Pluripotency Of Mouse Embryonic Stem Cells Research Articles

Embryonic stem cell models of development.

Pluripotent mouse embryonic stem (ES) cell lines have provided a means to analyze gene function in development via gene targeting. At the same time, they provide an opportunity to directly probe gene function by assessing the in vitro differentiation capacity of the ES cells themselves. In addition to providing direct data on lineage decisions not accessible in the complex three-dimensional milieu of the early mouse embryo, controlled differentiation of ES into specific lineages may provide a source of cells for transplantation and gene therapy.

Cellular differentiation and I-FABP protein expression modulate fatty acid uptake and diffusion.

The effect of cellular differentiation on fatty acid uptake and intracellular diffusion was examined in transfected pluripotent mouse embryonic stem (ES) cells stably expressing intestinal fatty acid binding protein (I-FABP). Control ES cells, whether differentiated or undifferentiated, did not express I-FABP. The initial rate and maximal uptake of the fluorescent fatty acid, 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-octadec anoic acid (NBD-stearic acid), was measured in single cells by kinetic digital fluorescence imaging. I-FABP expression in undifferentiated ES cells increased the initial rate and maximal uptake of NBD-stearic acid 1.7- and 1.6-fold, respectively, as well as increased its effective intracellular diffusion constant (Deff) 1.8-fold as measured by the fluorescence recovery after photobleaching technique. In contrast, ES cell differentiation decreased I-FABP expression up to 3-fold and decreased the NBD-stearic acid initial rate of uptake, maximal uptake, and Deff by 10-, 4.7-, and 2-fold, respectively. There were no significant differences in these parameters between the differentiated control and differentiated I-FABP-expressing ES cell lines. In summary, differentiation and expression of I-FABP oppositely modulated NBD-stearic acid uptake parameters and intracellular diffusion in ES cells.

7] Use of transgenic mice to study activation of retinoic acid-responsive promoters

Transgenic systems are invaluable in studies of function of retinoic acids (RAs). The spatial-temporal distribution of RAs within the embryo or a given tissue is difficult to assess because it involves laborious and tissue-disrupting analytical methods such as high-performance liquid chromatography (HPLC). As an alternative, relative RA concentrations can be measured in situ using RA-inducible promoter sequences fused to a reporter gene. Transgenic approaches may be also used to identify novel RA-dependent genes. Gene traps are used to identify novel genes that are important for development. In this strategy, pluripotent mouse embryonic stem (ES) cells are electroporated with a gene trap vector. The vector consists of a promoterless reporter gene (typically β -galactosidase), preceded by a splice-acceptor site. A selection marker provides means to select clones derived from ES cells that have integrated the construct into their genomes. Alternatively, the β -galactosidase and neomycin phosphotransferase activities can be contained in a single fusion protein. The activity of the reporter gene can be visualized by histological staining. Stained clones contain β -galactosidase-tagged genes, which are expressed in the ES cells. A modification of this technique allows the investigator to identify a group of genes that is inducible by a particular soluble factor.

Oxygen supply and oxygen-dependent gene expression in differentiating embryonic stem cells.

Blastocyst-derived pluripotent mouse embryonic stem cells can differentiate in vitro to form so-called embryoid bodies (EBs), which recapitulate several aspects of murine embryogenesis. We used this in vitro model to study oxygen supply and consumption as well as the response to reduced oxygenation during the earliest stages of development. EBs were found to grow equally well when cultured at 20% (normoxia) or 1% (hypoxia) oxygen during the first 5 days of differentiation. Microelectrode measurements of pericellular oxygen tension within 13- to 14-day-old EBs (diameter 510-890 micron) done at 20% oxygen revealed efficient oxygenation of the EBs' core region. Confocal laser scanning microscopy analysis of EBs incubated with fluorescent dyes that specifically stain living cells confirmed that the cells within an EB were viable. To determine the EBs' capability to sense low oxygen tension and to specifically respond to low ambient oxygen by modulating gene expression we quantified aldolase A and vascular endothelial growth factor (VEGF) mRNAs, since expression of these genes is upregulated by hypoxia in a variety of cells. Compared with the normoxic controls, we found increased aldolase A and VEGF mRNA levels after exposing 8- to 9-day-old EBs to 1% oxygen. We propose that EBs represent a powerful tool to study oxygen-regulated gene expression during the early steps of embryogenesis, where the preimplantation conceptus resides in a fluid environment with low oxygen tension until implantation and vascularization allow efficient oxygenation.

Expression of Retinoid X Receptors in P19 Embryonal Carcinoma Cells and Embryonic Stem Cells

The pluripotent mouse embryonal carcinoma cell line P19 provides an excellent model system to study the mechanisms by which retinoic acid (RA) exerts its biological effects. When aggregated and exposed to low concentrations of RA, P19 cells differentiate into neuron- and glial-like cells. The diverse biological effects of RA are mediated by two families of receptors localized in the cell nucleus, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Each family consists of three members designated α, β and γ. While the patterns of expression of the RARs have been studied in P19 cells, similar data for the RXRs have not been available. We demonstrate here that these receptors are expressed in P19 and are regulated during RA-induced differentiation. We also show that pluripotent mouse embryonic stem cells express the RXRs as well.

Gene targeting in normal somatic cells: inactivation of the interferon–γ receptor in myoblasts

Gene targeting in somatic cells represents a potentially powerful method for gene therapy, yet with the exception of pluripotent mouse embryonic stem (ES) cells, homologous recombination has not been reported for a well characterized, non-transformed mammalian cell. Applying a highly efficient strategy for targeting an integral membrane protein--the interferon gamma receptor--in ES cells, we have used homologous recombination to target a non-transformed somatic cell, the mouse myoblast, and to compare targeting efficiencies in these two cell types. Gene-targeted myoblasts display the properties of normal cells including normal morphology, ability to differentiate in vitro, stable diploid karyotype, inability to form colonies in soft agar and lack of tumorigenicity in nude mice.

Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca 2+ channel blockers

Abstract A defined cultivation system was developed for the differentiation of pluripotent embryonic stem cells of the mouse into spontaneously beating cardiomyocytes, allowing investigations of chronotropic responses, as well as electrophysiological studies of different cardioactive drugs in vitro. The β-adrenoceptor agonists (—)isoprenaline and clenbuterol, the mediators of cAMP metabolism, forsko-lin and isobutylmethylxanthine (IBMX), the α 1-adreno-ceptor agonist (—)phenylephrine, and the heart glyco-side digitoxine induced a positive, the muscarinic cholin-oceptor agonist carbachol and L-type Ca 2+ channel blockers nisoldipine, gallopamil and diltiazem induced a negative chronotropic response. In early differentiated cardiomyocytes β 1-, α 1-, but not β 2-adrenoceptors, cholinoceptors, as well as L-type Ca 2+ channels participated in the chronotropic response. In terminally differentiated cardiomyocytes β 2-adrenoceptors and digitoxine responses were also functionally expressed. The contractions of spontaneously beating cardiomyocytes were concommitant with rhythmic action potentials very similar to those described for embryonic cardiomyocytes and sinusnode cells. We conclude that cardiomyocytes differentiating from pluripotent embryonic stem cells are able to develop adrenoceptors and cholinoceptors and signal transduction pathways as well as L-type Ca 2+ channels as a consequence of cell-cell interactions during embryoid body formation in vitro, independent of the development in living organisms. The cellular system described may be useful as in vitro assay for toxicological investigations of chronotropic drugs and a model system for studying commitment and cellular differentiation in vitro.

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.