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An Extended Culture System that Supports Human Primordial Germ Cell-like Cell Survival and Initiation of DNA Methylation Erasure.

ABSTRACT: The development of an in vitro system in which human primordial germ cell-like cells (hPGCLCs) are generated from human pluripotent stem cells (hPSCs) has been invaluable to further our understanding of human primordial germ cell (hPGC) specification. However, the means to evaluate the next fundamental steps in germ cell development have not been well established. In this study we describe a two dimensional extended culture system that promotes proliferation of specified hPGCLCs, without reversion to a pluripotent state. We demonstrate that hPGCLCs in extended culture undergo partial epigenetic reprogramming, mirroring events described in hPGCs in vivo, including a genome-wide reduction in DNA methylation and maintenance of depleted H3K9me2. This extended culture system provides a new approach for expanding the number of hPGCLCs for downstream technologies, including transplantation, molecular screening, or possibly the differentiation of hPGCLCs into gametes by in vitro gametogenesis.

SUBMITTER: Gell JJ

PROVIDER: S-EPMC7066331 | biostudies-literature |

REPOSITORIES: biostudies-literature

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An ExtendedCulture System that Supports Human Primordial Germ Cell-Like Cells Survival and Initiation of DNA Methylation Erasure

Project description:In this study, we evalauted the transcriptionl profiles of human primordial germ cell-like cell (hPGCLCs) cultured for an extended period on STO feeders in FR10 media for an additional 10 days (D4C10). The hPGCLCs were obtained from two different human embryonic stem cell line (hESCs), UCLA 1 (XX) and UCLA2 (XY) for the RNA sequencing data. For whole genome bisulfite sequencing, we evaluate primed UCLA2 hESCs, directly out of the aggragate hPGCLCs (Day 4= D4) and extended culture hPGCLCs D4C10. Our D4C10 hPGCLCs were compared to prior RNA-sequencing data generated in our laboraty, which revealed that D4C10 hPGCLCs retain germline identy and closely resemble D4 hPGCLCs at the transcriptional level. Our WGBS data revealed that extended culture hPGCLCs at D4C10 have a decrease in CG methlyation, as compared to the starting hESCs and D4 hPGCLCs.

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Global profiling of DNA methylation erasure in mouse primordial germ cells.

Project description:Epigenetic reprogramming, characterized by loss of cytosine methylation and histone modifications, occurs during mammalian development in primordial germ cells (PGCs), yet the targets and kinetics of this process are poorly characterized. Here we provide a map of cytosine methylation on a large portion of the genome in developing male and female PGCs isolated from mouse embryos. We show that DNA methylation erasure is global and affects genes of various biological functions. We also reveal complex kinetics of demethylation that are initiated at most genes in early PGC precursors around embryonic day 8.0-9.0. In addition, besides intracisternal A-particles (IAPs), we identify rare LTR-ERV1 retroelements and single-copy sequences that resist global methylation erasure in PGCs as well as in preimplantation embryos. Our data provide important insights into the targets and dynamics of DNA methylation reprogramming in mammalian germ cells.

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Genome-wide erasure of DNA methylation in mouse primordial germ cells is affected by AID deficiency.

Project description:Epigenetic reprogramming including demethylation of DNA occurs in mammalian primordial germ cells (PGCs) and in early embryos, and is important for the erasure of imprints and epimutations, and the return to pluripotency. The extent of this reprogramming and its molecular mechanisms are poorly understood. We previously showed that the cytidine deaminases AID and APOBEC1 can deaminate 5-methylcytosine in vitro and in Escherichia coli, and in the mouse are expressed in tissues in which demethylation occurs. Here we profiled DNA methylation throughout the genome by unbiased bisulphite next generation sequencing in wild-type and AID-deficient mouse PGCs at embryonic day (E)13.5. Wild-type PGCs revealed marked genome-wide erasure of methylation to a level below that of methylation deficient (Np95(-/-), also called Uhrf1(-/-)) embryonic stem cells, with female PGCs being less methylated than male ones. By contrast, AID-deficient PGCs were up to three times more methylated than wild-type ones; this substantial difference occurred throughout the genome, with introns, intergenic regions and transposons being relatively more methylated than exons. Relative hypermethylation in AID-deficient PGCs was confirmed by analysis of individual loci in the genome. Our results reveal that erasure of DNA methylation in the germ line is a global process, hence limiting the potential for transgenerational epigenetic inheritance. AID deficiency interferes with genome-wide erasure of DNA methylation patterns, indicating that AID has a critical function in epigenetic reprogramming and potentially in restricting the inheritance of epimutations in mammals.

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Project description:The genome-wide depletion of 5-methylcytosines (5meCs) caused by passive dilution through DNA synthesis without daughter strand methylation and active enzymatic processes resulting in replacement of 5meCs with unmethylated cytosines is a hallmark of primordial germ cells (PGCs). Although recent studies have shown that in vitro differentiation of pluripotent stem cells (PSCs) to PGC-like cells (PGCLCs) mimics the in vivo differentiation of epiblast cells to PGCs, how DNA methylation status of PGCLCs resembles the dynamics of 5meC erasure in embryonic PGCs remains controversial. Here, by differential detection of genome-wide 5meC and 5-hydroxymethylcytosine (5hmeC) distributions by deep sequencing, we show that PGCLCs derived from mouse PSCs recapitulated the process of genome-wide DNA demethylation in embryonic PGCs, including significant demethylation of imprint control regions (ICRs) associated with increased mRNA expression of the corresponding imprinted genes. Although 5hmeCs were also significantly diminished in PGCLCs, they retained greater amounts of 5hmeCs than intragonadal PGCs. The genomes of both PGCLCs and PGCs selectively retained both 5meCs and 5hmeCs at a small number of repeat sequences such as GSAT_MM, of which the significant retention of bisulfite-resistant cytosines was corroborated by reanalysis of previously published whole-genome bisulfite sequencing data for intragonadal PGCs. PSCs harboring abnormal hypermethylation at ICRs of the Dlk1-Gtl2-Dio3 imprinting cluster diminished these 5meCs upon differentiation to PGCLCs, resulting in transcriptional reactivation of the Gtl2 gene. These observations support the usefulness of PGCLCs in studying the germline epigenetic erasure including imprinted genes, epimutations, and erasure-resistant loci, which may be involved in transgenerational epigenetic inheritance.

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An ExtendedCulture System that Supports Human Primordial Germ Cell-Like Cells Survival and Initiation of DNA Methylation Erasure

Project description:An ExtendedCulture System that Supports Human Primordial Germ Cell-Like Cells Survival and Initiation of DNA Methylation Erasure

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Oct4 is required for primordial germ cell survival.

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Active DNA demethylation is required for complete imprint erasure in primordial germ cells.

Project description:In mammalian primordial germ cells (PGCs), DNA demethylation is indispensible for parental imprint erasure, which is a reprogramming process essential for normal developmental potential. Thus, it is important to elucidate how DNA demethylation occurs in each imprinted region in PGCs and to determine which DNA demethylation pathway, passive or active, essentially contributes to the erasure of the imprint. Here, we report that active DNA demethylation via a putative Poly(ADP-ribose) polymerase (PARP) pathway is involved in H19-DMR imprint erasure in PGCs, as shown by an in vivo small molecule inhibitor assay. To the best of our knowledge, this is the first direct demonstration of a DNA replication-independent active DNA demethylation pathway in the erasure process of genomic imprinting in PGCs in vivo. The data also suggest that active DNA demethylation plays a significant role in the complete erasure of paternal imprinting in the female germ line.

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Long-term in vitro culture and preliminary establishment of chicken primordial germ cell lines.

Project description:Primordial germ cells (PGCs) are precursors of functional gametes and can be used as efficient transgenic tools and carriers in bioreactors. Few methods for long-term culture of PGCs are available. In this study, we tested various culture conditions for PGCs, and used the optimum culture system to culture chicken gonad PGCs for about three hundred days. Long-term-cultured PGCs were detected and characterized by karyotype analysis, immunocytochemical staining of SSEA-1, c-kit, Sox2, cDAZL, and quantitative RT-PCR for specific genes like Tert, DAZL, POUV, and NANOG. Cultured PGCs labeled with PKH26 were reinjected into Stage X recipient embryos and into the dorsal aorta of Stage 14-17 embryos to assay their ability of migration into the germinal crescent and gonads, respectively. In conclusion, the most suitable culture system for PGCs is as follows: feeder layer cells treated with 20 μg/mL mitomycin C for 2 hours, and with 50% conditioned medium added to the factor culture medium. PGCs cultured in this system retain their pluripotency and the unique ability of migration without transformation, indicating the successful preliminary establishment of chicken primordial germ cell lines and these PGCs can be considered for use as carriers in transgenic bioreactors.

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