Germline competency of human embryonic stem cells depends on eomesodermin
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE93126 | GEO | 2018/11/10
REPOSITORIES: GEO
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Project description:The successful segregation of germ cells from somatic lineages is vital for sexual reproduction and species survival. In the mouse, primordial germ cells (PGCs), precursors of all germ cells, are induced from the post-implantation epiblast. Induction requires BMP4 signalling to prospective PGCs and the intrinsic action of PGC transcription factors (TFs). However, the molecular mechanisms connecting BMP4 action to induction of PGC TFs that are responsible for segregation of PGCs from somatic lineages are unknown. Here we show that the transcription factor OTX2 is a key regulator of these processes. Down-regulation of Otx2 precedes the initiation of the PGC programme both in vitro and in vivo. Deletion of Otx2 in vitro dramatically increases PGCLC differentiation efficiency and prolongs the period of PGC competence. In the absence of Otx2 activity, PGCLC differentiation becomes independent of the otherwise essential cytokine signals, with germline entry initiating even in the absence of the PGC TF Blimp1. Deletion of Otx2 in vivo increases PGC numbers. These data demonstrate that Otx2 acts repressively upstream of PGC TFs and functions as a roadblock to prevent the untimely entry of pluripotent stem cells into the PGC lineage, thereby ensuring correct spatio-temporal segregation of the germline and soma.
2018-07-06 | GSE116640 | GEO
Project description:The DND microRNA-mediated repression inhibitor 1 (DND1) is a conserved RNA binding protein (RBP) and plays an important role in survival and maintenance of primordial germ cells (PGCs) and the development of the male germline in zebrafish and mice. It was shown to be expressed in human pluripotent stem cells (PSCs), PGCs, and spermatogonia, but little is known about its specific role in pluripotency and human germline development. Here we use CRISPR/Cas mediated knockout and PGC-like cell (PGCLC) differentiation in human iPSCs to analyse if DND1 (1) plays a role in maintaining pluripotency and (2) in specification of PGCLCs. We generated several clonal lines with biallelic loss of function mutations and analysed their potential to differentiate towards PGCLCs and their gene expression on RNA and protein level via bulk RNA sequencing and mass spectrometry. The generated knockout iPSCs showed no differences in pluripotency gene expression, proliferation nor trilineage differentiation potential, but yielded reduced numbers o PGCLCs compared to their parental iPSCs. RNAseq analysis in PGCLCs showed significantly reduced expression of genes associated with cellular developmental processes and cell differentiation in knockout cells, including known markers for PGCs (NANOS3, SOX17, PRDM1, EPCAM) and naïve pluripotency (TFCP2L, DNMT3L).
2021-12-10 | PXD023802 | Pride
Project description:Generating primordial germ cells (PGCs) from human pluripotent stem cells (hPSCs) advances studies of human reproduction and development of infertility treatments, but currently entails complex 3D aggregates. Here we develop a simplified, monolayer method to differentiate hPSCs into PGCs within 3.5 days, with higher efficiencies and improved consistency across multiple hPSC lines. We used our simplified differentiation platform and single-cell RNA-sequencing to uncover new insights into PGC specification. Transient WNT activation for 12 hours followed by WNT inhibition specified PGCs; by contrast, sustained WNT instead induced primitive streak. Thus, somatic (primitive streak) and PGCs are related—yet distinct—lineages segregated by temporally-dynamic signaling. Pluripotency factors are continuously expressed during the transition from pluripotency to posterior epiblast to PGCs, thus bridging pluripotent and germline states. Finally, hPSC-derived PGCs can be easily purified by virtue of their CXCR4+PDGFRA-GARP- surface-marker profile and single-cell RNA-sequencing reveals that they harbor strong transcriptional similarities with fetal PGCs. We report single-cell RNA-sequencing data of in vitro differentiated hPSCs (H9) cells into human Primordial germ cell-like cells (PGCLCs), profiled at different time-points of differentiation - Day 0 (H9), D0.5, D3.5 (sorted) and D3.5 unsorted population and a negative control of hPSCs differentiated into definitive endoderm (DE) (using the protocol described in (Loh et al., 2014, Cell Stem Cell 14, 237-252)). The data comprises of 5 single-cell RNA-sequencing libraries (H9, D0.5, D3.5S, D3.5U and DE) generated using 10xChromium Single cell RNA-expression platform and 10xGenomics Chromium version 2 chemistry with a targeted capture of ~6000 cells each. Single cell libraries were sequenced on Illumina HiSeq4000 platform as paired-end 150bp reads.
2023-08-09 | GSE157475 | GEO
Project description:Generating primordial germ cells (PGCs) from human pluripotent stem cells (hPSCs) advances studies of human reproduction and development of infertility treatments, but currently entails complex 3D aggregates. Here we develop a simplified, monolayer method to differentiate hPSCs into PGCs within 3.5 days, with higher efficiencies and improved consistency across multiple hPSC lines. We used our simplified differentiation platform, bulk RNA-seq and single-cell RNA-sequencing to uncover new insights into PGC specification. Transient WNT activation for 12 hours followed by WNT inhibition specified PGCs; by contrast, sustained WNT instead induced primitive streak. Thus, somatic (primitive streak) and PGCs are related—yet distinct—lineages segregated by temporally-dynamic signaling. Pluripotency factors are continuously expressed during the transition from pluripotency to posterior epiblast to PGCs, thus bridging pluripotent and germline states. Finally, hPSC-derived PGCs can be easily purified by virtue of their CXCR4+PDGFRA-GARP- surface-marker profile and single-cell RNA-sequencing reveals that they harbor strong transcriptional similarities with fetal PGCs. We report single-cell RNA-sequencing data of in vitro differentiated hPSCs (H9) cells into human Primordial germ cell-like cells (PGCLCs), profiled at different time-points of differentiation - Day 0 (H9), D0.5, D3.5 (sorted) and D3.5 unsorted population and a negative control of hPSCs differentiated into definitive endoderm (DE) (using the protocol described in (Loh et al., 2014, Cell Stem Cell 14, 237-252)). The data comprises of 5 single-cell RNA-sequencing libraries (H9, D0.5, D3.5S, D3.5U and DE) generated using 10xChromium Single cell RNA-expression platform and 10xGenomics Chromium version 2 chemistry with a targeted capture of ~6000 cells each. Single cell libraries were sequenced on Illumina HiSeq4000 platform as paired-end 150bp reads. Bulk RNA seq libraries were sequnced as paired-end reads.
2023-08-09 | GSE210711 | GEO
Project description:Early mammalian development entails a series of cell fate transitions that includes transit through naïve pluripotency to post-implantation epiblast. This can subsequently give rise to primordial germ cells (PGC), the founding population of the germline lineage. To investigate the gene regulatory networks that control these critical cell fate decisions, we developed a compound-reporter system to track cellular identity in a model of PGC specification (PGC-like cells; PGCLC), and coupled it with unbiased genome-wide CRISPR screening. This enabled identification of key genes both for exit from pluripotency and for acquisition of PGC fate, with further characterisation revealing a central role for the transcription regulators Nr5a2 and Zfp296 in germline ontogeny. Abrogation of these genes results in significantly impaired PGCLC development accompanied with widespread activation (Nr5a2-/-) or inhibition (Zfp296-/-) of WNT pathway components. This leads to aberrant upregulation of the somatic programme or failure to appropriately activate germline genes in PGCLC, respectively, and consequently loss of germ cell identity. Overall our study places Zfp296 and Nr5a2 as key components of an expanded PGC gene regulatory network, and outlines a transferable strategy for identifying critical regulators of complex cell fate transitions.
2018-10-01 | GSE117473 | GEO
Project description:The nuanced mechanisms driving primordial germ cells (PGC) specification remain incompletely understood since genome-wide transcriptional regulation in developing PGCs has previously only been defined indirectly. Here, using SLAMseq analysis, we determined genome-wide transcription rates during the differentiation of embryonic stem cells (ESCs) to form epiblast-like (EpiLC) cells and ultimately PGC-like cells (PGCLCs). This revealed thousands of genes undergoing bursts of transcriptional induction and rapid shut-off not detectable by RNAseq analysis. Our SLAMseq datasets also allowed us to infer RNA turnover rates, which revealed thousands of mRNAs stabilized and destabilized during PGCLC specification. mRNAs tend to be unstable in ESCs and then are progressively stabilized as they differentiate. For some classes of genes, mRNA turnover regulation collaborates with transcriptional regulation, but these processes opposed each other in a surprisingly high frequency of genes. To test whether regulated mRNA turnover has a physiological role in PGC development, we examined 3 genes that we found were regulated by RNA turnover: Sox2, Klf2, and Ccne1. Circumvention of their regulated RNA turnover severely impaired the ESC-to-EpiLC and EpiLC-to-PGCLC transitions. Our study demonstrates the functional importance of regulated RNA stability in germline development and provides a roadmap of transcriptional and post-transcriptional regulation during germline specification.
2022-06-10 | GSE189543 | GEO
Project description:Xenopus primordial germ cells (PGCs) are determined by the presence of maternally derived germ plasm. Germ plasm components both protect PGCs from somatic differentiation and begin a unique gene expression program. Segregation of the germline from the endodermal lineage occurs during gastrulation and PGCs subsequently initiate zygotic transcription. However, the gene-networks that operate to both preserve the potential for totipotency and promote germline differentiation are poorly understood. Here, we utilized RNA-sequencing analysis to comprehensively interrogate PGC and neighboring endoderm cell RNAs after lineage segregation. We identified 1,865 transcripts enriched in PGCs compared to endoderm cells. Over 50% of maternal, vegetally-enriched transcripts were enriched in the PGC transcriptome, including sox7. PGC-directed sox7 knockdown and over-expression studies revealed an early requirement for sox7 in proper germ plasm localization, zygotic transcription, and PGC number. We identified oct60 as the most highly expressed and enriched OCT3/4 homologue in PGCs. Lastly, we compared the Xenopus PGC transcriptome with human PGC transcripts and showed that 80% of transcripts are conserved, identifying Xenopus as a relevant model system for understanding the gene-networks necessary for human germline development.
2017-08-01 | GSE102047 | GEO
Project description:Generation of haploid gametes in vitro can potentially address gamete failure-based infertility.This study reports complete in vitro meiosis from murine ESC-derived PGCLCs resulting in the formation of male spermatid-like cells (SLCs) capable of producing viable fertile offspring via intracytoplasmic sperm injection (ICSI).Our findings provide the basis for generation of haploid spermatids in vitro in human, the generation of transgenic animals, and the use of this system to investigate mechanisms of meiosis. We used microarrays to compare gene expression profiles of in vivo and in vitro derived PGC cells and round spermatids. We collected E12.5 male fatal PGCs, PGCLC in vitro, round spermatids and spermatids like cells produced in vitro, each sample has 3 replications.
2016-02-26 | E-GEOD-71478 | biostudies-arrayexpress
Project description:Analysis of genes enriched in MIXL1+ sorted cells during primitive streak induction identified Apelin receptor (APLNR), a highly conserved member of the G-protein coupled receptor family. Depending upon the growth factor conditions used for differentiation, APLNR+ cells identified both posterior mesodermal and anterior mesendodermal components of the primitive streak. APLNR+ cells isolated from mesodermal differentiated cultures were enriched in hematopoietic blast colony forming cells (Bl-CFCs) and the addition of Apelin peptide enhanced the frequency and growth of both hematopoietic colonies and hESC-derived endothelial cells. These studies identified APLNR as a marker of primitive streak-like cells differentiated from hESCs and defined a novel role for Apelin as a regulator of early human hematopoiesis.
2012-02-12 | E-MEXP-3380 | biostudies-arrayexpress
Project description:Germline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naïve pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1 deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions.
2020-07-27 | GSE155069 | GEO