Project description:SAGE identification of differentiation responsive genes in P19 embryonic cells induced to form cardiomyocytes in vitro. P19 embryonic carcinoma (EC) cells, induced to form cardiomyocytes in vitro - undifferentiated cells, day 3+0.5 and day 3+3.0 of differentiation protocol. Keywords = EC cells, P19, differentiation, cardiomyocytes Keywords: time-course
Project description:Pluripotent stem cell lines derived from embryos of different stages have distinct pluripotent ground states, but similar levels of the transcription factor Oct4. Epiblast-derived pluripotent stem cells (EpiSCs), in contrast to embryonic stem (ES) cells, cannot form chimeras. We show that EpiSCs express lower levels of the transcription factors Sox2 and Klf4 than ES cells and have limited reprogramming potential, as shown by cell fusion. Sox2 overexpression dramatically increases the reprogramming potential, chimera formation, and germline contribution of EpiSCs. Therefore, although Oct4 is essential for reprogramming, the level of Sox2 defines both the reprogramming capability and the pluripotent ground states. RNA samples to be analyzed on microarrays were prepared using Qiagen RNeasy columns with on-column DNA digestion. 300 ng of total RNA per sample was used as input into a linear amplification protocol (Ambion), which involved synthesis of T7-linked double-stranded cDNA and 12 hrs of in-vitro transcription incorporating biotin-labelled nucleotides. Purified and labelled cRNA was then hybridized for 18 hrs onto MouseRef-8 v2 expression BeadChips (Illumina) according to the manufacturer's instructions. After washing, as recommended, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were hybridized as biological replicates. 12 sample types were analyzed, each of them in duplicate. ESCm: Mouse ESC male; ESCf: Mouse ESC OG2 female; F9 EC: F9 EC (mouse embryonic carcinoma cell); F9-Sox2: F9 EC (mouse embryonic carcinoma cell) overexpressing wild type Sox2; EpiSCf: Mouse EpiSC OG2 female; Epi-Sox2f: Mouse EpiSC Sox2 (OG2 female) overexpressing wild type Sox2; P19 EC: P19 EC (mouse embryonic carcinoma cell); P19-Sox2: P19 EC (mouse embryonic carcinoma cell) overexpressing wild type Sox2; EpiSCm: Mouse EpiSC (GOF18 male) (duplicates); EpiSox2mL2: Mouse EpiSC Sox2 (GOF18 male) overexpressing wild type Sox2 cultured in condition EpiSC medium (CM); EpiSox2mE1: Mouse EpiSC Sox2 (GOF18 male) overexpressing wild type Sox2 cultured in ESC medium (ESC like1); EpiSox2mE2: Mouse EpiSC Sox2 (GOF18 male) overexpressing wild type Sox2 cultured in ESC medium (ESC like2).
Project description:Gene expression profiles of undifferentiated mouse embryonal carcinoma cell strains (P19, P19CL6, and 4 P19CL6 sublines) were obtained, using Affymetrix GeneChip Mouse Genome 430A and 430B. Heart diseases such as cardiac infarction damage cardiomyocytes and consequently lead to significant loss of the contractile capacity of the heart. To repair functions of the injured heart, a great deal of research has attempted to develop regenerative medicine using pluripotent stem cell-based cardiomyocytes as cell therapy products. However, the efficiency of the current methods available for the cardiac differentiation of stem cells is insufficient for clinical settings. A comprehensive understanding of the mechanism involved in the cardiac differentiation of stem cells is necessary to improve the differentiation efficiency. To identify genes assosiated with cardiomyogenic potential, we isolated P19CL6 cell sublines possessing distinct properties in cardiomyogenesis and comprared their transcriptome profiles with those of mouse embryonal carcinoma P19 and P19CL6 cells. Total RNA isolated from undifferentiated EC cell strains (P19 cells, P19CL6 cells, and 4 P19CL6 sublines) using Affymetrix chips MOE430A and MOE430AB. CEL files unavailable.
Project description:Valproic acid (VPA) is a potent inducer of neural tube defects (NTDs), but its mechanism of teratogenicity is not known. To study the transcriptional response of VPA during the susceptible period, i.e. when VPA is likely to exert most of its teratogenic effect, RNA was extracted from 8.25-dpc embryos (6 h post treatment) from control and VPA-treated dams, and subjected to microarray analysis (four arrays). To be more useful for risk assessment, in vitro models, using cells, may bridge biomarkers discovered by gene expression profiling in an animal in vivo model. Since pluripotent embryocarcinoma (EC) cell lines may be relevant models for early embryonic cells, P19 mouse EC cells were treated with or without 1 mM sodium valproate for 24 h and alos subjected to microarray analysis (four arrays).
Project description:As previously reported (Neuroscience letters 444(2008)127-131), we established embryonic carcinoma P19 cell line expressing the intracellular domain of APP (AICD). Although neurons could be differentiated from these cell lines with RA treatment, expression of AICD gave rise to neuron-specific apoptosis. To identify the genes that are involved in this cell death, we evaluated changes of gene expressions that was induced by AICD through this process of cell death. The expression profiles of almost forty thousands transcripts were monitored by DNA microarrays. AICD-expressing P19 cell lines (AICD/P19) and control P19 cell lines (pCDNA/P19) that carry vector alone were used and cultured. To induce neural differentiation of these P19 cell lines, cells were allowed to aggregate in culture medium with 5×10-7 M all-trans-retinoic acid (RA) for 4 days (aggregated state). After aggregation, cells were replated onto cell culture grade dishes or poly-L-Lysine coated cell-disks for two days (differentiated state). RNAs were isolated at each states.
Project description:microRNAs (or miRNAs) are small noncoding RNAs (21 to 25 nucleotides) that are processed from longer hairpin RNA precursors and are believed to be involved in a wide range of developmental and cellular processes, by either repressing translation or triggering mRNA degradation (RNA interference). Expression analysis of miRNAs is necessary to understand their complex role in the regulation of gene expression during the development, differentiation and proliferation of cells. Here we report on the expression profile analysis of miRNAs in mouse P19 embryonic carcinoma (EC) cell,which were used to get efficient derivation of a high percentage (∼95%) of neural stem cells from in N2B27 serum-free medium, in vitro simulated the process in vivo neural induction.The data presented here indicate marked changes in the expression of miRNAs between the P19 EC cell and the neural stem cells derived from them. Among ~160 mouse miRNAs, 37 miRNAs are increased (2.0-fold up-regulated) and 41 miRNAs are decreased (2.0-fold down-regulated) in their expression level after the differentiation. MiR-124a is dramatically up-regulated (above 20-fold). However, miR-302 cluster and miR290-295 cluster are down-regulated strongly. Further analyses show that some important factors, such as Oct4 and Sox2, are involved in the regulation system of these miRNAs. In brief, our results might contribute to a greater understanding of the many biological events in neural induction of early mouse embryos regulated by miRNAs. Keywords: microRNA, mouse, neural differentiation
Project description:Here we perfomed the Teratoma assay for a normal human embryonic stem cell line (H9(+Dox)), a human embryonic stem cell line with a mesendodermal differentiation bias (H9Hyb), a normal human induced pluripotent stem cell line (LU07), a human induced pluripotent stem cell line with reactivated transgenes (LU07+Dox) and a human embryonal carcinoma cell line (EC) and anayzed their gene expression. The ability to form teratomas in vivo containing multiple somatic cell types is regarded as functional evidence of pluripotency for human pluripotent stem cells (hPSCs). Since the Teratoma assay is animal-dependent, laborious and only qualitative, the PluriTest and the hPSC ScoreCard assay have been developed as in vitro alternatives. Here we compared normal hPSCs, induced hPSCs (hiPSCs) with reactivated reprogramming transgenes and human embryonal carcinoma (hEC) cells in these assays. While normal hPSCs gave rise to typical teratomas, the xenografts of the hEC cells and the hiPSCs with reactivated reprogramming transgenes were largely undifferentiated and malignant. The hPSC ScoreCard assay confirmed the line-specific differentiation propensities in vitro. However, when undifferentiated cells were analysed with PluriTest only hEC cells were identified as abnormal whereas all other cell lines were indistinguishable and resembled normal hPSCs. Our results indicate that pluripotency assays are best selected on the basis of intended downstream applications.