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Expanding highly homogenous population of human primordial germ cell like cells in long-term and feeder-free culture condition

ABSTRACT: This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Homo sapiens

PROVIDER: GSE174485 | GEO | 2021/05/15

REPOSITORIES: GEO

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Expanding highly homogenous population of human primordial germ cell like cells in long-term and feeder-free culture condition [WGBS]

Project description:The culture methods for human primordial germ cell like cells (hPGCLCs) has not been established. Here we report the culture method to expand long-term culture hPGCLCs (LTC-hPGCLCs) representing migrating morphology and early hPGC transcriptome and methylome. Key components of the culture media are stem cell factor, STO-conditioned media and absence of fetal calf serum. In this condition, these LTC-hPGCLCs were highly homogenous population without feeder cells and expanded at least 150 days. They did not express senescent phenotype such as β-galactosidase activity and shortening of telomere length at the 150-day culture, indicating that the cell might be a perpetual cell line. LTC-hPGCLCs still have potent pluripotency indicating the phenotype that the cells become human embryonic germ cells which were distinguishable from human iPS cells in strong DLK1 expression and H19 biallelic expression. In summary this simple culture method and highly homogenous LTC-hPGCLCs would be a useful resource that supports investigations on human germline cells.

2021-05-15 | GSE174484 | GEO

Expanding highly homogenous population of human primordial germ cell like cells in long-term and feeder-free culture condition [RNA-Seq]

2021-05-15 | GSE174465 | GEO

Expanding highly homogenous population of human primordial germ cell like cells in long-term and feeder-free culture condition [scRNA-seq]

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Expanding highly homogenous population of human primordial germ cell like cells in long-term and feeder-free culture condition

Project description:Expanding highly homogenous population of human primordial germ cell like cells in long-term and feeder-free culture condition

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Expanding highly homogenous population of human primordial germ cell like cells in long-term and feeder-free culture condition [WGBS]

Project description:Expanding highly homogenous population of human primordial germ cell like cells in long-term and feeder-free culture condition [WGBS]

| PRJNA730031 | ENA

Expanding highly homogenous population of human primordial germ cell like cells in long-term and feeder-free culture condition [RNA-Seq]

Project description:Expanding highly homogenous population of human primordial germ cell like cells in long-term and feeder-free culture condition [RNA-Seq]

| PRJNA730019 | ENA

Expanding highly homogenous population of human primordial germ cell like cells in long-term and feeder-free culture condition [scRNA-seq]

Project description:Expanding highly homogenous population of human primordial germ cell like cells in long-term and feeder-free culture condition [scRNA-seq]

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Expanding homogeneous culture of human primordial germ cell-like cells maintaining germline features without serum or feeder layers.

Project description:In vitro expansion of human primordial germ cell-like cells (hPGCLCs), a pluripotent stem cell-derived PGC model, has proved challenging due to rapid loss of primordial germ cell (PGC)-like identity and limited cell survival/proliferation. Here, we describe long-term culture hPGCLCs (LTC-hPGCLCs), which actively proliferate in a serum-free, feeder-free condition without apparent limit as highly homogeneous diploid cell populations maintaining transcriptomic and epigenomic characteristics of hPGCLCs. Histone proteomics confirmed reduced H3K9me2 and increased H3K27me3 marks in LTC-hPGCLCs compared with induced pluripotent stem cells (iPSCs). LTC-hPGCLCs established from multiple human iPSC clones of both sexes were telomerase positive, senescence-free cells readily passaged with minimal cell death or deviation from the PGC-like identity. LTC-hPGCLCs are capable of differentiating to DAZL-positive M-spermatogonia-like cells in the xenogeneic reconstituted testis (xrTestis) organ culture milieu as well as efficiently producing fully pluripotent embryonic germ cell-like cells in the presence of stem cell factor and fibroblast growth factor 2. Thus, LTC-hPGCLCs provide convenient access to unlimited amounts of high-quality and homogeneous hPGCLCs.

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Derivation and long-term culture of an embryonic stem cell-like line from zebrafish blastomeres under feeder-free condition.

Project description:Existing zebrafish embryonic stem (ES) cell lines are derived and maintained using feeder layers. We describe here the derivation and long-term culture of an ES cell-like line derived from zebrafish blastomeres without the use of feeder cells. This line, designated as ZES1, has been maintained for more than 800 days in defined Dulbecco's modified Eagle's medium supplemented with fetal bovine serum, zebrafish embryo extract, trout serum, and human basic fibroblast growth factor. ZES1 cells possessed a morphology typical of ES cells, being round or polygonal in shape with a large nucleus and sparse cytoplasm and were mostly diploid. The cells formed individual colonies consisting of tightly packed cells that stained positively for alkaline phosphatase. ZES1 cells also formed embryoid bodies when transferred onto uncoated wells. The pluripotent nature of ZES1 cells was confirmed when they could be induced to differentiate in vitro into several cell types, through low- or high-density culture conditions. Treatment with retinoic acid also induced the differentiation of ZES1 cells into primarily neuronal cells. Using immunostaining and real-time polymerase chain reaction, we showed that Sox2, a known pluripotent marker in mammalian ES cells, was also present in ZES1 cells. Chimera experiments revealed that fluorescent-labeled ZES1 cells microinjected into zebrafish blastulas participated in the formation of all three germ layers. Using GFP-labeled ZES1 cells, chimera germline transmission was also demonstrated at the F1 generation. In conclusion, ZES1 cells possess both in vitro and in vivo pluripotency characteristics, indicating that nonmammalian ES cells can be readily derived and maintained for a long term under feeder-free culture conditions.

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DJ-1 Can Replace FGF-2 for Long-Term Culture of Human Pluripotent Stem Cells in Defined Media and Feeder-Free Condition.

Project description:Conventional human pluripotent stem cell (hPSC) cultures require high concentrations of expensive human fibroblast growth factor 2 (hFGF-2) for hPSC self-renewal and pluripotency in defined media for long-term culture. The thermal instability of the hFGF-2 mandates media change every day, which makes hPSC culture costly and cumbersome. Human DJ-1 (hDJ-1) can bind to and stimulate FGF receptor-1. In this study, for the first time, we have replaced hFGF-2 with hDJ-1 in the essential eight media and maintained the human embryonic stem cells (hESCs), H9, in the defined media at feeder-free condition. After more than ten passages, H9 in both groups still successfully maintained the typical hESC morphology and high protein levels of pluripotency markers, SSEA4, Tra1-60, Oct4, Nanog, and ALP. DNA microarray revealed that more than 97% of the 21,448 tested genes, including the pluripotency markers, Sox2, Nanog, Klf4, Lin28A, Lin28B, and Myc, have similar mRNA levels between the two groups. Karyotyping revealed no chromosome abnormalities in both groups. They also differentiated sufficiently into three germ layers by forming in vitro EBs and in vivo teratomas. There were some variations in the RT-qPCR assay of several pluripotency markers. The proliferation rates and the mitochondria of both groups were also different. Taken together, we conclude that hDJ-1 can replace hFGF-2 in maintaining the self-renewal and the pluripotency of hESCs in feeder-free conditions.

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