Integrative analysis of the acquisition of pluripotency in PGCs
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Shigeru Saito
PROVIDER: E-GEOD-67616 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:To elucidate the molecular mechanisms by which PGCs become pluripotent cells, we performed whole transcriptome analysis during the conversion of PGCs into EGCs.
2015-04-08 | GSE67616 | GEO
Project description:The male germ cell differentiation is a subtle and complex regulation processes, currently its regulatory mechanism is not fully understood. In our experiment, we performed the first comprehensive genome wide analyses of the crucial genes in three kinds of crucial cells (ESCs, PGCs and SSCs) associated with the male germ cells differentiation. We identified thousands of differentially expressed genes (DEGs) in this process andwe choosed 173 candidate genes involved in the differentiation and metabolic processes, like GAL9, AMH, PLK1, PSMD7 and so on. Further exploration found that the candidate genes express patterns were the same between in vitro induction experiments and transcriptome results. Our results clues to the mechanistic basis of male germ cell differentiation and provides an important reference for future studies. Gene expression in chicken germ stem cells was measured at different stages of development. Three independent experiments were performed at each stage (ESCs, PGCs and SSCs).
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Project description:Specification to primordial germ cells (PGCs) occurs under the mesoderm induction signals during gastrulation. Here, we found that Akt activation in embryonic stem (ES) cells generated self-renewing spheres during mesodermal differentiation induction and that the differentiation status of the sphere cells was in between ES cells and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, showing that the cells of the sphere commenced the differentiation to germ lineage. However, the spheres could not proceed to spermatogenesis after transplantation to testes. Meanwhile, the transfer of the spheres to the original feeder-free ES cell culture conditions induced chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, the reversion to the ES cell-like cell states was induced. These results indicate that the Akt signaling brings about a novel metastable and pluripotent state between ES cells and PGCs. Five samples were analyzed, which included the Akt-Mer-expressing ES cell (ESC) line #21 treated with or without 4OHT (4-hydroxytamoxifen), the #21 ESC-derived primordial germ cell (PGC)-like sphere cells and the ESC-like cells reverted from #21 PGC-like sphere cells. The PGC-like sphere cells derived from another Akt-Mer ESC line #42 was also examined.
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Project description:Gene-expression profile of human embryonic germ cells (EGCs), primordial germ cells (PGCs), embryonal carcinoma cells (ECCs), pluripotent embryonic stem cells (ESCs) and induced pluripotent stem cells (IPSCs)
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Project description:Naive pluripotent embryonic stem cells (ESCs) and embryonic germ cells (EGCs) are derived from the preimplantation epiblast and primordial germ cells (PGCs), respectively. We investigated whether differences exist between ESCs and EGCs, in view of their distinct developmental origins. PGCs are programmed to undergo global DNA demethylation; however, we find that EGCs and ESCs exhibit equivalent global DNA methylation levels. Importantly, inhibition of Erk and Gsk3b by 2i conditions leads to pronounced reduction in DNA methylation in both cell types. This is driven by Prdm14 and is associated with downregulation of Dnmt3a and Dnmt3b. However, genomic imprints are maintained in 2i, and we report derivation of EGCs with intact genomic imprints. Collectively, our findings establish that culture in 2i instills a naive pluripotent state with a distinctive epigenetic configuration that parallels molecular features observed in both the preimplantation epiblast and nascent PGCs.
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Project description:The growth factors and signaling pathways that are essential for the inducing proliferation of chicken PGCs is unclear. We investigated the effect of basic fibroblast growth factor (bFGF) on the survival and proliferation of PGCs under feeder-free conditions. We used microarrays to examine the genes regulated by bFGF treatment in chicken PGCs. Cultured PGCs were collected before and after withdrawal of bFGF for 24 h, and 24 h after bFGF replacement. The RNA was extracted and hybridized on Affymetrix microarrays. Three replicates each.
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Project description:Primordial germ cells (PGCs) are fate restricted to differentiate into gametes in vivo. However when removed from their embryonic niche PGCs undergo reversion to generate pluripotent embryonic germ cells (EGCs) in vitro. One of the major differences between EGCs and embryonic stem cells (ESCs) involves variable methylation at imprinting control centers (ICCs), a phenomenon that is poorly understood. In the current study we show that reverting PGCs to EGCs involves ICC methylation erasure, which remain stably hypomethylated at Snrpn, Igf2r and Kcnqot1. In contrast, the H19/Igf2 ICC undergoes almost complete de novo remethylation. Using the same approach for PGCs differentiated in vitro from ESCs we show that the Snrpn ICC is erased however the hypomethylated state is highly unstable. We also discovered that when the H19/Igf2 ICC is abnormally hypermethylated in ESCs, ICC methylation is not erased with differentiation into PGCs. This highlights the importance of not only launching germline differentiation with correctly methylated ESC lines but also the need to better stabilize the hypomethylated state in the in vitro derivatives following ICC erasure.
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Project description:Naive pluripotent embryonic stem cells (ESCs) and embryonic germ cells (EGCs) are derived from the preimplantation epiblast and primordial germ cells (PGCs), respectively. We investigated whether differences exist between ESCs and EGCs, in view of their distinct developmental origins. PGCs are programmed to undergo global DNA demethylation; however, we find that EGCs and ESCs exhibit equivalent global DNA methylation levels. Importantly, inhibition of Erk and Gsk3b by 2i conditions leads to pronounced reduction in DNA methylation in both cell types. This is driven by Prdm14 and is associated with downregulation of Dnmt3a and Dnmt3b. However, genomic imprints are maintained in 2i, and we report derivation of EGCs with intact genomic imprints. Collectively, our findings establish that culture in 2i instills a naive pluripotent state with a distinctive epigenetic configuration that parallels molecular features observed in both the preimplantation epiblast and nascent PGCs. EGC lines were derived from E8.5 mouse embryos using three different protocols: FCS+LIF on MEFs (FCS, n = 4)), FCS+LIF on MEFs for 48 hours followed by 2i+LIF (2is, n = 4), and direct derivation into 2i+LIF (2i, n = 4). ESC lines were derived in either FCS+LIF on MEFs or in 2i+LIF conditions and once established the cell lines were also switched between the two culture environments (n = 5 for each culture condition). All cell lines were derived from genetically identical embryos.
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Project description:Epigenetic reprogramming, characterized by loss of cytosine methylation and histone modifications, occurs during mammalian development in primordial germ cells (PGCs). Here we provide a detailed map of cytosine methylation on a large portion of the genome in developing male and female PGCs isolated from mouse embryos. We mapped DNA methylation in E13.5 PGCs isolated by FACS sorting from Oct4-GFP male and female embryos. As a control, we also mapped DNA methylation in E7.5 epiblasts from wich PGCs derive from. MeDIP and Input samples were hybridized to Nimblegen HD2 MM8 promoter deluxe arrays covering 12 kb of all gene promoters. Experiments were performed in duplicates for E7.5 epiblasts and triplicates for E13.5 PGCs.
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