CRISPR/Cas9-mediated activation of NR5A1 steers female human embryonic stem cell-derived bipotential gonadal-like cells towards a steroidogenic cell fate.
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA991975 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR25179300_1.fastq.gz | Fastqsanger.gz | |||
| SRR25179300_2.fastq.gz | Fastqsanger.gz | |||
| SRR25179301_1.fastq.gz | Fastqsanger.gz | |||
| SRR25179301_2.fastq.gz | Fastqsanger.gz | |||
| SRR25179302_1.fastq.gz | Fastqsanger.gz |
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Project description:The nuclear receptor subfamily 5 group A member 1 (NR5A1), encoding steroidogenic factor 1 (SF-1), has been identified as a critical factor in gonadal development in animal studies. A previous study of ours suggested that upregulation of NR5A1 during early gonadal differentiation in male (46,XY) human pluripotent stem cells steers the cells into a more mature gonadal cell type. However, the detailed role of NR5A1 in female gonadal differentiation has yet to be determined. In this study, by combining the processes of gonadal differentiation and conditional gene activation, we show that NR5A1 induction predominantly upregulates the female gonadal marker inhibin subunit α (INHA) and steroidogenic markers steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), cytochrome P450 family 17 subfamily A member 1 (CYP17A1), hydroxy-delta-5-steroid dehydrogenase (HSD3B2) and hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1). In contrast, NR5A1 induction did not seem to affect the bipotential gonadal markers gata binding protein 4 (GATA4) and Wilms tumour suppressor 1 (WT1) nor the female gonadal markers r-spondin 1 (RSPO1) and wnt family member 4 (WNT4). Differentially expressed genes were highly associated with adrenal and ovarian steroidogenesis pathways. Moreover, time-series analysis revealed different dynamic changes between male and female -induced samples, where continuously upregulated genes in female gonadal differentiation were mostly associated with adrenal steroidogenesis. Thus, in contrast to male gonadal differentiation, NR5A1 is necessary but not sufficient to steer human embryonic stem cell (hESC)-derived bipotential gonadal-like cells towards a more mature somatic, female cell fate. Instead, it seems to direct bipotential gonadal-like cells more towards a steroidogenic-like cell population. The information obtained in this study helps in elucidating the role of NR5A1 in gonadal differentiation of a female stem cell line.
2023-09-27 | GSE236653 | GEO
Project description:Nuclear receptor subfamily 5 group A member 1 (NR5A1) encodes steroidogenic factor 1 (SF1), a key regulatory factor that determines gonadal development and coordinates endocrine functions. Here, we have established a stem cell-based model of human gonadal development and applied it to evaluate the effects of NR5A1 during the transition from bipotential gonad to testicular cells. We combined directed differentiation of human induced pluripotent stem cells (46,XY) with activation of endogenous NR5A1 expression by conditionally-inducible CRISPR activation. The resulting male gonadal-like cells expressed several Sertoli cell transcripts, secreted anti-Müllerian hormone and responded to follicle-stimulating hormone by producing sex steroid intermediates. These characteristics were not induced without NR5A1 activation. A total of 2691 differentially expressed genetic elements, including both coding and non-coding RNAs, were detected immediately following activation of NR5A1 expression. Of those, we identified novel gonad-related putative NR5A1 targets, such as SCARA5, which we validated also by immunocytochemistry. In addition, NR5A1 activation was associated with dynamic expression of multiple gonad- and infertility-related differentially expressed genes. In conclusion, by combining targeted differentiation and endogenous activation of NR5A1 we have for the first time, been able to examine in detail the effects of NR5A1 in early human gonadal cells. The model and results obtained provide a useful resource for future investigations exploring the causative reasons for gonadal dysgenesis and infertility in humans.
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Project description:Steroidogenic factor 1 (NR5A1) induces multiple transcriptional changes during differentiation of human gonadal-like cells
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Project description:The nuclear receptor subfamily 5 group A member 1 (NR5A1), encoding steroidogenic factor 1 (SF-1), has been identified as a critical factor in gonadal development in animal studies. A previous study of ours suggested that upregulation of NR5A1 during early gonadal differentiation in male (46,XY) human pluripotent stem cells steers the cells into a more mature gonadal cell type. However, the detailed role of NR5A1 in female gonadal differentiation has yet to be determined. In this study, by combining the processes of gonadal differentiation and conditional gene activation, we show that NR5A1 induction predominantly upregulates the female gonadal marker inhibin subunit α (INHA) and steroidogenic markers steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), cytochrome P450 family 17 subfamily A member 1 (CYP17A1), hydroxy-delta-5-steroid dehydrogenase (HSD3B2) and hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1). In contrast, NR5A1 induction did not seem to affect the bipotential gonadal markers gata binding protein 4 (GATA4) and Wilms' tumour suppressor 1 (WT1) nor the female gonadal markers r-spondin 1 (RSPO1) and wnt family member 4 (WNT4). Differentially expressed genes were highly associated with adrenal and ovarian steroidogenesis pathways. Moreover, time-series analysis revealed different dynamic changes between male and female induced samples, where continuously upregulated genes in female gonadal differentiation were mostly associated with adrenal steroidogenesis. Thus, in contrast to male gonadal differentiation, NR5A1 is necessary but not sufficient to steer human embryonic stem cell (hESC)-derived bipotential gonadal-like cells towards a more mature somatic, female cell fate. Instead, it seems to direct bipotential gonadal-like cells more towards a steroidogenic-like cell population. The information obtained in this study helps in elucidating the role of NR5A1 in gonadal differentiation of a female stem cell line.
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Project description:NR5A1 and FOXL2 Induce Human Fibroblasts into Steroidogenic Ovarian Granulosa-Like Cells
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Project description:We developed a gradual differentiation protocol to differentiate mESC towards the EpiSC lineage, followed by early mesoderm cells. These were later differentiated to intermediate mesoderm and we found a condition (IM3) that best generates early gonadal progenitor cells which are normally present at the bipotential gonad at E11.5. Forced expression of 2 transcription factors: Nr5A1 (SF1) and Dmrt1 induced a specific-Sertoli marker (TESCO-CFP) and the cells started to self-aggregate and form tubule-like structures, highly resembling the testis cords. There was proper induction of relevant markers at the correct time-points as shown by both qPCR, immuno-staining and RNA-Seq that includes comparison to in-vivo gonads.
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Project description:Granulosa cells provide essential niche for ovarian folliculogenesis by communicating with oocytes and secreting steroids at different follicular stages, but current cellular models for studying human granulosa cells mainly rely on immortal cancer cell lines. Here, we established a reprogramming method to collect early and late granulosa cells with different steroidogenic abilities. We applied AMH-fluorescence-reporter system to screen six candidate factors for cellular reprogramming and generated human induced granulosa like cells (hiGC) by overexpressing FOXL2 and NR5A1. AMH-EGFP+ hiGC resembled human cumulus cells in transcriptome profiling and secreted high levels of estrogen as well as progesterone, similar to late-stage granulosa cells at antral or preovulatory stage. Moreover, we identified a cell surface marker CD55 which can be used to isolate early-stage granulosa cells. CD55+AMH-EGFP- hiGC secreted high levels of estrogen but low levels of progesterone, and the transcriptome profiles were more similar to early-stage granulosa cells. Both types of the hiGC promoted oocyte maturation when cocultured with immature oocytes. More importantly, when CD55+ hiGC were transplanted to mouse model with Polycystic ovary syndrome (PCOS), the number of cystic follicles was significantly reduced, showing alleviation of PCOS. Hence, hiGC provides a cellular model to study developmental program of human granulosa cells and has potential to treat PCOS.
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Project description:The nuclear receptor steroidogenic factor-1 (SF-1, NR5A1) is a key regulator of adrenal and gonadal biology. We aimed to identify a novel subset of SF-1 target genes in the adrenal by performing ChIP-on-chip in NCI-H295R human adrenocortical cells using promoter tiling arrays. Analysis of ChIP-on-chip experiments with CisGenome identified 738 SF-1-binding regions that met criteria of an MA score more than 3.5 mean ± S.D. and a false discovery rate of <5%. Subsequent analysis focused on those regions that were located between 10 kb upstream and 3 kb downstream of the TSS of known genes, in keeping with the design of the Human Promoter 1.0R arrays. Using this approach, binding regions were annotated to 445 gene loci. The supplementary bed file contains all 946 SF-1 binding sites identified by analysis with CisGenome using standard settings (MA>3.0)
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Project description:The ovarian reserve of follicles comprises all oocytes for lifetime fertility and once formed, it is non renewing. The orphan nuclear receptor steroidogenic factor 1 (SF-1; Nr5a1) is essential for steroidogenesis, but its role in the earliest stages of follicular development is not known. We therefore developed a model of conditional depletion of SF-1 from prenatal ovaries and performed RNAsequencing to identify SF-1 regulated genes.
2023-07-10 | GSE216420 | GEO
Project description:Gonadal sex differentiation – testis versus ovary formation – is a fundamental process required for reproduction and evolution. Reflecting this importance, the embryonic gonads of vertebrate species comprise the same key cell types; germ cells, supporting cells and interstitial steroidogenic cells. Remarkably, the genetic triggers for gonadal sex differentiation vary across species (the SRY gene in mammals, DMRT1 in birds and some turtles, temperature in many reptiles, AMH and various other genes in fishes). Despite this variation, the cell biology of gonadal development was long thought to be largely conserved. Here, we present a comprehensive analysis of gonadal sex differentiation, using the chicken embryo as a model and considering the entire gonad. We sampled over 30,000 cells across several developmental stages, prior, during and after the onset of gonadal sex differentiation. The data provide several new insights into cell lineage specification during vertebrate gonadogenesis. Combining lineage tracing with single cell transcriptomics, the data show that somatic supporting cells of the embryonic chicken gonad do not derive from the coelomic epithelium, in contrast to other vertebrates studied. Instead, the early somatic precursors cells of the gonads in both sexes derive from a DMRT1+/PAX2+/WNT4+/OSR1+ mesenchymal cell population. In particular, PAX2 marks immigrating mesenchymal cells that give rise to the supporting cell lineage. We find a greater complexity of gonadal cell types than previously thought, including the identification of two distinct sub-populations of Sertoli cells in developing testes, and derivation of embryonic steroidogenic cells from a differentiated supporting cell lineage. We provide significantly improved resolution of gonadal cell types and identify several new gonadal marker genes. Altogether, these results indicate that, just as the genetic trigger for sex differs across vertebrate groups, cell lineage specification in the gonad may also vary substantially.
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