Unknown,Transcriptomics,Genomics,Proteomics

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Characterization of Human Induced Pluripotent Stem Cell Lines Derived from Neonatal and Adult Human Dermal Fibroblasts

ABSTRACT: Somatic cells can be reprogrammed to Induced Pluripotent Stem Cells (iPSCs) by expressing four transcription factors, Oct4, Sox2, Klf4 and c-Myc. Co-expressing Rarg (retinoic acid receptor gamma) and Lrh-1 (liver receptor homolog 1, Nr5a2) with the four factors greatly accelerated reprogramming so that reprogramming of mouse embryonic fibroblast cells (MEFs) to ground state iPSCs requires only four days’ induction of these six factors. The six-factor combination readily reprogrammed primary human neonatal and adult fibroblast cells to exogenous-factor-independent iPSCs, which resembled ground state mouse ES cells in growth properties, gene expression and signalling dependency. Our findings demonstrate that signalling through RARs has critical roles in molecular reprogramming and the synergistic interaction between Rarg and Lrh1 directs reprogramming towards ground state pluripotency. SH-iPS20-1, -3 and -9 lines were derived from HDFa (439656), SH-iPS24-1, -2 and -11 lines were derived from HDFa (709590), and SH-iPS28-23, -25 and -27 lines were derived from HDFn (617769). Parental HDF lines were used as control. SH-iPS15-5 and -10 lines were derived from HDFn (617769, Invitrogen). H1 hESC were obtained from Wicell (Madison, USA). SH-iPSCs were cultured in both 2i+LIF and FGFconditions. Expression pattern of SH-iPSCs was compared to H1 hESC cultured in FGF conditions. S1, S2 and S3 are three indpendent subcultures of a cell line.

ORGANISM(S): Homo sapiens

SUBMITTER: Jack Chen

PROVIDER: E-GEOD-32581 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Our goal is to find new genes regulated by p21 in human primary cells . To get it we carried out a gene expression profiling in two different models, human myeloid leukemia K562 cells and human keratinocytes both of them with conditional expression of p21. In order to find genes regulated by p21 in human primary cells we carried out a gene expression profiling in human myeloid leukemia K562 cells with conditional expression of p21. We previously described a K562 derivative, termed Kp21-4, that carries a zinc-inducible p21 gene (Munoz-Alonso MJ et at., 2005). We performed a kinetic study to identify the expression peak of p21 in this system. This transient induction of p21 was accompanied by proliferation arrest and an increase in polyploid cells after 48-72 h (Munoz-Alonso MJ et at., 2005). Actually, 6-12 h of p21 induction with ZnSO4 is sufficient to irreversibly trigger proliferation arrest. Therefore, we chose 12 h as the induction time to analyse p21 effects on the transcriptome of these cells, as gene expression changes later on may be indirect due to other phenotypic effects. We next carried out the gene expression profiling of Kp21-4 cells upon p21 induction by ZnSO4. In order to identify genes specifically modulated by p21 we compared with the cell line Kp27-5, which carries a Zn2+-inducible p27 allele (Munoz-Alonso MJ et at., 2005). p27 is a close relative to p21 that also inhibits CDKs and induce cell cycle arrest . Thus, the comparison serves to identify genes specifically regulated by p21 in our analysis. We subtracted the gene expression changes occurring at 72 h in Kp21-4 cells those genes regulated by p27 in the Kp27-5 cells and genes changed by ZnSO4 treatment in parental K562 cells. So, our intention is to identify only genes regulated at short time of induction by p21 and not by p27. In order to confirm these results we studied the p21-dependent repression of mitotic genes in a different cellular system. A dramatic increase in p21 and p27 in Kp21 and Kp27 were demonstrated by RT-qPCR and immunoblot (as we show in the manuscript). We prepared RNA 12 h and 72h after induction with ZnSO4 and performed large-scale expression assay using the Afftymetrix platform. The clustering analysis revealed that p21 provoked the down-regulation of a number genes involved in cell cycle control not shared by cells expressing p27 (as we show in the manuscript). Our goal, has been getting genes regulated more strongly by p21 and not by p27, in cell cycle and itosis. Our result are supported because we have found the same genes in two different models and also we have validated (by RT-qPCR) more than 20 cell cycle and mitotic genes, found in our affymetrix arrays. Also we have found the region of p21 that is sufficient for gene regulation and for one gene we have described as p21 bind to the promoter. Finally, we have discussed in our manuscript how p21 can do this regulation by bioinformatic analysis of p21-target genes. The sucess of this study is describe a new role of p21 as a transcriptional co-repressor in some systems.

Project description:The mammalian FoxO transcription factors - FoxO1, FoxO3, FoxO4 - function in the nucleus to direct transcription of specific gene targets governing cellular survival, proliferation, metabolism, differentiation and oxidative defense. Activation of PI3K by extracellular growth factors leads to AKT-mediated phosphorylation of FoxO1, FoxO3 and FoxO4, resulting in their sequestration in the cytoplasm such that they are unable to regulate their gene targets. Our study identified FoxOs as novel tumor suppressors in kidney cancer (Gan et al, 2010, Cancer Cell). To understand the tumor suppression function of FoxOs in kidney cancer cells, we performed gene expression profiling in human kidney cancer cells upon FoxO1 or FoxO3 reactivation in order to identify the key transcriptomic alterations mediating FoxO tumor suppression function in kidney cancer cells. We generated RCC4 and UMRC2 cell lines (two human kidney cancer cells with low endogenous FoxO1 and FoxO3 expression) with stable expression of FoxO1(TA)ERT2 or FoxO3(TA)ERT2 construct, which expressed a fusion protein consisting of FoxO(TA) (containing three Ser/Thr AKT phosphorylation sites mutated to alanine) fused to the T2-modified estrogen receptor (ERT2) moiety. We documented that the FoxO(TA)ERT2 fusion protein sequestered FoxO(TA) in the cytoplasm and that 4OHT treatment resulted in rapid translocation of FoxO(TA)ERT2 into the nucleus. We also established stable cell lines with ERT2 expression as control cell lines. (For simplicity, ERT2, FoxO1(TA)ERT2 and FoxO3(TA)ERT2 cell lines will be referred to as EV (empty vector), FoxO1 and FoxO3, respectively, hereafter). We then conducted comparative transcriptome analysis (using the Human Genome U133 Plus 2.0 Array) of EV, FoxO1, or FoxO3-expressing RCC4 and UMRC2 cells at 12 hours with or without 100 nm 4OHT treatment (cultured in DMEM+10% FBS with puromycin selection). To enrich for more proximal actions of FoxO, we selected the 12 hour time point as time course studies revealed dramatic transcriptional changes of known FoxO targets (such as Cyclin D1), yet no discernable cellular phenotypes (apoptosis and cell cycle arrest). We generated 4 transcriptome datasets: FoxO1 RCC4, FoxO3 RCC4, FoxO1 UMRC2, and FoxO3 UMRC2 (by comparing transcriptome data with or without 4OHT treatment), and normalized these transcriptome data against 4OHT-treated EV cells, which show modest 4OHT-induced transcriptional changes.

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Characterization of Human Induced Pluripotent Stem Cell Lines Derived from Neonatal and Adult Human Dermal Fibroblasts

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