiating, EBs were put into 24-well tissue culture dish and differentiate into cells with different forms. On day 7 to day 11 acid fibroblast growth factor (aFGF) with terminal concentration of 100 ng/ml was added, and on day 11 to day 15 hepatocyte growth factor (HGF) terminal concentration of 20 ng/ml was added. EBs in culture without growth factor was used as controls. The development and differentiation status was observed by inversion microscope dynamically. The concentrations of alpha-fetoprotein (AFP) and albumin (ALB) in the supernatants were determined by radioimmunoassay on the 1st to 15th day since the ES cells were inoculated. Immunocytochemistry (ICC) was used to detect the expression of hepatic proteins such as AFP, ALB, cytokeratin 8 (CK8) and cytokeratin 18 (CK18) in the cells on the days 7, 8, 9, 10, 11, 12, 13, 14, and 15. The number of ALB positive cells was counted. The ALB positive rate was calculated as the differentiation ratio of hepatic cells. RESULTS: ES cells remained undifferentiated and grew to ES clones in the DMEM containing LIF and developed to EBs in the medium without LIF. AFP was detected firstly in the medium on day 8 with a concentration of 3.4 ng/ml and the concentration increased to be 22.8 ng/ml at day 15; ALB was detected firstly in the medium on day 11 with a concentration of 0.2 microg/ml and the concentration increased to be 2.2 microg/ml on day15. ICC showed that AFP, ALB, CK8, and CK18 began to be expressed on day 8, day 10, day 10, and day 11 respectively. The ICC-positive cells have the ultrastructures consistent with those of the normal mouse hepatocytes. The ratio of hepatic differentiation was 30%. CONCLUSION: ES cells can differentiate directionally into hepatocytes when cultured in gelatin-coated dish and medium with aFGF and HGF. This ES cell differentiating system provides enough functional hepatocytes and may be a good new source of differentiated cell types for hepatocytes replacement therapies in treatment of hepatic failure.

Albumins↗
PubMed2006

Mouse embryonic stem cells are not susceptible to cytomegalovirus but acquire susceptibility during differentiation.

BACKGROUND: Cytomegalovirus (CMV) is the most significant infectious cause of congenital anomalies of the central nervous system caused by intrauterine infection in humans. The timing of infection and the susceptibility of cells in early gestational stages are not well understood. In this study we investigated the susceptibility of embryonic stem (ES) cells to CMV infection during differentiation. METHODS: ES cell lines were established from transgenic mice integrated with the murine CMV (MCMV) immediate-early (IE) promoter connected with a reporter lacZ gene. The susceptibility of the ES cells was analyzed in terms of viral gene expression and viral replication after induction of differentiation. RESULTS: ES cells were nonpermissive to MCMV infection in the undifferentiated state. Upon differentiation, permissive cells appeared approximately 2 weeks after the leukemia inhibitory factor was removed. Upon neural differentiation by retinoic acid (RA), glial cells showed specific susceptibility in terms of expression of the viral antigen. The MCMV IE promoter was not activated in ES cells from the transgenic mice. Activation of the IE promoter was detected approximately 2 weeks after induction of differentiation and observed predominantly in glial cells. Upon MCMV infection of the ES cells, viral infection was correlated with the activation of the IE promoter. CONCLUSIONS: ES cells are nonpermissive to MCMV infection and acquire permissiveness about 2 weeks after induction of differentiation, especially in glial cells. Acquisition of permissiveness in differentiated ES cells may be associated with activation of the IE promoter.

Animals↗
PubMed2004

Detection of tissue-specific effects by methotrexate on differentiating mouse embryonic stem cells.

BACKGROUND: Pluripotent embryonic stem (ES) cells offer a unique possibility to monitor the differentiation of several cell types in vitro. This study attempts to identify marker genes during in vitro cell differentiation of murine ES cells and allow a prediction of chemical effects on cell differentiation of specific target tissues. The study focused on the expression pattern of key genes involved in cardiomyocyte and osteoblast differentiation: Oct-4, Brachyury, Nkx2.5, alpha myosin heavy chain, Cbfa1, and Osteocalcin. METHODS: Methotrexate was selected due to its well-characterized teratogenic effects. Several in vivo studies have demonstrated the specific interactions of methotrexate with bone formation whereas the cardiovascular system is not specifically affected after exposure to low concentration. The capability of murine ES cells to differentiate in vitro into cardiomyocytes as well as into osteoblasts have been used to demonstrate the target cell specificity in vitro, at non-cytotoxic concentration. RESULTS: Exposure of differentiating ES cells did not result in any gene profile modification of the selected cardiomyocyte specific genes, whereas the expression of osteoblast specific key genes, Cbfa1 and Osteocalcin, decreased. At the latter stages of skeletal differentiation we observed a 30% decrease in gene expression for Cbfa1 and a 60% decrease for Osteocalcin, with reference to the control. Early marker genes for undifferentiated cells and mesodermal cells were not modified after methotrexate treatment. CONCLUSIONS: These results show the possibility to integrate specific in vitro tests for teratogenicity in a test strategy for developmental toxicity.

Animals↗
PubMed2003

Oct4 RNA interference induces trophectoderm differentiation in mouse embryonic stem cells.

We examined whether suppression of Oct4 via RNA interference (RNAi) would affect embryonic stem (ES) cell lineage choice. Cells were transfected with plasmids containing an independently expressed reporter gene and an RNA polymerase type III promoter to constitutively express small stem-loop RNA transcripts corresponding to Oct4 mRNA. Cells transfected with Oct4 RNAi constructs demonstrated reduced levels of Oct4 mRNA and exhibited characteristics of trophectodermal differentiation. These findings support the critical role of Oct4 in regulating stem cell identity and suggest that future experiments using RNAi in ES cells can elucidate the roles of other genes affecting lineage specification during differentiation.

Animals↗
PubMed2004

Germline transmission and efficient DNA recombination in mouse embryonic stem cells mediated by adenoviral-Cre transduction.

Following gene targeting, a loxP-neo-loxP cassette was introduced into ES cells. The presence of a selectable marker such as neo in the targeted allele may result in gene interference in flox mice or unexpected phenotypes due to genetic ambiguity in direct knockout mice. Typically, the neo cassette is selectively removed by transient expression of the Cre recombinase in targeted ES cell. However, this method involves a tedious process of selecting, expanding, and screening ES cell clones which may compromise germline competency. Here, we describe a novel method of combining adenovirus-Cre mediated gene recombination with ES gene targeting to facilitate efficient loxP-neo-loxP removal in ES cells. We demonstrate that adenovirus-Cre infected ES cells can retain their germline competency. The procedures described here facilitate a rapid genetic manipulation of ES cells to obtain neo-free knockout animals, multiple gene targeting, homozygous mutant ES cells ideal for in vitro characterization, or Rag-deficient blastocyst complementation.

Adenoviridae↗
PubMed2005

Direct hepatic fate specification from mouse embryonic stem cells.

The molecules responsible for hepatic differentiation from embryonic stem (ES) cells have yet to be elucidated. Here we have identified growth factors that allow direct hepatic fate-specification from ES cells by using simple adherent monolayer culture conditions. ES cell-derived hepatocytes showed liver-specific characteristics, including several metabolic activities, suggesting that ES cells can differentiate into functional hepatocytes without the requirement for embryoid body (EB) formation, in vivo transplantation, or a coculture system. Most importantly, transplantation of ES cell-derived hepatocytes in mice with cirrhosis showed significant therapeutic effects. In conclusion, this novel system for hepatic fate specification will help elucidate the precise molecular mechanisms of hepatic differentiation in vitro and could represent an attractive approach for developing stem cell therapies for treatment of hepatic disease in humans. Supplementary material for this article can be found on the HEPATOLOGY website ( http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

Animals↗
PubMed1987

Mouse embryonic stem cells exhibit indefinite proliferative potential.

The proliferative potential of embryonic stem cells was examined. In contrast to the current concept of the finite life-span being the hallmark of normal cells, we have been able to maintain these embryonic stem cells in vitro up to about 250 cumulative doublings with no indication of "crisis" or transformation. These cells could be considered normal on the basis of: (1) their apparently normal diploid karyotype, (2) their ability to extensively colonize embryos without causing tumors and developmental anomalies, and (3) their ability to form normal gametes when differentiated into the germ-line. These results suggest that embryonic stem cells prior to differentiation into germ and somatic cells are indeed immortal.

Animals↗
PubMed2004

Retinoic acid inhibits leukemia inhibitory factor signaling pathways in mouse embryonic stem cells.

Retinoic acid (RA) induces the differentiation of murine embryonic stem (ES) cells to cell types resembling those found in the early embryo. When cultured in the presence of leukemia inhibitory factor (LIF), ES cells are maintained in an undifferentiated (self-renewing) state. Addition of RA to the culture media overrides the self-renewing effects of LIF to induce ES cell differentiation. Therefore, we hypothesized that RA-induced differentiation of ES cells may be accomplished by antagonism of LIF-induced signaling pathways. We demonstrate that RA-induced differentiation of CCE ES cells is associated with (1) downregulation of the LIF receptor (LIFR); (2) decreased tyrosine phosphorylation of signal transducer and activator of transcription 3 protein (Stat3); and (3) increased activation of extracellular regulated kinase (Erk1/2). We conclude that RA induces CCE ES cell differentiation in the presence of LIF, in part, by disrupting signaling between the LIFR/gp130 receptor and nuclear targets that are required to prevent ES cell differentiation. Our data indicate that RA-induced inhibition of LIF signaling does not involve Erk1/2-dependent actions.

Animals↗
PubMedDate not supplied

Neuronal differentiation of mouse embryonic stem cells: lineage selection and forced differentiation paradigms.

Primitive embryonic stem cells are an ideal starting cell population for studies of gene expression and lineage segregation during development. Despite their potential, it has been difficult to determine culture conditions that cause single-lineage differentiation of these pluripotent cells. Both genetic and epigenetic approaches have been taken to promote neuronal differentiation of embryonic stem cells, including aggregation, exposure to the nonspecific teratogen/morphogen retinoic acid, low-density culture, exposure to growth/differentiation factors, and forced differentiation following expression of lineage-restricted "developmental control" genes. In the current investigation, a hybrid approach involving genetic techniques of "lineage selection" or "forced differentiation" has been employed to develop primitive neural progenitor cell lines. These lines form an important starting point to examine the cascades of gene expression (and inhibition) during neuronal and glial lineage segregation, to study growth factor effects on neural differentiation, and ultimately to provide a source of cells for transplantation to a damaged nervous system.

Animals↗
PubMed1996

Differential regulation of gap junction protein (connexin) genes during cardiomyocytic differentiation of mouse embryonic stem cells in vitro.

Using an in vitro system for differentiation of embryonic stem (ES) cells into cardiac myocytes, we analyzed the expression of connexin (Cx) genes by RT-PCR to learn what changes in the expression of multiple connexin genes occur during the early stage of heart development. We also examined gap junctional intercellular communication by using Lucifer Yellow dye microinjection transfer, studied intracellular Ca2+ transients by confocal laser image analysis using fluo 3, and determined localization of Cx43 by immunofluorescence during in vitro differentiation of ES cells into cardiac myocytes. The transcripts for Cx43 and Cx45 were detected in undifferentiated ES cells and in embryoid bodies before and after the appearance of beating cardiomyocytes. In contrast, Cx40 transcripts were not observed in undifferentiated ES cells and were barely detectable in 3- and 5-day-old embryoid bodies. Cx40 transcripts significantly increased with the appearance of beating cells similar to those of cardiac-specific genes. Dye coupling was present among undifferentiated ES cells, prebeating cells of embryoid body outgrowth and ES cell-derived beating cardiomyocytes. When dye was injected into a beating cell, dye spread was restricted to neighboring beating cells. Immunofluorescence demonstrated that Cx43 protein was localized not only in beating cells but also in surrounding nonbeating cells, whereas myosin heavy chain alpha/beta was exclusively positive in the beating cells. These data suggest that the expression of multiple connexins is differentially regulated during the cardiomyocytic differentiation of ES cells in vitro and that Cx40 expression may be linked to early stages in cardiomyocytic differentiation.

Animals↗
PubMed1996

Expression of the 25-kDa heat-shock protein (HSP27) correlates with resistance to the toxicity of cadmium chloride, mercuric chloride, cis-platinum(II)-diammine dichloride, or sodium arsenite in mouse embryonic stem cells transfected with sense or antisense HSP27 cDNA.

Embryonic stem (ES) cells were transfected with the protein-coding region of rat HSP27 cDNA placed in sense or antisense orientation in vector pcDNA3 under the control of the constitutive cytomegalovirus (CMV) promoter. Compared with nontransfected ES cells, five sense HSP27 cDNA-transfected ES cell clones displayed up to fourfold increases in expression of HSP27 mRNA and up to sixfold increases in expression of HSP27 protein, whereas four antisense HSP27 cDNA-transfected ES cell lines exhibited synthesis of antisense HSP27 RNA and a 50-85% decrease in HSP27 protein expression. Compared to the parental ES cell lines or ES cells transfected with the vector lacking any HSP27 sequence, all ES cell lines overexpressing HSP27 were resistant to killing by cadmium chloride (CdCl2), mercuric chloride (HgCl2), cis-platinum(II)-diammine dichloride (cDDP), sodium arsenite (NaAsO2), and heat while ES cell lines expressing reduced HSP27 were more sensitive to metal toxicity and heat. The relative toxicities of the tested metals to ES cells were cDDP > NaAsO2 > HgCl2 > CdCl2. Protection of ES cells against metal or heat toxicity was positively correlated with the level of HSP27. These data confirm in ES cells previous reports of the ability of HSP27 to protect other cell types against heat and demonstrate that HSP27 protects mammalian cells against the toxic effects of diverse metals.

Animals↗
PubMed1990

Differentiation-promoting effects of mammary-derived growth inhibitor (MDGI) on pluripotent mouse embryonic stem cells.

Pluripotent embryonic stem cells (line BLC6), when cultivated in vitro as embryoid bodies and injected subcutaneously into syngeneic mice, form teratocarcinomas consisting of embryonal carcinoma cells and differentiated tissues of all three primary germ layers. In order to study the possible effects of the mammary-derived growth inhibitor (MDGI) on the differentiation pattern of the tumors developing in the mice, BLC6 cell-derived embryoid bodies were treated in vitro for 4 days with either MDGI or a synthetic peptide composed of the C-terminal 11 amino acids of MDGI. In those tumors, significantly more differentiated neural tissue and lesser proportions of undifferentiated embryonic carcinoma cells (ECC) were found in the MDGI- and peptide-treated groups, compared with controls. The results are discussed with respect to a possible differentiation-promoting capacity of MDGI.

Animals↗
PubMed2007

Passage number affects the pluripotency of mouse embryonic stem cells as judged by tetraploid embryo aggregation.

The aim of this study was to determine whether the number of passages affected the developmental pluripotency of embryonic stem (ES) cells as measured by the attainment of adult fertile mice derived from embryonic stem (ES) cell/tetraploid embryo complementation. Thirty-six newborns were produced by the aggregation of tetraploid embryos and hybrid ES cells after various numbers of passages. These newborns were entirely derived from ES cells as judged by microsatellite DNA, coat-color phenotype, and germline transmission. Although 15 survived to adulthood, 17 died of respiratory failure, and four were eaten by their foster mother. From the 15 mice that reached adulthood and that could reproduce, none arose from ES cells at passage level 15 or more. All 15 arose from cells at passages 3-11. Our results demonstrate that the number of passages affects the developmental pluripotency of ES cells.

Animals↗
PubMedDate not supplied

Self-renewal and differentiation of mouse embryonic stem cells as measured by Oct 4 gene expression: effects of lif, serum-free medium, retinoic acid, and dbcAMP.

In this study we examined the interplay between serum, leukemia inhibitory factor (LIF), retinoic acid, and dibutyrl cyclic adenosine monophosphate (dbcAMP) in affecting IOUD2 embryonic stem cell self-renewal and differentiation as assessed by Oct 4 expression, and cell proliferation as measured by total cell protein. Removal of LIF, reduced levels of fetal calf serum (FCS), and addition of retinoic acid all induced embryonic stem cell differentiation as measured by reduced Oct 4 expression. Lower levels of retinoic acid (0.1-10 nM) promoted the formation of epithelial-like cells, whereas higher levels (100-10,000 nM) favored differentiation into fibroblastic-like cells. The effects of dbcAMP varied with the presence or absence of FCS and LIF and the concentration of dbcAMP. In FCS-containing media, a low level of dbcAMP (100 microM) increased self-renewal in the absence of LIF, but it had no effect in its presence. In contrast, at higher concentrations (1,000 microM dbcAMP), regardless of LIF, differentiation was promoted. A similar effect of dbcAMP was seen in the presence of retinoic acid. In media without FCS but with serum replacement supplements, there was no effect of dbcAMP. This study shows that the Oct 4 expression system of IOUD2 cells provides a novel, simple method for quantifying cellular differentiation.

Analysis of Variance↗
PubMed1990

Androgenetic mouse embryonic stem cells are pluripotent and cause skeletal defects in chimeras: implications for genetic imprinting.

The inviability of diploid androgenetic and parthenogenetic embryos suggests imprinting of paternal and maternal genes during germ cell development, and differential expression of loci depending on parental inheritance appears to be involved. To facilitate identification of imprinted genes, we have derived diploid androgenetic embryonic stem (ES) cell lines. In contrast to normal ES cells, they form tumors composed almost entirely of striated muscle when injected subcutaneously into adult mice. They also form chimeras following blastocyst injection, although many chimeras die at early postnatal stages. Surviving chimeras develop skeletal abnormalities, particularly in the rib cartilage. These results demonstrate that androgenetic ES cells are pluripotent and point to stage- and cell-specific expression of developmentally important imprinted genes.

Animals↗