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Stage-specific germ-cell marker genes are expressed in all mouse pluripotent cell types and emerge early during induced pluripotency.

ABSTRACT: Embryonic stem cells (ESCs) generated from the in-vitro culture of blastocyst stage embryos are known as equivalent to blastocyst inner cell mass (ICM) in-vivo. Though several reports have shown the expression of germ cell/pre-meiotic (GC/PrM) markers in ESCs, their functional relevance for the pluripotency and germ line commitment are largely unknown. In the present study, we used mouse as a model system and systematically analyzed the RNA and protein expression of GC/PrM markers in ESCs and found them to be comparable to the expression of cultured pluripotent cells originated from the germ line. Further, siRNA knockdown experiments have demonstrated the parallel maintenance and independence of pluripotent and GC/PrM networks in ESCs. Through chromatin immunoprecipitation experiments, we observed that pluripotent cells exhibit active chromatin states at GC marker genes and a bivalent chromatin structure at PrM marker genes. Moreover, gene expression analysis during the time course of iPS cells generation revealed that the expression of GC markers precedes pluripotency markers. Collectively, through our observations we hypothesize that the chromatin state and the expression of GC/PrM markers might indicate molecular parallels between in-vivo germ cell specification and pluripotent stem cell generation.

SUBMITTER: Xu X

PROVIDER: S-EPMC3143132 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Stage-specific germ-cell marker genes are expressed in all mouse pluripotent cell types and emerge early during induced pluripotency.

Xu Xingbo X Pantakani D V Krishna DV Lührig Sandra S Tan Xiaoying X Khromov Tatjana T Nolte Jessica J Dressel Ralf R Zechner Ulrich U Engel Wolfgang W

PloS one 20110725 7

Embryonic stem cells (ESCs) generated from the in-vitro culture of blastocyst stage embryos are known as equivalent to blastocyst inner cell mass (ICM) in-vivo. Though several reports have shown the expression of germ cell/pre-meiotic (GC/PrM) markers in ESCs, their functional relevance for the pluripotency and germ line commitment are largely unknown. In the present study, we used mouse as a model system and systematically analyzed the RNA and protein expression of GC/PrM markers in ESCs and fo ...[more]

PMID: 21799849

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Project description:BackgroundDespite years of research, porcine-induced pluripotent stem cells (piPSCs) with germline chimeric capacity have not been established. Furthermore, the key transcription factors (TFs) defining the naïve state in piPSCs also remain elusive, even though TFs in the inner cell mass (ICM) are believed to be key molecular determinants of naïve pluripotency. In this study, interferon regulatory factor 1 (IRF-1) was screened to express higher in ICM than trophectoderm (TE). But the impact of IRF-1 on maintenance of pluripotency in piPSCs was not determined.MethodsTranscriptome profiles of the early ICM were analyzed to determine highly interconnected TFs. Cells carrying these TFs' reporter were used to as donor cells for somatic cell nuclear transfer to detect expression patterns in blastocysts. Next, IRF1-Flag was overexpressed in DOX-hLIF-2i piPSCs and AP staining, qRT-PCR, and RNA-seq were conducted to examine the effect of IRF-1 on pluripotency. Then, the expression of IRF-1 in DOX-hLIF-2i piPSCs was labeled by GFP and qRT-PCR was conducted to determine the difference between GFP-positive and GFP-negative cells. Next, ChIP-Seq was conducted to identify genes target by IRF-1. Treatment with IL7 in wild-type piPSCs and STAT3 phosphorylation inhibitor in IRF-1 overexpressing piPSCs was conducted to confirm the roles of JAK-STAT3 signaling pathway in IRF-1's regulation of pluripotency. Moreover, during reprogramming, IRF-1 was overexpressed and knocked down to determine the change of reprogramming efficiency.ResultsIRF-1 was screened to be expressed higher in porcine ICM than TE of d6~7 SCNT blastocysts. First, overexpression of IRF-1 in the piPSCs was observed to promote the morphology, AP staining, and expression profiles of pluripotency genes as would be expected when cells approach the naïve state. Genes, KEGG pathways, and GO terms related to the process of differentiation were also downregulated. Next, in the wild-type piPSCs, high-level fluorescence activated by the IRF-1 promoter was associated with higher expression of naïve related genes in piPSCs. Analysis by ChIP-Seq indicated that genes related to the JAK-STAT pathway, and expression of IL7 and STAT3 were activated by IRF-1. The inhibitor of STAT3 phosphorylation was observed could revert the expression of primed genes in IRF-1 overexpressing cells, but the addition of IL7 in culture medium had no apparent change in the cell morphology, AP staining results, or expression of pluripotency related genes. In addition, knockdown of IRF-1 during reprogramming appeared to reduce reprogramming efficiency, whereas overexpression exerted the converse effect.ConclusionThe IRF-1 expressed in the ICM of pigs' early blastocyst enhances the pluripotency of piPSCs, in part through promoting the JAK-STAT pathway.

Dataset Information

Stage-specific germ-cell marker genes are expressed in all mouse pluripotent cell types and emerge early during induced pluripotency.

Publications

Stage-specific germ-cell marker genes are expressed in all mouse pluripotent cell types and emerge early during induced pluripotency.

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