Whole-genome single-cell 5mC sequencing of human primordial germ cell-like cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE138982 | GEO | 2019/10/21
REPOSITORIES: GEO
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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.
2020-01-31 | GSE139115 | GEO
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
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 | GSE174465 | GEO
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.
2021-06-25 | GSE178885 | GEO
Project description:Specification of primordial germ cells (PGCs) marks the beginning of the totipotent state. However, without a tractable experimental model, the mechanism of human PGC (hPGC) specification remains unclear. Here, we demonstrate specification of hPGC-like cells (hPGCLCs) from germline competent pluripotent stem cells. The characteristics of hPGCLCs are consistent with the embryonic hPGCs and a germline seminoma that share a CD38 cell-surface marker, which collectively defines likely progression of the early human germline. Remarkably, SOX17 is the key regulator of hPGC-like fate, whereas BLIMP1 represses endodermal and other somatic genes during specification of hPGCLCs. Notable mechanistic differences between mouse and human PGC specification could be attributed to their divergent embryonic development and pluripotent states, which might affect other early cell-fate decisions. We have established a foundation for future studies on resetting of the epigenome in hPGCLCs and hPGCs for totipotency and the transmission of genetic and epigenetic information. RNA-Seq analysis to investigate transcriptomes of hPGC-like cells (hPGCLCs), fetal hPGCs, TCam-2 and hESCs
2014-12-26 | E-GEOD-60138 | biostudies-arrayexpress
Project description:In human and mouse stem cells and brain, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) can occur outside of CG dinucleotides. Using protein binding microarrays (PBMs) containing 60-mer DNA probes, we evaluated the effect of 5mC and 5hmC on one DNA strand on the double-stranded DNA binding of the mouse B-ZIP transcription factors (TFs) CREB1, ATF1, and JUND. 5mC inhibited CREB1 binding to the canonical CRE half-site |GTCA, but increased binding to the C/EBP half-site |GCAA. 5hmC inhibited CREB1 binding to all 8-mers except TGAT|GCAA, where binding is enhanced. We observed similar DNA binding patterns with the closely related TF: ATF1. In contrast, both 5mC and 5hmC inhibited binding of JUND. These results identify new DNA sequences that are well-bound by CREB1 and ATF1 only when they contain 5mC or 5hmC. Analysis of two x-ray structures examines the consequences of 5mC and 5hmC on DNA binding by CREB and FOS|JUN.
2016-10-19 | GSE88897 | GEO
Project description:The Epstein-Barr virus (EBV) bZIP transcription factor (TF) Zta is a sequence-specific DNA binding protein that recognizes both unmethylated and methylated DNA. To study the contribution of the conserved N182 amino acid to sequence specific Zta DNA binding, we replaced it with five other amino acids: serine (S), glutamine (Q), threonine (T), isoleucine (I), or valine (V). We used protein binding microarrays (PBMs) to evaluate sequence-specific DNA binding to four types of double-stranded DNA: 1) DNA with cytosine in both strands (DNA(C|C), 2) DNA with 5-methylcytosine (5mC, M) in one strand and cytosine in the second strand (DNA(5mC|C)), 3) DNA with 5-hydroxymethylcytosine (5hmC, H) in one strand and cytosine in the second strand (DNA(5hmC|C)), and 4) DNA with methylated cytosine in both strands in all CG dinucleotides (DNA(5mCG)). With unmethylated DNA, Zta(N182S) binds variants of the consensus TRE (TGA-G/C-TCA) motif, such as TGA-G/C-TGA and TCA-G/C-TGA where C at position 3 is replaced with G in one or both half sites of the motif. Zta(N182S) also binds stronger to DNAs containing modified cytosines compared to wildtype. Zta(N182Q) binds new sequences containing GTAA with DNA(C|C), DNA(5mC|C) and DNA(5hmC|C) where C at position 3 is replaced with A. Zta(N182I) and Zta(N182V) bind sequence specifically to DNA(5mC|C), and weakly with all other types of DNA examined. Zta(N182T) DNA binding is weaker to all types of DNA examined. Our data highlight that mutation of ZtaN182 with the hydrophilic amino acids serine and glutamine alters Zta sequence specific DNA binding, while mutation with hydrophobic amino acids (I and V) increases binding to DNA(5mC|C).
2019-06-04 | GSE115351 | GEO
Project description:In vitro gametogenesis is the process of making germline cells from human pluripotent stem cells. The foundation of this model is the quality of the first progenitors called primordial germ cells (PGCs), which in vivo are specified during the peri-implantation window of human development. Here, we show using human embryo attachment culture that hPGC specification begins at day 12 post fertilization. Using single cell RNA-sequencing of hPGC-like cells (hPGCLCs) differentiated from pluripotent stem cells we discovered that hPGCLC specification involves resetting pluripotency towards a transitional state with shared characteristics between naïve and primed pluripotency followed by differentiation into lineage primed TFAP2A+ progenitors. Applying the germline trajectory to TFAP2C mutants reveals that TFAP2C functions in the TFAP2A+ progenitors upstream of PRDM1 to regulate the expression of SOX17. This serves to protect hPGCLCs from crossing the Weismann’s barrier to adopt somatic cell fates and therefore is an essential mechanism for successfully initiating in vitro gametogenesis.
2019-12-29 | GSE140021 | GEO
Project description:Oxidized 5mC modifications in fungi
| PRJNA769605 | ENA
Project description:Specification of primordial germ cells (PGCs) marks the beginning of the totipotent state. However, without a tractable experimental model, the mechanism of human PGC (hPGC) specification remains unclear. Here, we demonstrate specification of hPGC-like cells (hPGCLCs) from germline competent pluripotent stem cells. The characteristics of hPGCLCs are consistent with the embryonic hPGCs and a germline seminoma that share a CD38 cell-surface marker, which collectively defines likely progression of the early human germline. Remarkably, SOX17 is the key regulator of hPGC-like fate, whereas BLIMP1 represses endodermal and other somatic genes during specification of hPGCLCs. Notable mechanistic differences between mouse and human PGC specification could be attributed to their divergent embryonic development and pluripotent states, which might affect other early cell-fate decisions. We have established a foundation for future studies on resetting of the epigenome in hPGCLCs and hPGCs for totipotency and the transmission of genetic and epigenetic information.
2014-12-26 | GSE60138 | GEO