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Human Primordial Germ Cell Heterogeneity in vitro is Associated with Distinctive Biological States [WGBS]

ABSTRACT: Primordial germ cells (PGCs) exhibit molecular heterogeneity during development, but whether this reflects distinct biological states remains unclear. Using human PGC-like cells (hPGCLCs) in extended culture with dual fluorescent reporters for TFAP2C and DND1, we investigated the functional significance of DND1 expression heterogeneity. Single-cell RNA sequencing revealed DND1+ hPGCLCs are enriched in RNA regulatory genes including those associated with P-bodies and translational control. DND1+ cells exhibited reduced proliferation, cell cycle exit, and elevated P27 (CDKN1B) expression compared to DND1- cells. Whole-genome bisulfite sequencing demonstrated enhanced DNA demethylation in DND1+ cells, particularly at gene bodies. DND1+ hPGCLCs showed stable expression of male germ cell differentiation markers including PIWIL1, PIWIL2, and DMRT1. BMP2 supplementation increased DND1 translation without affecting mRNA levels or proliferation rates. These findings demonstrate that DND1 marks a post-transcriptionally regulated developmental state in human germ cells, highlighting translational control mechanisms in germline development.

ORGANISM(S): Homo sapiens

PROVIDER: GSE313386 | GEO | 2026/09/17

REPOSITORIES: GEO

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Human Primordial Germ Cell Heterogeneity in vitro is Associated with Distinctive Biological States [scRNA-seq]

Project description:Primordial germ cells (PGCs) exhibit molecular heterogeneity during development, but whether this reflects distinct biological states remains unclear. Using human PGC-like cells (hPGCLCs) in extended culture with dual fluorescent reporters for TFAP2C and DND1, we investigated the functional significance of DND1 expression heterogeneity. Single-cell RNA sequencing revealed DND1+ hPGCLCs are enriched in RNA regulatory genes including those associated with P-bodies and translational control. DND1+ cells exhibited reduced proliferation, cell cycle exit, and elevated P27 (CDKN1B) expression compared to DND1- cells. Whole-genome bisulfite sequencing demonstrated enhanced DNA demethylation in DND1+ cells, particularly at gene bodies. DND1+ hPGCLCs showed stable expression of male germ cell differentiation markers including PIWIL1, PIWIL2, and DMRT1. BMP2 supplementation increased DND1 translation without affecting mRNA levels or proliferation rates. These findings demonstrate that DND1 marks a post-transcriptionally regulated developmental state in human germ cells, highlighting translational control mechanisms in germline development.

2026-09-17 | GSE313385 | GEO

Human Primordial Germ Cell Heterogeneity in vitro is Associated with Distinctive Biological States [RNA-seq]

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RNA-binding proteins DND1 and NANOS3 coordinately suppress SOX4 in processing bodies for restricting the entry of germ cell lineage

Project description:Specification of germ cell fate establishes the germline development during early embryogenesis, yet the underlying mechanisms remain largely unknown in humans. Here we focus on the functional roles of the RNA-binding protein (RBP) DND1 in human germline specification. We deleted the whole genomic region of DND1 in human embryonic stem cells (hESCs), based on which we generated human primordial germ cell-like cells (hPGCLCs). Interestingly, we discovered an increased percentage of hPGCLCs induced from DND1 deleted hESCs, suggesting that DND1 may restrict the specification of human germ cell lineage. Mechanistic investigation reveals that DND1 forms a complex with another RBP NANOS3, in which DND1 facilitates the binding of NANOS3 to their target mRNAs. Furthermore, by analyzing the mRNAs bound by DND1 and NANOS3, we identified SOX4 mRNAs as the key downstream factor for DND1 and NANOS3 complex to restrict the induction of hPGCLCs. Interestingly, DND1 and NANOS3 function in processing bodies (P-bodies) to repress the translation of SOX4 mRNAs, where NANOS3 bridges the interaction between DND1 and the translational repressor 4E-T. Altogether, these findings identify the RBPs DND1 and NANOS3 as “break system” to restrict the entry of germ cell fate in humans.

2025-02-01 | GSE268946 | GEO

Piwi proteins and piRNAs in mammalian oocytes and early embryos

Project description:Germ cells of most animals critically depend on piRNAs and Piwi proteins. Surprisingly, piRNAs in mouse oocytes are relatively rare and dispensable. We present compelling evidence for strong Piwi-piRNA expression in oocytes of other mammals. Human fetal oocytes express PIWIL2 and transposon-enriched piRNAs. Oocytes in adult human ovary express PIWIL1 and PIWIL2, while those in bovine ovary just express PIWIL1. In human, macaque and bovine ovaries we find piRNAs that resemble testis-borne pachytene piRNAs. Isolated bovine follicular oocytes were shown to contain abundant, relatively short piRNAs that preferentially target transposable elements. Using label-free quantitative proteome analysis we show that these maturing oocytes strongly and specifically express the thus-far uncharacterized PIWIL3 protein, alongside other known piRNA-pathway components. In bovine early embryos these piRNAs are still abundant, revealing a potential impact of piRNAs on mammalian embryogenesis. Our results reveal unexpected, highly dynamic piRNA pathways in mammalian oocytes and early embryos. Analyses of multiple small RNA libraries obtained from fetal/adult oocytes, cumulus cells, ovary, testis and 2-4 cell stage ivf embryos of multiple mammalian species.

2015-03-27 | E-GEOD-64942 | biostudies-arrayexpress

Piwi proteins and piRNAs in mammalian oocytes and early embryos

2015-03-27 | GSE64942 | GEO

The RNA Binding Protein Dead End1 Is Differentially Expressed in Male Germ Cells During Late Gestation and Targets Chromatin Modifiers and Translational Machinery

Project description:DND1 is essential to maintain germ cell identity. Loss of Dnd1 function results in trans-differentiation of germ cells to somatic fates in zebrafish or the formation of teratomas in mice. To explore the mechanistic role of DND1, we recently developed a transgenic mouse line in which a functional fusion protein between DND1 and GFP is expressed from the endogenous locus (Dnd1GFP). Surprisingly, we found that this reporter distinguishes two male germ cell populations (MGCs) during late gestation cell cycle arrest (G0). Most MGCs express low levels of DND1-GFP, but 5-12% of the population express high levels of DND1-GFP. An RNA-seq time course during late gestation revealed that Dnd1 transcript levels as well as transcript levels for multiple epigenetic regulators are 5-10-fold higher in DND1-GFP-hi cells. Furthermore, using antibodies against DND1-GFP for RNA immunoprecipitation (RIP) time course sequencing during late gestation, we identified multiple epigenetic and translational regulators that are binding targets of DND1 during G0. Among these targets are DNA methyltransferases (Dnmts), the enzyme Setdb1 that imposes the nuclear lamina associated repressive histone mark (H3K9me3), five Tudor domain proteins (Tdrds), four actin dependent regulators (Smarcs), and a group of ribosomal and Golgi proteins. These data suggest that DND1 binds to transcripts of a group of epigenetic enzymes and gates their translation during MGC G0 arrest in late gestation.

2022-10-18 | GSE197610 | GEO

Reconstitution of human fetal ovaries reveals niche requirements for primordial germ cell-like cell progression

Project description:Human primordial germ cell-like cells (hPGCLCs) can be specified from human induced pluripotent stem cells (hiPSCs), offering a valuable model for human germ cell development. However, further maturation steps of hPGCLCs rely on mouse feeders, or co-culture with mouse gonadal somatic cells. Exposure of hPGCLCs to human embryonic niche has not been attempted. Here, we co-cultured female hPGCLCs in two distinct somatic compartments. In reconstituted ovary (rOv) culture, human fetal germ cells proliferate and initiate meiosis, while hPGCLCs upregulate gonadal germ cell markers such as DDX4. Additionally, hPGCLCs can be supported in 3D culture by the amnion-like cells (AMLC) generated during PGCLC differentiation. Compared to rOV, hPGCLCs are less prone to dedifferentiation in PGCLC/AMLC aggregates. Finally, we demonstrated that SCF is crucial for the survival of hPGCLCs but not second trimester fetal germ cells. Together, this work highlights a shift in niche is required in human germ cell development.

2026-03-02 | GSE309100 | GEO

SOX17 Is a Critical Specifier of Human Primordial Germ Cell Fate

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

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Project description:Piwi proteins are a subfamily of Argonaute proteins that maintain germ cells in eukaryotes. However, the role of their human homologues in cancer stem cells and more broadly in cancer is poorly understood. Here, we report that the Piwi-like family members, including Piwil1 (Hiwi), are overexpressed in glioblastoma (GBM), with Piwil1 levels most frequently elevated. Piwil1 is enriched in glioma stem cells (GSCs) and helps to maintain their self-renewal. GSCs were transduces with control non-targeting shRNAs (shNT) and shPiwil1 (#1 and #2) and global gene expression was analyzed to identify Piwil1 downscream singalings.

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Dmrt1 (doublesex and mab-3 related transcription factor 1) knockout expression analyses in E13.5 testes in S6 background

Project description:Dmrt1 (doublesex and mab-3 related transcription factor 1) is a conserved transcriptional regulator of male differentiation required for testicular development in vertebrates. In mice of the 129Sv strain, loss of Dmrt1 causes a high incidence of teratomas. Mutant 129Sv germ cells undergo apparently normal differentiation up to embryonic day 13.5 (E13.5), but some cells fail to arrest mitosis and ectopically express pluripotency markers. Expression analysis and chromatin immunoprecipitation identified DMRT1 target genes whose misexpression may underly teratoma formation. Comparison of E13.5 testes gene expression between Dmrt1(+/-) and Dmrt1(-,-) animals that show high incidence of teratoma formation.

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