Mapping developmental paths of monkey primordial germ-like cell differentiation from pluripotent stem cells by single cell RNA sequencing analysis
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PROVIDER: PRJNA579767 | ENA |
REPOSITORIES: ENA
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Project description:The induction of the primordial germ-like cells (PGCLCs) from primate pluripotent stem cells (PSCs)provides a powerful system to study the cellular and molecular mechanism underlying germline specification, which are difficult to study in vivo. However a comprehensive single cell RNA sequencing(scRNA-seq) analysis of the developmental trajectory of PGCLCs differentiation that also dissects the required niche has not been achieved. Here, we applied the embryoid body (EB) differentiation system to induce PGCLCs specification from male and female monkey naïve-state PSCs and then performed high throughput scRNA-seq analysis of approximately 40000 of naïve cells and cells within EBs. We found that EBs provided a niche for PGCLCs differentiation by secreting growth factors critical for PGCLCs specification, such as BPM2, BMP4 and WNT3. Moreover, the developmental trajectory of PGCLCs was reconstituted and gene expression dynamics were revealed. Our study outlines the roadmap of PGCLCs specification from PSCs and provides insights that will improve the differentiation efficiency of PGCLCs from PSCs.
2021-12-31 | GSE139432 | GEO
Project description:Primordial germ cell like cell competent populations
| PRJEB48665 | ENA
Project description:Primordial germ cells (PGCs), the embryonic precursors of eggs and sperm, are a unique model for identifying and studying regulatory mechanisms in singly migrating cells. From their time of specification to eventual colonization of the gonad, mouse PGCs traverse through and interact with many different cell types, including epithelial cells and mesenchymal tissues. Work in drosophila and zebrafish have identified many genes and signaling pathways involved in PGC migration, but little is known about this process in mammals. We have generated a point mutation in the Ror2 gene that we know disrupts primordial germ cell migration in the developing mouse embryo. We used microarray analysis to determine if this defect is mediated through genome-wide or pathway-specific transcriptional changes. We analyzed primordial germ cells (PGCs) from 4 wild-type (WT) and 4 Ror2Y324C/Y324C mutant embryos using Oct4-DPE-EGFP. PGCs were collected during their active migratory state at embryonic day 9.5 (somite range 20-25).
2014-08-08 | E-GEOD-60179 | biostudies-arrayexpress
Project description:To investigate the role of NRF1 in regulating primordial germ cell development, We established conditional knockout mice of Nrf1 in primordial germ cell to observe the effect of Nrf1 knockout on the development of primordial germ cell. At the same time, we utilized a pluripotent stem cell differentiation system in vitro to obtain PGCL cells for chip_ Seq, analyze which genes Nrf1 directly binds to. Meanwhile, we established a pluripotent stem cell line induced by Nrf1 overexpression and performed RNA_seq analysis on PGCL cells overexpressing Nrf1 obtained in vitro
2023-08-05 | GSE231661 | GEO
Project description:To investigate the role of NRF1 in regulating primordial germ cell development, We established conditional knockout mice of Nrf1 in primordial germ cell to observe the effect of Nrf1 knockout on the development of primordial germ cell. At the same time, we utilized a pluripotent stem cell differentiation system in vitro to obtain PGCL cells for chip_ Seq, analyze which genes Nrf1 directly binds to. Meanwhile, we established a pluripotent stem cell line induced by Nrf1 overexpression and performed RNA_seq analysis on PGCL cells overexpressing Nrf1 obtained in vitro
2023-08-05 | GSE231662 | GEO
Project description:Transgenic StellaGFP ESCs were used to derive primordial germ cells during embryoid body (EB) differentiation, and microarry analysis used to compared FACS sorted Stella-positive cells of day 7 Ebs with the parental ESCs and Stella-negative cells of day 7 Ebs. Keywords: in vitro, primordial germ cells, embryonic stem cells, stella
2009-06-22 | GSE7948 | GEO
Project description:The molecular mechanisms underlying the commitment of cells to the germ cell lineage and the subsequent formation of germ cells during mammalian development remain poorly understood due to an inability to obtain sufficient amounts of cellular materials to conduct thorough in-vitro analyses. Although primordial germ cells (PGCs) have been generated and isolated from embryonic stem cells (ESCs) in vitro, concurrent expression of several cell surface receptors and transcription factors, at various levels, confounds the identification of these cells. To date, only a few genes that mark the onset of germ cell commitment to the epiblast, including tissue nonspecific alkaline phosphatase (TNAP), Blimp1, Stella, and Fragilis, have been used with some degree of success to detect PGC formation in in-vitro model systems. In light of the current literature, the generation of an unlimited supply of germ cells and gametes from pluripotent stem cells would greatly facilitate studies into reproductive development. To accomplish this, we would need to discover novel markers that are specifically expressed in germ cells. Here we identified four genes that are specifically expressed in male and female germ cells, both in vivo and in vitro, but are not expressed in somatic tissues or ESCs. Expression of these genes therefore allows us to distinguish committed germ cells from undifferentiated pluripotent cell populations, a prerequisite for the successful derivation of germ cells and gametes in vitro. For RNA isolation, PGCs were sorted by FACS, collected by centrifugation, lysed in RLT buffer (QIAGEN), and processed using RNeasy Micro kits with on-column DNase digestion as per the manufacturer's instructions. Integrity of RNA samples was quality-checked using a 2100 Bioanalyzer (Agilent). If possible, 300 ng of total RNA per sample was used as starting material for linear amplification (Illumina TotalPrep RNA amplification kit, Ambion), which involved synthesis of T7-linked double-stranded cDNA and 14 hrs of in-vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then quality-checked on a 2100 Bioanalyser, and hybridized as biological or technical duplicates for 18 hrs onto MouseRef-8 v2 gene expression BeadChips (Illumina), following the manufacturer's instructions. After being washed, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader and accompanying software. 44 samples were analyzed. ESCm: OG2 male Embryonic Stem Cell, +/+ Oct4-GFP ES cells, 1 biological rep 11.5: 11.5 embryonic day PGC (Primordial Germ Cell), 2 biological reps 12.5F: 12.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps 13.5F: 13.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps 14.5F: 14.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps 15.5F: 15.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps 16.5F: 16.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps 17.5F: 17.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps 18.5F: 18.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps 12.5M: 12.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps 13.5M: 13.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps 14.5M: 14.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps 15.5M: 15.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps 16.5M: 16.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps 17.5M: 17.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps 18.5M: 18.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps 8M: 8 day after birth male PGC (Primordial Germ Cell), 2 biological reps 10M: 10 day after birth male PGC (Primordial Germ Cell), 2 biological reps 12M: 12 day after birth male PGC (Primordial Germ Cell), 2 biological reps 14M: 14 day after birth male PGC (Primordial Germ Cell), 1 biological rep 16M: 16 day after birth male PGC (Primordial Germ Cell), 2 biological reps 18M: 18 day after birth male PGC (Primordial Germ Cell), 2 biological reps 20M: 20 day after birth male PGC (Primordial Germ Cell), 2 biological reps
2010-11-16 | E-GEOD-23322 | biostudies-arrayexpress
Project description:We sequenced embryoid bodies at two time points (18h, 96h) following a differentiation protocol to induce Primordial Germ Cell-like Cells (PGCLC) in a TFAP2A KO line and parental line
2023-05-14 | E-MTAB-11305 | biostudies-arrayexpress
Project description:Embryo development of post-implantation and gastrulation is a milestone of embryogenesis. However, the fundamental mechanisms underlying this process remain elusive as technological and ethical limitations. Here, we we improved an in vitro culture system that cynomolgus monkey embryos could develop to 20 days without any maternal tissues, which clearly reveal lineage specification, bi-laminar disc generation, amniotic and yolk sac formation, and primordial germ cell like cells (PGCLC). By applying single cell-omics analysis, unsupervised dimensionality reduction and clustering algorithm data show clear, distinct, stepwise development routes for the epiblast, primitive endoderm, trophectoderm and extra-embryonic mesenchyme lineage. We also found development path of PGCLCs, suggesting origin of PGLCs in amnion instead of EPI. Furthermore, signal pathways analysis showed existence of intimate interactions among lineages. By sequencing (scATAC-seq ) assay for transposase-accessible chromatin, we identify transcription factor grammar specifying of each cell type (EPI, PE and TE) and infer potential master transcriptional regulators. Collectively, the results provide the new insight into understanding molecular and cellular dynamics that underlie monkey post-implantation development, especially beyond E13. Our study provides a basic knowledge about regulation of primate pluripotency in vitro for cell replacement therapy.
2020-05-07 | GSE150004 | GEO
Project description:Embryo development of post-implantation and gastrulation is a milestone of embryogenesis. However, the fundamental mechanisms underlying this process remain elusive as technological and ethical limitations. Here, we we improved an in vitro culture system that cynomolgus monkey embryos could develop to 20 days without any maternal tissues, which clearly reveal lineage specification, bi-laminar disc generation, amniotic and yolk sac formation, and primordial germ cell like cells (PGCLC). By applying single cell-omics analysis, unsupervised dimensionality reduction and clustering algorithm data show clear, distinct, stepwise development routes for the epiblast, primitive endoderm, trophectoderm and extra-embryonic mesenchyme lineage. We also found development path of PGCLCs, suggesting origin of PGLCs in amnion instead of EPI. Furthermore, signal pathways analysis showed existence of intimate interactions among lineages. By sequencing (scATAC-seq ) assay for transposase-accessible chromatin, we identify transcription factor grammar specifying of each cell type (EPI, PE and TE) and infer potential master transcriptional regulators. Collectively, the results provide the new insight into understanding molecular and cellular dynamics that underlie monkey post-implantation development, especially beyond E13. Our study provides a basic knowledge about regulation of primate pluripotency in vitro for cell replacement therapy.
2020-05-07 | GSE149974 | GEO