ascorbic-acid and Teratoma

Human parthenogenetic embryonic stem cells (hpESCs) are generated from artificially activated oocytes, however, the issue of whether hpESCs have equivalent differentiation ability to human fertilized embryonic stem cells remains controversial.. hpESCs were injected into male severe combined immunodeficiency (SCID) mice and the efficiency of teratoma formation was calculated. Then the gene expression and methylation modification were detected by real time-PCR and bisulfate methods.. Comparison of five hpESCs with different differentiation abilities revealed that levels of paternal genes in the Dlk1-Dio3 region on chromosome 14 in the hpESCs with high differentiation potential are enhanced, but strictly methylated and silenced in the hpESCs with lower differentiation potential. Treatment with ascorbic acid, rescued their ability to support teratoma formation and altered the expression profiles of paternally expressed genes in hpESCs that could not form teratoma easily. No differences in the expression of other imprinting genes were evident between hpESCs with higher and lower differentiation potential, except for those in the Dlk1-Dio3 region.. The Dlk1-Dio3 imprinting gene cluster distinguishes the differentiation ability of hpESCs. Moreover, modification by ascorbic acid may facilitate application of hpESCs to clinical settings in the future by enhancing their pluripotency.

Topics: Animals; Ascorbic Acid; Calcium-Binding Proteins; Cell Differentiation; DNA Methylation; Embryo Culture Techniques; Embryonic Stem Cells; Gene Expression; Gene Expression Profiling; Humans; Intercellular Signaling Peptides and Proteins; Iodide Peroxidase; Male; Membrane Proteins; Mice; Mice, SCID; Multigene Family; Parthenogenesis; Pluripotent Stem Cells; Teratoma

Induced pluripotent stem cells (iPSCs) are capable of unlimited self-renewal and can give rise to all three germ layers, thereby providing a new platform with which to study mammalian development and epigenetic reprogramming. However, iPSC generation may result in subtle epigenetic variations, such as the aberrant methylation of the Dlk1-Dio3 locus, among the clones, and this heterogeneity constitutes a major drawback to harnessing the full potential of iPSCs. Vitamin C has recently emerged as a safeguard to ensure the normal imprinting of the Dlk1-Dio3 locus during reprogramming. Here, we show that vitamin C exerts its effect in a manner that is independent of the reprogramming kinetics. Moreover, we demonstrate that reprogramming cells under 2i conditions leads to the early upregulation of Prdm14, which in turn results in a highly homogeneous population of authentic pluripotent colonies and prevents the abnormal silencing of the Dlk1-Dio3 locus.

Topics: Animals; Ascorbic Acid; Calcium-Binding Proteins; Cells, Cultured; DNA-Binding Proteins; Gene Expression; Genetic Loci; Induced Pluripotent Stem Cells; Intercellular Signaling Peptides and Proteins; Iodide Peroxidase; Mice, Inbred NOD; Mice, SCID; Mice, Transgenic; RNA-Binding Proteins; Teratoma; Transcription Factors

We have previously reported the isolation of an osteogenic clonal cell line (C1) derived from mouse teratocarcinoma and immortalized by the SV 40 oncogenes. In this report we describe the kinetics of osteogenic differentiation of aggregated C1 cells by following the matrix deposition and mineralization and the expression of alkaline phosphatase. We show that after addition of beta-glycerophosphate and ascorbic acid, more than 95% of C1 aggregates synthesize a bone matrix which is deposited as early as 2 days and increases progressively with time in culture. Matrix calcification is evidenced by von Kossa staining and tetracycline incorporation into the mineral whereas no calcification appears in control cultures. Calcium is detectable in mineralizing aggregates at 2 days and calcium content increases linearly with time in culture, being 125-fold higher in mineralizing nodules than in control aggregates at 30 days. Aggregated C1 cells are characterized by a high activity of the bone type isoenzyme of alkaline phosphatase, a marker of osteoblast phenotype. Upon addition of inducers, alkaline phosphatase activity decreases by five-fold after the onset of mineralization and remains stable thereafter. The down-regulation of alkaline phosphatase activity is confirmed at the cellular level by histochemical staining. The mRNA levels for alkaline phosphatase decline during osteogenesis, following a pattern similar to the decrease in protein activity. Analysis of DNA synthesis by (3H)-thymidine incorporation and quantification of labelled nuclei on autoradiographs shows that C1 cells proliferation is not down-regulated during the time course of differentiation and that proliferating C1 cells still express alkaline phosphatase activity during osteogenic differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Biomarkers; Cell Differentiation; Cell Transformation, Viral; Clone Cells; DNA; Extracellular Matrix; Glycerophosphates; Isoenzymes; Kinetics; Mice; Minerals; Osteoblasts; Osteogenesis; RNA, Messenger; Simian virus 40; Teratoma; Tumor Cells, Cultured

We have selected a mutant F9 teratocarcinoma stem cell line, RA-5-1, which does not exhibit normal differentiation into parietal endoderm in the presence of retinoic acid, dibutyryl cyclic AMP, and theophylline (RACT). In this report, we demonstrate that the RA-5-1 mutant possesses a prolyl-4-hydroxylase enzyme with a higher Km for a synthetic collagen substrate and that this alteration results in a 6-7-fold reduction in the amount of collagen IV in the medium of RACT-treated mutant cells, as compared to wild type F9 cells. In addition, the collagen IV that is secreted by RACT-treated RA-5-1 cells has an abnormally low molecular weight and contains 6-9-fold less 4-hydroxyproline than the collagen IV secreted by RACT-treated wild type F9 cells. A brief ascorbate treatment can increase the hydroxyproline content of the collagen IV secreted by RACT-treated RA-5-1 cells. A large reduction in the amount of laminin in the medium of RACT-treated RA-5-1 mutant cells is also observed. Concomitant with the reduction in collagen IV and laminin polypeptides in the medium, the expression of several other differentiation-specific mRNAs is delayed in the RACT-treated RA-5-1 cells relative to wild type F9 cells. Moreover, the mutant cells do not exhibit the morphology or the complete growth arrest of wild type terminally differentiated parietal endoderm cells in the presence of RACT. These results suggest that a defect in the post-translational modification of collagen IV in the mutant RA-5-1 prevents the complete expression of the differentiation program in response to RACT. These experiments also demonstrate that the expression of certain differentiation-specific genes is compatible with continued proliferation in the mutant line.

Topics: Animals; Ascorbic Acid; Bucladesine; Cell Differentiation; Cell Division; Cell Line; Collagen; Hydroxylation; Kinetics; Laminin; Mutation; Procollagen-Proline Dioxygenase; Protein Processing, Post-Translational; RNA, Messenger; Teratoma; Theophylline; Transcription, Genetic; Tretinoin

The nutritional status of 14 patients with metastatic testicular teratomas was measured longitudinally through four courses of treatment with vinblastine and bleomycin, or vinblastine, bleomycin and cis platinum regimens. On each regimen patients lost weight during each course and did not entirely regain it between courses. The nutritional status with respect to retinol and vitamins E, B1 and B6 also fell during each course. The fall in plasma retinol levels was correlated with a fall in the plasma level of retinol-binding protein (RBP). In patients treated with the vinblastine and bleomycin regimen, plasma retinol levels were higher at the beginning of the fourth course than at the start of the treatment, possibly due to improved synthesis of RBP. In contrast, there was no overall improvement in B1 status.

Topics: Adult; Ascorbic Acid; Avitaminosis; Bleomycin; Cisplatin; Drug Therapy, Combination; Humans; Longitudinal Studies; Male; Middle Aged; Retinol-Binding Proteins; Retinol-Binding Proteins, Plasma; Teratoma; Testicular Neoplasms; Vinblastine; Vitamin A; Vitamin E; Vitamins

The embryonal carcinoma mouse cell line F-9 was used as a convenient model for a quantitative study of the production of the basement membrane proteins laminin and type IV collagen. Both proteins could be identified in the culture medium and cell layer by radioimmuno assays, metabolic labeling and immunofluorescence. More than 95% of the material is secreted into the medium. Lack of ascorbic acid inhibits secretion of type IV collagen but not of laminin. Induction of differentiation into endoderm-like cells by retinoic acid consistently caused after a lag period of 2-3 days a 5-10 fold increase in the production of basement membrane proteins but not of total protein. Dibutyryl cyclic AMP further potentiated this specific effect particularly with respect to type IV collagen synthesis. Insulin, epidermal growth factor and nerve growth factor produced only moderate increases (10-60%) in the amount of laminin and type IV collagen. Effects of these hormones were only observed with certain doses and were quite variable between different experiments.

Topics: Animals; Ascorbic Acid; Basement Membrane; Bucladesine; Cell Line; Collagen; Electrophoresis, Polyacrylamide Gel; Fluorescent Antibody Technique; Glycoproteins; Growth Substances; Kinetics; Laminin; Mice; Teratoma; Tretinoin