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PubMed2004

Proteomic signatures and aberrations of mouse embryonic stem cells containing a single human chromosome 21 in neuronal differentiation: an in vitro model of Down syndrome.

Neurodegeneration in fetal development of Down syndrome (DS) patients is proposed to result in apparent neuropathological abnormalities and to contribute to the phenotypic characteristics of mental retardation and premature development of Alzheimer disease. In order to identify the aberrant and specific genes involved in the early differentiation of DS neurons, we have utilized an in vitro neuronal differentiation system of mouse ES cells containing a single human chromosome 21 (TT2F/hChr21) with TT2F parental ES cells as a control. The paired protein extracts from TT2F and TT2F/hChr21 cells at several stages of neuronal differentiation were subjected to two-dimensional polyacrylamide gel electrophoresis protein separation followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry to identify the proteins differentially expressed between TT2F and TT2F/hChr21 cells. We provide here a novel set of specific gene products altered in early differentiating DS neuronal cells, which differs from that identified in adult or fetal brain with DS. The aberrant protein expression in early differentiating neurons, due to the hChr21 gene dosage effects or chromosomal imbalance, may affect neuronal outgrowth, proliferation and differentiation, producing developmental abnormalities in neural patterning, which eventually leads to formation of a suboptimal functioning neuronal network in DS.

Animals↗

PubMed2005

Rudhira is a cytoplasmic WD40 protein expressed in mouse embryonic stem cells and during embryonic erythropoiesis.

We describe a novel murine gene rudhira that is expressed at high levels in embryonic stem cells and is restricted to blood islands and the erythroid lineage during embryonic development. Rudhira is expressed in angiogenic precursors but is excluded from the differentiated endothelium. Rudhira-expressing cells are seen in close proximity to endothelial cells in angiogenic blood vessels. Rudhira encodes a predicted cytoplasmic WD40 protein that is 98% identical to human BCAS3. The gene encoding BCAS3 maps to a breakpoint of hematological neoplasms on human chromosome 17q23, but its expression and function remain to be determined. We demonstrate that mouse Rudhira is a novel marker for analysis of the erythroid lineage.

Alternative Splicing↗

PubMed2003

Development of electrical activity in cardiac myocyte aggregates derived from mouse embryonic stem cells.

Embryonic stem cells differentiate into cardiac myocytes, repeating in vitro the structural and molecular changes associated with cardiac development. Currently, it is not clear whether the electrophysiological properties of the multicellular cardiac structure follow cardiac maturation as well. In long-term recordings of extracellular field potentials with microelectrode arrays consisting of 60 substrate-integrated electrodes, we examined the electrophysiological properties during the ongoing differentiation process. The beating frequency of the growing preparations increased from 1 to 5 Hz concomitant to a decrease of the action potential duration and action potential rise time. A developmental increase of the conduction velocity could be attributed to an increased expression of connexin43 gap junction channels. Whereas isoprenalin elicited a positive chronotropic response from the first day of spontaneous beating onward, a concentration-dependent negative chronotropic effect of carbachol only developed after approximately 4 days. The in vitro development of the three-dimensional cardiac preparation thus closely follows the development described for the mouse embryonic heart, making it an ideal model to monitor the differentiation of electrical activity in embryonic cardiomyocytes.

Adrenergic beta-Agonists↗

PubMed2003

Growth restricted in vitro culture conditions alter the imprinted gene expression patterns of mouse embryonic stem cells.

Embryonic stem (ES) cell-derived clones and chimeras are often associated with growth abnormalities during fetal development, leading to the production of over/under-weight offspring that show elevated neonatal mortality and morbidity. Due to the role played by imprinted genes in controlling fetal growth, much of the blame is pointed at improper epigenetic reprogramming of cells used in the procedures. We have analyzed the expression pattern of two growth regulatory imprinted genes, namely insulin like growth factor II (Igf2) and H19, in mouse ES cells cultured under growth restricted conditions and after in vitro aging. Culture of cells with serum-depleted media (starvation) and at high cell density (confluence) increased the expression of both imprinted genes and led to aberrant methylation profiles of differentially methylated regions in key regulatory sites of Igf2 and H19. These findings confirm that growth constrained cultures of ES cells are associated with alterations to methylation of the regulatory domains and the expression patterns of imprinted genes, suggesting a possible role of epigenetic factors in the loss of developmental potential.

Animals↗

PubMed2004

Behavioral changes in unilaterally 6-hydroxy-dopamine lesioned rats after transplantation of differentiated mouse embryonic stem cells without morphological integration.

OBJECTIVE: Transplantation of fetal mesencephalic cells into the striatum has been performed in about 350 patients with Parkinson's disease and has been intensively studied in rat models of Parkinson's disease. Limited access to this material has shifted the focus toward embryonic stem (ES) cells. The grafting of undifferentiated ES cells to 6-hydroxy-dopamine (6-OHDA)-lesioned rats leads to behavioral improvements but may induce teratoma-like structures. This risk might be avoided by using more differentiated ES cells. In this study, we aimed to investigate differentiated mouse ES cells regarding their in vivo development and fate after transplantation in the striatum in the 6-OHDA rat model and the behavioral changes induced after transplantation. METHODS: Mouse ES cells were differentiated on PA6 feeder cells for 14 days before grafting. Twenty to twenty-five percent of the neurons obtained were positive for tyrosine-hydroxylase (TH). PKH26-labeled cells were transplanted in the striata of unilaterally 6-OHDA-lesioned rats. RESULTS: Direct PKH26 fluorescence visualization and TH staining proved the existence of cell deposits in the striata of all grafted animals, indicating cell survival for at least 5 weeks posttransplantation. There was no evidence of tumor formation. Immunocytochemical staining showed glial immunoreactivity surrounding the grafted cell deposits, probably inhibiting axonal outgrowth into the surrounding host tissue. There was a significant reduction in amphetamine-induced rotational behavior seen in grafted animals, which was not observed in sham-operated animals. CONCLUSIONS: The findings of this study suggest that the amphetamine-induced rotational behavioral test without histological confirmation is not proof of morphological integration with axonal outgrowth within the first 4 weeks posttransplantation.

Animals↗

PubMed2006

Differentiation of mouse embryonic stem cells into endothelial cells: genetic selection and potential use in vivo.

Embryonic stem (ES) cells are pluripotent cells derived from blastocyst-stage embryos. They are characterized by their infinite self-renewal capacity and their ability to differentiate into many cell types in vitro as well as in vivo. The present protocol describes culture conditions that allow efficient differentiation of mouse ES cells toward the endothelial lineage, both in two-dimensional cultures and in three-dimensional, multicellular embryoid bodies. We also provide a protocol for establishing recombinant ES cell clones, giving the example of cells expressing green fluorescent protein and an antibiotic resistance gene under the control of an endothelial-specific promoter. These transgenes allow the visualization and the genetic selection of endothelial cells from a heterogeneous population of differentiating ES cells. Potential applications for these differentiation models are given, including the study of the endothelial cell lineage and its derivatives, the testing of molecules involved in angiogenesis, and the potential use of selected endothelial cells in vivo.

Animals↗

PubMed2006

Comparative proteomic analysis of mouse embryonic stem cells and neonatal-derived cardiomyocytes.

Pluripotent embryonic stem cells (ESCs) spontaneously differentiate via embryo-like aggregates into cardiomyocytes. A thorough understanding of the molecular conditions in ESCs is necessary before other potential applications of these cells such as cell therapy can be materialized. We applied two dimensional electrophoresis to analyze and compare the proteome profiling of spontaneous mouse ESC-derived cardiomyocytes (ESC-DCs), undifferentiated mouse ESCs, and neonatal-derived cardiomyocytes (N-DCs). Ninety-five percent of the proteins detected on the ESC-DCs and N-DCs could be precisely paired with one other, whereas only twenty percent of the ESC proteins could be reliably matched with those on the ESC-DCs and N-DCSs, suggesting a striking similarity between them. Having identified sixty proteins in the said three cell types, we sought to provide possible explanations for their differential expression patterns and discuss their relevance to cell biology. This study provides a new insight into the gene expression pattern of differentiated cardiomyocytes and is further evidence for a close relation between ESC-DCs and N-DCSs.

Animals↗

PubMed2005

Chromatin-related proteins in pluripotent mouse embryonic stem cells are downregulated after removal of leukemia inhibitory factor.

Embryonic stem (ES) cells have generated enormous interest due to their capacity to self-renew and the potential for growing many different cell types in vitro. Leukemia inhibitory factor (LIF), bone morphogenetic proteins, octamer-binding protein 3 or 4, and Nanog are important factors in the maintenance of pluripotency in mouse ES cells. However, the mechanisms by which these factors regulate the pluripotency remain poorly understood. To identify other proteins involved in this process, we did a proteomic analysis of mouse ES cells that were cultured in the presence or absence of LIF. More than 100 proteins were found to be involved specifically in either the differentiation process or the maintenance of undifferentiated state. Among these, chromatin-related proteins were identified as the major proteins in nuclear extracts of undifferentiated cells. Analysis with real-time RT-PCR revealed that enrichment of these proteins in pluripotent ES cells was regulated at the transcriptional levels. These results suggest that specific chromatin-related proteins may be involved in maintaining the unique properties of pluripotent ES cells.

Animals↗

PubMed2004

Enhanced in vitro midbrain dopamine neuron differentiation, dopaminergic function, neurite outgrowth, and 1-methyl-4-phenylpyridium resistance in mouse embryonic stem cells overexpressing Bcl-XL.

Embryonic stem (ES) cells provide a potentially unlimited source of specialized cells for regenerative medicine. The ease of inducing stable genetic modifications in ES cells allows for in vitro manipulations to enhance differentiation into specific cell types and to optimize in vivo function of differentiated progeny in animal models of disease. We have generated mouse ES cells that constitutively express Bcl-XL, an antiapoptotic protein of Bcl-2 family. In vitro differentiation of Bcl-XL overexpressing ES (Bcl-ES) cells resulted in higher expression of genes related to midbrain dopamine (DA) neuron development and increased the number of ES-derived neurons expressing midbrain DA markers compared with differentiation of wild-type ES cells. Moreover, DA neurons derived from Bcl-ES cells were less susceptible to 1-methyl-4-phenylpyridium, a neurotoxin for DA neurons. On transplantation into parkinsonian rats, the Bcl-ES-derived DA neurons exhibited more extensive fiber outgrowth and led to a more pronounced reversal of behavioral symptoms than wild-type ES-derived DA neurons. These data suggest a role for Bcl-XL during in vitro midbrain DA neuron differentiation and provide an improved system for cell transplantation in a preclinical animal model of Parkinson's disease.

1-Methyl-4-phenylpyridinium↗

PubMed2005

Culture method for the induction of neurospheres from mouse embryonic stem cells by coculture with PA6 stromal cells.

Embryonic stem (ES) cells proliferate and maintain their pluripotency for over 1 year in vitro and may therefore provide a sufficient source for cell therapies. However, most of the previously reported methods for obtaining a source for cell therapies have not been simple. We describe here a novel method for induction of neurospheres from mouse ES cells by coculturing on PA6 cells instead of the formation of embryoid bodies. The ES cells cocultured with the PA6 stromal cell line for at least 3 days were capable of differentiating into spheres. The cells in the spheres were all green fluorescent protein (GFP) positive, showing that they were derived from GFP-expressing D3-ES cells. The spheres contained nestin-positive cells. The number of spheres increased when they were cocultured with PA6 for a longer period. Sphere formation was observed even after 10 mechanical dissociations and subculturings, showing its self-renewal ability. The cells differentiated into microtubule-associated protein-2 (MAP2)-positive neuronal cells and glial fibrillary acidic protein (GFAP)-positive glial cells. gamma-Aminobutyric acid-positive cells and tyrosine hydroxylase-positive cells were also observed in the spheres. The percentages of the MAP2- or GFAP-positive cells in the sphere changed according to the period of coculture on PA6 cells. At an early stage of coculture, more neurons were generated and, at a later period, more glial cells were generated. These results suggested that neurosphere could be generated from ES cells by coculturing with PA6, and that these cells resembled neural stem cells derived from mouse fetal brain tissue.

Animals↗

PubMed2003

Role of ERas in promoting tumour-like properties in mouse embryonic stem cells.

Embryonic stem (ES) cells are pluripotent cells derived from early mammalian embryos. Their immortality and rapid growth make them attractive sources for stem cell therapies; however, they produce tumours (teratomas) when transplanted, which could preclude their therapeutic usage. Why ES cells, which lack chromosomal abnormalities, possess tumour-like properties is largely unknown. Here we show that mouse ES cells specifically express a Ras-like gene, which we have named ERas. We show that human HRasp, which is a recognized pseudogene, does not contain reported base substitutions and instead encodes the human orthologue of ERas. This protein contains amino-acid residues identical to those present in active mutants of Ras and causes oncogenic transformation in NIH 3T3 cells. ERas interacts with phosphatidylinositol-3-OH kinase but not with Raf. ERas-null ES cells maintain pluripotency but show significantly reduced growth and tumorigenicity, which are rescued by expression of ERas complementary DNA or by activated phosphatidylinositol-3-OH kinase. We conclude that the transforming oncogene ERas is important in the tumour-like growth properties of ES cells.

3T3 Cells↗

PubMed2006

Collagen synthesis is required for ascorbic acid-enhanced differentiation of mouse embryonic stem cells into cardiomyocytes.

Ascorbic acid has been reported to promote the differentiation of embryonic stem (ES) cells into cardiomyocytes; however, the specific functions of ascorbic acid have not been defined. A stable form of ascorbic acid, namely, l-ascorbic acid 2-phosphate (A2-P), significantly enhanced cardiac differentiation; this was assessed by spontaneous beating of cardiomyocytes and expression of cardiac-specific markers obtained from mouse ES cells. This effect of ascorbic acid was observed only when A2-P was present during the early phase of differentiation. Treatment with two types of collagen synthesis inhibitors, l-2-azetidine carboxylic acid and cis-4-hydroxy-d-proline, significantly inhibited the A2-P-enhanced cardiac differentiation, whereas treatment with the antioxidant N-acetyl cysteine showed no effect. These findings demonstrated that ascorbic acid enhances differentiation of ES cells into cardiomyocytes through collagen synthesis and suggest its potential in the modification of cardiac differentiation of ES cells.

Animals↗

PubMed2004

Retinoic acid induces Pdx1-positive endoderm in differentiating mouse embryonic stem cells.

We have generated an embryonic stem (ES) cell line in which sequences encoding green fluorescent protein (GFP) were targeted to the locus of the pancreatic-duodenal homeobox gene (Pdx1). Analysis of chimeric embryos derived from blastocyst injection of Pdx1(GFP/w) ES cells demonstrated that the pattern of GFP expression was consistent with that reported for the endogenous Pdx1 gene. By monitoring GFP expression during the course of ES cell differentiation, we have shown that retinoic acid (RA) can regulate the commitment of ES cells to form Pdx1(+) pancreatic endoderm. RA was most effective at inducing Pdx1 expression when added to cultures at day 4 of ES differentiation, a period corresponding to the end of gastrulation in the embryo. RT-PCR analysis showed that Pdx1-positive cells from day 8 cultures expressed the early endoderm markers Ptf1a, Foxa2, Hnf4alpha, Hnf1beta, and Hnf6, consistent with the notion that they corresponded to the early pancreatic endoderm present in the embryonic day 9.5 mouse embryo. These results demonstrate the utility of Pdx1(GFP/w) ES cells as a tool for monitoring the effects of factors that influence pancreatic differentiation from ES cells.

Animals↗

PubMed2002

[p16INK4a exon 1 alpha knockout in mouse embryonic stem cells].

INK4a/ARF locus distinguishes itself by its unusual structure and function. It contains 2 overlapping genes with exons 1 alpha, 2 and 3 encoding p16INK4a and exons 1 beta, 2 and 3 encoding p19ARF. Mice with their exons 2 and 3 of the INK4a/ARF knocked out are viable and fertile but develop spontaneous tumors at an early age and highly sensitive to carcinogenic treatment. However, mice with their exon 1 beta knocked out, without interference the expression of p16INK4a, show almost the same phenotype as those with their exons 2 and 3 knocked out. This raises a question of whether the mouse p16INK4a plays a role in tumor suppression. To investigate this problem, a targeting vector pointing to p16INK4a exon 1 alpha with 1.5 kb Eco81 I/Acc II fragment as short arm and 5.9 kb Xba I/Xho I fragment as long arm was built. After linearlization and purification, the targeting vector was introduced into ES cells through electroporation. Thirty-seven G418- and gancyclovir-resistant colonies were picked out and one of them was confirmed as positive by Southern hybridization.

Animals↗

PubMed1995

Gene targeting of DT-diaphorase in mouse embryonic stem cells: establishment of null mutant and its mitomycin C-resistance.

It is generally accepted that DT-diaphorase is primarily involved in the detoxification of quinone compounds and is capable of metabolically activating some cancer chemotherapeutic quinones including mitomycin C. However, these conclusions have mainly been drawn from the experiments using the DT-diaphorase inhibitor, dicoumarol. To understand directly the roles of this enzyme in quinone metabolism, we have established heterozygously and homozygously DT-diaphorase-targeted mutant embryonic stem (ES) cells by homologous recombination. Cytotoxicity experiments using these cells clearly demonstrated that DT-diaphorase acts as an activator of mitomycin C in ES cells. These mutant cell lines seem to be very useful for investigating the functions of DT-diaphorase including the bioactivation and detoxification of quinone species. The generation of a DT-diaphorase-targeted mouse is under investigation.

Animals↗

PubMed2006

Kinetic properties of extracted lactate dehydrogenase and creatine kinase from mouse embryonic stem cell- and neonatal-derived cardiomyocytes.

Embryonic stem cells (ESCs), representing a population of undifferentiated pluripotent cells with both self-renewal and multilineage differentiation characteristics, are capable of spontaneous differentiation into cardiomyocytes. The present study sought to define the kinetic characterization of lactate dehydrogenase (LDH) and creatine kinase (CK) of ESC- and neonatal-derived cardiomyocytes. Spontaneously differentiated cardiomyocytes from embryoid bodies (EBs) derived from mouse ESC line (Royan B1) and neonatal cardiomyocytes were dispersed in a buffer solution. Enzymes were extracted by sonication and centrifugation for kinetic evaluation of LDH and CK with spectrophotometric methods. While a comparison between the kinetic properties of the LDH and CK of both groups revealed not only different Michaelis constants and optimum temperatures for LDH but also different Michaelis constants and optimum pH for CK, the pH profile of LDH and optimum temperature of CK were similar. In defining some kinetic properties of cardiac metabolic enzymes of ESC-derived cardiomyocytes, our results are expected to further facilitate the use of ESCs as an experimental model.

Animals↗

PubMed2006

DNA damage response and mutagenesis in mouse embryonic stem cells.

Mutation in embryonic stem (ES) cells can potentially compromise multiple cell lineages and affect the well-being of subsequent generations. Thus, ES cells require sensitive mechanisms to maintain genomic integrity. One mechanism involves suppression of mutation. A complementary mechanism is to regulate the cell cycle checkpoint and facilitate cell death. Here, we describe the detailed protocols we have used to investigate DNA damage response and mutagenesis in mouse ES cells.

Adenine Phosphoribosyltransferase↗

PubMed2006

A stable analogue of glucose-dependent insulinotropic polypeptide, GIP(LysPAL16), enhances functional differentiation of mouse embryonic stem cells into cells expressing islet-specific genes and hormones.

Embryonic stem (ES) cells can be differentiated into insulin-producing cells by conditioning the culture media. However, the number of insulin-expressing cells and amount of insulin released is very low. Glucose-dependent insulinotropic polypeptide (GIP) enhances the growth and differentiation of pancreatic beta-cells. This study examined the potential of the stable analogue GIP(LysPAL16) to enhance the differentiation of mouse ES cells into insulin-producing cells using a five-stage culturing strategy. Semi-quantitative PCR indicated mRNA expression of islet development markers (nestin, Pdx1, Nkx6.1, Oct4), mature pancreatic beta-cell markers (insulin, glucagon, Glut2, Sur1, Kir6.1) and the GIP receptor gene GIP-R in undifferentiated (stage 1) cells, with increasing levels in differentiated stages 4 and 5. IAPP and somatostatin genes were only expressed in differentiated stages. Immunohistochemical studies confirmed the presence of insulin, glucagon, somatostatin and IAPP in differentiated ES cells. After supplementation with GIP(LysPAL16), ES cells at stage 4 released insulin in response to secretagogues and glucose in a concentration-dependent manner, with 35-100% increases in insulin release. Cellular C-peptide content also increased by 45% at stages 4 and 5. We conclude that the stable GIP analogue enhanced differentiation of mouse ES cells towards a phenotype expressing specific beta-cell genes and releasing insulin.

Animals↗