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Directed Differentiation of Human iPSCs to Functional Ovarian Granulosa-Like Cells via Transcription Factor Overexpression [RNA-seq]

ABSTRACT: An in vitro model of human ovarian follicles would greatly benefit the study of female reproduction. Ovarian development requires the combination of germ cells and their supporting somatic cells, known as granulosa cells. Whereas efficient protocols exist for generating human primordial germ cell-like cells (hPGCLCs) from human iPSCs, a method of generating granulosa cells has been elusive. Here we report that simultaneous overexpression of two transcription factors (TFs) can direct the differentiation of human iPSCs to granulosa-like cells. We elucidate the regulatory effects of several granulosa-related TFs, and establish that overexpression of NR5A1 and either RUNX1 or RUNX2 is necessary and sufficient to generate granulosa-like cells. Our granulosa-like cells have transcriptomes similar to human fetal ovarian cells, and recapitulate key ovarian phenotypes including follicle formation and steroidogenesis. When aggregated with hPGCLCs, our cells form ovary-like organoids (ovaroids) and support hPGCLC development from the premigratory to the gonadal stage as measured by induction of DAZL expression. This model system will provide unique opportunities for studying human ovarian biology, and may enable the development of therapies for female reproductive health.

ORGANISM(S): Homo sapiens

PROVIDER: GSE213156 | GEO | 2023/02/21

REPOSITORIES: GEO

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Directed Differentiation of Human iPSCs to Functional Ovarian Granulosa-Like Cells via Transcription Factor Overexpression [scRNA-seq]

Project description:An in vitro model of human ovarian follicles would greatly benefit the study of female reproduction. Ovarian development requires the combination of germ cells and their supporting somatic cells, known as granulosa cells. Whereas efficient protocols exist for generating human primordial germ cell-like cells (hPGCLCs) from human iPSCs, a method of generating granulosa cells has been elusive. Here we report that simultaneous overexpression of two transcription factors (TFs) can direct the differentiation of human iPSCs to granulosa-like cells. We elucidate the regulatory effects of several granulosa-related TFs, and establish that overexpression of NR5A1 and either RUNX1 or RUNX2 is necessary and sufficient to generate granulosa-like cells. Our granulosa-like cells have transcriptomes similar to human fetal ovarian cells, and recapitulate key ovarian phenotypes including follicle formation and steroidogenesis. When aggregated with hPGCLCs, our cells form ovary-like organoids (ovaroids) and support hPGCLC development from the premigratory to the gonadal stage as measured by induction of DAZL expression. This model system will provide unique opportunities for studying human ovarian biology, and may enable the development of therapies for female reproductive health.

2023-02-21 | GSE213462 | GEO

In vitro differentiation of human embryonic stem cells into ovarian follicle-like cells

Project description:Understanding the unique mechanisms of human oogenesis necessitates the development of an in vitro system of stem cell differentiation into oocytes. Specialized cell types and organoids have been derived from human pluripotent stem cells in vitro, but generating a human ovarian follicle remains a challenge. Here we report that human embryonic stem cells (hESCs) can be induced to differentiate into ovarian follicle-like cells in vitro. First, we find that two RNA-binding proteins specifically expressed in germ cells, DAZL and BOULE, regulate the exit from pluripotency and entry into meiosis. By expressing DAZL and BOULE with recombinant human GDF9 and BMP15, these meiotic germ cells are further induced to form ovarian follicle-like cells (FLCs), including oocytes and granulosa cells. This robust in vitro differentiation system will allow the study of the unique molecular mechanisms underlying human pluripotent stem cell differentiation into late PGCs, meiotic germ cells, and ovarian follicles.

2017-07-04 | GSE97388 | GEO

Derivation of functional oocytes from granulosa cells

Project description:Limited oocyte and ovarian reserve in vivo or chemo-therapy leads to reproductive aging or premature aging and associated diseases including infertility. Excitingly, oocytes have been successfully derived from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) by ectopic expression of transcription factors, showing great potential in fertility preservation or restoration. The accessible granulosa cells are a type of somatic cells that interact and evolve with oocyte development during folliculogenesis. Further, with stem cells-like property, granulosa cells are amenable to reprogramming to generate iPSCs and have been the first used for clone animals. These prompted us to explore the potential of granulosa cells in derivation of germ cells. Meanwhile, the strict genome fidelity required for germ cells inspired us to test reprograming by complete small chemicals, which avoids genetic manipulation, cell transfection and destruction of embryos. Here we show that somatic granulosa cells of adult mouse ovaries can be converted to germ cells and functional oocytes that reproduce fertile pups. We are able to consistently induce granulosa cells to pluripotent state (gPSCs) like ESCs in both developmental competence and molecular signatures. Notably, crotonic sodium-facilitated crotonylation is critical not only for pure small chemicals-based reprogramming of granulosa cells to gPSCs, but also confers the gPSCs with high germline capacity. Consequently, the gPSCs and the derived primordial germ-cell like cells hold longer telomeres and maintain high genomic stability which is critical for germ cells. Taken together, we efficiently generate high quality gPSCs and functional oocytes from adult granulosa cells by significantly improving chemical reprograming approach.

2019-03-06 | GSE127833 | GEO

Efficient differentiation of steroidogenic and germ-like cells from epigenetically-related iPSCs derived from ovarian granulosa cells

Project description:To explore restoration of ovarian function using epigenetically-related, induced pluripotent stem cells (iPSCs), we functionally evaluated the epigenetic memory of novel iPSC lines, derived from mouse and human ovarian granulosa cells (GCs) using c-Myc, Klf4, Sox2 and Oct4 retroviral vectors. The stem cell identity of the mouse and human GC-derived iPSCs (mGriPSCs, hGriPSCs) was verified by demonstrating embryonic stem cell (ESC) antigen expression using immunocytochemistry and RT-PCR analysis, as well as formation of embryoid bodies (EBs) and teratomas that are capable of differentiating into cells from all three germ layers. GriPSCs' gene expression profiles associate more closely with those of ESCs than of the originating GCs as demonstrated by genome-wide analysis of mRNA and microRNA. A comparative analysis of EBs generated from three different mouse cell lines (mGriPSCs; fibroblast-derived iPSC, mFiPSCs; G4 embryonic stem cells, G4 mESCs) revealed that differentiated mGriPSC-EBs synthesize 10-fold more estradiol (E2) than either differentiated FiPSC- or mESC-EBs under identical culture conditions. By contrast, mESC-EBs primarily synthesize progesterone (P4) and FiPSC-EBs produce neither E2 nor P4. Differentiated mGriPSC-EBs also express ovarian markers (AMHR, FSHR, Cyp19a1, ER and Inha) as well as markers of early gametogenesis (Mvh, Dazl, Gdf9, Boule and Zp1) more frequently than EBs of the other cell lines. These results provide evidence of preferential homotypic differentiation of mGriPSCs into ovarian cell types. Collectively, our data support the hypothesis that generating iPSCs from the desired tissue type may prove advantageous due to the iPSCs' epigenetic memory.

2016-12-21 | GSE62565 | 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

2014-12-26 | E-GEOD-60138 | biostudies-arrayexpress

single cell RNAseq of mouse E12.5 fetal ovarian somatic cells

Project description:A major technology for generating oocytes by in vitro gametogenesis involves generating ovary models called reconstituted ovaries (rOvaries). Widespread use of rOvaries depends on the reliable manufacturing of primordial germ cell like cells (PGCLCs) and fetal ovarian somatic cells (FOSCs). Using scRNA-seq, we identify that upon thaw, FOSCs are composed largely of Foxl2+ pre-granulosa cells, KRT19+ epithelial cells and Nr2f2+ mesenchymal cells which self-assemble together with PGCLCs into disc-like organoids containing multiple follicles embedded in NR2F2 stroma without an ovarian surface epithelium and no steroidogenic theca. The absence of an ovarian surface epithelium combined with the small size of GV-oocytes indicates that follicle production in the rOvary corresponds to first-wave folliculogenesis.

2023-12-14 | GSE238263 | GEO

GATA transcription factors, SOX17 and TFAP2C, drive the human germ-cell specification program [Bisulfite-Seq]

Project description:The in vitro reconstitution of human germ-cell development provides a robust framework for clarifying key underlying mechanisms. Here, we explored transcription factors (TFs) that engender the germ-cell fate in their pluripotent precursors. Unexpectedly, SOX17, TFAP2C, and BLIMP1, which act under the BMP signaling and are indispensable for human primordial germ-cell-like cell (hPGCLC) specification, failed to induce hPGCLCs. In contrast, GATA3 or GATA2, immediate BMP effectors, combined with SOX17 and TFAP2C, generated hPGCLCs. GATA3/GATA2 knockouts dose-dependently impaired BMP-induced hPGCLC specification, whereas GATA3/GATA2 expression remained unaffected in SOX17, TFAP2C, or BLIMP1 knockouts. In cynomolgus monkeys, a key model for human development, GATA3, SOX17, and TFAP2C were co-expressed exclusively in early PGCs. Crucially, the TF-induced hPGCLCs acquired a hallmark of bona fide hPGCs to undergo epigenetic reprogramming and mature into oogonia/gonocytes in xenogeneic reconstituted ovaries. By uncovering a TF circuitry driving the germ line program, our study provides a paradigm for TF-based human gametogenesis.

2021-02-16 | GSE154690 | GEO

GATA transcription factors, SOX17 and TFAP2C, drive the human germ-cell specification program [RNA-Seq]

2021-02-16 | GSE154688 | GEO

GATA transcription factors, SOX17 and TFAP2C, drive the human germ-cell specification program

2021-02-15 | GSE154691 | GEO

Small RNA sequencing of human preovulatory cumulus and mural granulosa cells

Project description:The granulosa cells in the mammalian ovarian follicle respond to gonadotropin signalling and are involved in the processes of folliculogenesis and oocyte maturation. Studies on gene expression and regulation in human granulosa cells are of interest due to their potential for estimating the oocyte viability and IVF success. However, the post-transcriptional gene expression studies on miRNA level in the human ovary have been scarce. The current study determined the miRNA profile by deep sequencing of the two intrafollicular somatic cell types: mural and cumulus granulosa cells isolated from women undergoing controlled ovarian stimulation and in vitro fertilization. Paired cumulus and mural granulosa samples were analysed from 3 women participating in IVF procedure. Libraries of all 6 samples were sequenced twice, generating 2 technical replicates for each sample. Differential gene expression study was performed on the pooled results of technical replicates.

2013-08-05 | E-GEOD-46489 | biostudies-arrayexpress

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