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Cyclosporin A and FGF signaling support the proliferation/survival of mouse primordial germ cell-like cells in vitro [bis-seq]

ABSTRACT: Primordial germ cells (PGCs) are the founding population of the germ cell lineage that undergo a multistep process to generate spermatozoa or oocytes. Establishing an appropriate culture system for PGCs is a key challenge in reproductive biology. By a chemical screening using mouse PGC-like cells (mPGCLCs), which were induced from mouse embryonic stem cells, we reported previously that forskolin and rolipram synergistically enhanced the proliferation/survival of mPGCLCs with an average expansion rate of ∼20-fold. In the present study, we evaluated other chemicals or cytokines to see whether they would improve the current mPGCLC culture system. Among the chemicals and cytokines examined, in the presence of forskolin and rolipram, cyclosporin A (CsA) and fibroblast growth factors (FGFs: FGF2 and FGF10) effectively enhanced the expansion of mPGCLCs in vitro (∼50-fold on average). During the expansion by CsA or FGFs, mPGCLCs comprehensively erased their DNA methylation to acquire a profile equivalent to that of gonadal germ cells in vivo, while maintaining their highly motile phenotype as well as their transcriptional properties as sexually uncommitted PGCs. Importantly, these mPGCLCs robustly contributed to spermatogenesis and produced fertile offspring. Furthermore, mouse PGCs (mPGCs) cultured with CsA ex vivo showed transcriptomes and DNA methylomes similar to those of cultured mPGCLCs. The improved culture system for mPGCLCs/mPGCs would be instructive for addressing key questions in PGC biology, including the mechanisms for germ cell migration, epigenetic reprogramming, and sex determination of the germline.

ORGANISM(S): Mus musculus

PROVIDER: GSE151445 | GEO | 2021/02/16

REPOSITORIES: GEO

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Cyclosporin A and FGF signaling support the proliferation/survival of mouse primordial germ cell-like cells in vitro [RNA-seq]

Project description:Primordial germ cells (PGCs) are the founding population of the germ cell lineage that undergo a multistep process to generate spermatozoa or oocytes. Establishing an appropriate culture system for PGCs is a key challenge in reproductive biology. By a chemical screening using mouse PGC-like cells (mPGCLCs), which were induced from mouse embryonic stem cells, we reported previously that forskolin and rolipram synergistically enhanced the proliferation/survival of mPGCLCs with an average expansion rate of ∼20-fold. In the present study, we evaluated other chemicals or cytokines to see whether they would improve the current mPGCLC culture system. Among the chemicals and cytokines examined, in the presence of forskolin and rolipram, cyclosporin A (CsA) and fibroblast growth factors (FGFs: FGF2 and FGF10) effectively enhanced the expansion of mPGCLCs in vitro (∼50-fold on average). During the expansion by CsA or FGFs, mPGCLCs comprehensively erased their DNA methylation to acquire a profile equivalent to that of gonadal germ cells in vivo, while maintaining their highly motile phenotype as well as their transcriptional properties as sexually uncommitted PGCs. Importantly, these mPGCLCs robustly contributed to spermatogenesis and produced fertile offspring. Furthermore, mouse PGCs (mPGCs) cultured with CsA ex vivo showed transcriptomes and DNA methylomes similar to those of cultured mPGCLCs. The improved culture system for mPGCLCs/mPGCs would be instructive for addressing key questions in PGC biology, including the mechanisms for germ cell migration, epigenetic reprogramming, and sex determination of the germline.

2021-02-16 | GSE151444 | GEO

In vitro expansion of mouse primordial germ cell‐like cells recapitulates an epigenetic blank slate [ChIP-Seq]

Project description:The expansion of primordial germ cells (PGCs), the precursors for the oocytes and spermatozoa, is a key challenge in reproductive biology/medicine. Using a chemical screening exploiting PGC‐like cells (PGCLCs) induced from mouse embryonic stem cells (ESCs), we here identify key signaling pathways critical for PGCLC proliferation. We show that the combinatorial application of Forskolin and Rolipram, which stimulate cAMP signaling via different mechanisms, expands PGCLCs up to ~50‐fold in culture. The expanded PGCLCs maintain robust capacity for spermatogenesis, rescuing the fertility of infertile mice. Strikingly, during expansion, PGCLCs comprehensively erase their DNA methylome, including parental imprints, in a manner that precisely recapitulates genome‐wide DNA demethylation in gonadal germ cells, while essentially maintaining their identity as sexually uncommitted PGCs, apparently through appropriate histone modifications. By establishing a paradigm for PGCLC expansion, our system reconstitutes the epigenetic “blank slate” of the germ line, an immediate precursory state for sexually dimorphic differentiation.

2017-05-30 | GSE87645 | GEO

In vitro expansion of mouse primordial germ cell‐like cells recapitulates an epigenetic blank slate [RNA-Seq]

2017-05-30 | GSE87644 | GEO

In Vitro Reconstitution of the Whole Male Germ-Cell Development from Mouse Pluripotent Stem Cells [PBAT]

Project description:Mammalian male germ-cell development consists of three distinct phases: primordial germ cell (PGC) development, male germ-cell specification for spermatogonium development, and ensuing spermatogenesis. Here, we show an in vitro reconstitution of whole male germ-cell development by pluripotent stem cells (PSCs). Mouse embryonic stem cells (mESCs) are induced into PGC-like cells (mPGCLCs), which are expanded for epigenetic reprogramming. In reconstituted testes under an optimized condition, such mPGCLCs differentiate into spermatogonium-like cells with proper developmental transitions, gene expression, and cell-cycle dynamics and are expanded robustly as germline stem cell-like cells (GSCLCs) with an appropriate androgenetic epigenome. Importantly, GSCLCs show vigorous spermatogenesis, not only upon transplantation into testes in vivo but also under an in vitro culture of testis transplants, and the resultant spermatids contribute to fertile offspring. By uniting faithful recapitulations of the three phases of male germ-cell development, our study creates a paradigm for the in vitro male gametogenesis by PSCs.

2021-09-30 | GSE168221 | GEO

In Vitro Reconstitution of the Whole Male Germ-Cell Development from Mouse Pluripotent Stem Cells [RNA-Seq]

2021-09-30 | GSE168220 | GEO

A cell-matrix interaction regulates the undifferentiated state and self-renewal capacity of avian primordial germ cells

Project description:Primordial germ cells (PGCs) are lineage restricted precursor cells of sperm and eggs. While avian PGCs from chicken can be cultured and modified to produce genome-edited chickens, the long-term culture of PGCs from other bird species has not been achieved. Here, we explored the effects of a cell-matrix interaction on the in vitro propagation of chicken PGCs. Blocking integrin signaling severely reduced the self-renewal of the PGC, indicating that a PGC-matrix interaction is an essential process for self-renewal. We investigated the properties of somatic cell differentiated PGCs and expression analyses suggested that the PGCs undergo a partial epithelial-to-mesenchymal transition caused by excess PGC-matrix interactions. Finally, we conducted a long-term culture of chicken PGCs with matrix components, resulting in significant induction of their somatic conversion. These results suggested that a high level of PGC-matrix interaction can cause somatic conversion, while a moderate level is essential for their proliferation. Overall, we identified a molecular aspect of self-renewal and maintaining undifferentiated states of avian PGCs.

2024-10-29 | GSE280091 | GEO

MCM8 interacts with DDX5 to promote R-loop resolution in primordial germ cells

Project description:MCM8 has emerged as a core gene in reproductive aging and is crucial for meiotic homologous recombination repair. It also safeguards genome stability by coordinating the replication stress response during mitosis, but its function in mitotic germ cells remains elusive. Here we found that the number of primordial germ cells (PGCs) decreased dramatically after MCM8 deficiency due to proliferation defects. We further demonstrated that MCM8 interacted with two known helicases DDX5 and DHX9 in PGCs, and loss of MCM8 led to R-loop accumulation by reducing the retention of these helicases at R-loops, thus inducing genome instability. Cells expressing premature ovarian insufficiency-causative mutants of MCM8 with decreased interaction with DDX5 displayed increased R-loop levels. These results suggest that MCM8 interacts with R-loop-resolving factors to prevent DNA damage in PGCs, thus maintaining PGC proliferation and establishing the reproductive reserve. Our findings thus reveal a novel role for MCM8 in PGC development and improve our understanding of reproductive aging caused by genome instability in mitotic germ cells.

2024-05-31 | PXD050727 | Pride

Characteristic energy metabolism in mouse primordial germ cells

Project description:Primordial germ cells (PGCs), undifferentiated embryonic germ cells, are the only cells that have the ability to become gametes and to reacquire totipotency upon fertilization. It is generally understood that the development of PGCs proceeds through the expression of germ cell-specific transcription factors and characteristic epigenomic changes. However, little is known about the properties of PGCs at the metabolite and protein levels, which are directly responsible for the control of cell function. Here, we report the metabolic profiles of PGCs through the first comprehensive analyses of the metabolome and proteome, including remarkable activation of oxidative phosphorylation in mitochondria. Moreover, conversion of energy metabolism is involved in the control of PGC reprogramming and differentiation of pluripotential stem cells (PSCs) into PGCs in culture. Our findings about the unique metabolic property of PGCs provide novel insights into our understanding of the importance of distinct energy metabolism for switching PGC and PSC status.

2018-12-31 | GSE89738 | GEO

The Xenopus Primordial Germ Cell Transcriptome: Unexpected Role for sox7 in Early PGC Development

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

Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

Project description:Specification to primordial germ cells (PGCs) occurs under the mesoderm induction signals during gastrulation. Here, we found that Akt activation in embryonic stem (ES) cells generated self-renewing spheres during mesodermal differentiation induction and that the differentiation status of the sphere cells was in between ES cells and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, showing that the cells of the sphere commenced the differentiation to germ lineage. However, the spheres could not proceed to spermatogenesis after transplantation to testes. Meanwhile, the transfer of the spheres to the original feeder-free ES cell culture conditions induced chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, the reversion to the ES cell-like cell states was induced. These results indicate that the Akt signaling brings about a novel metastable and pluripotent state between ES cells and PGCs. Five samples were analyzed, which included the Akt-Mer-expressing ES cell (ESC) line #21 treated with or without 4OHT (4-hydroxytamoxifen), the #21 ESC-derived primordial germ cell (PGC)-like sphere cells and the ESC-like cells reverted from #21 PGC-like sphere cells. The PGC-like sphere cells derived from another Akt-Mer ESC line #42 was also examined.

2010-04-30 | E-GEOD-18813 | biostudies-arrayexpress

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