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W4321462022 abstract "Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics 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 several types of somatic cells. Among these, granulosa cells play a key role in follicle formation and support for oogenesis. Whereas efficient protocols exist for generating human primordial germ cell-like cells (hPGCLCs) from human induced pluripotent stem cells (hiPSCs), 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 hiPSCs 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 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. Editor's evaluation This manuscript addresses a fundamental issue in ovarian biology of deriving granulosa cells from human iPS cells. These findings are important to treat female infertility in the future and may prove valuable in the Ob and Gyn clinical practice. The authors provide compelling evidence by developing and validating their model using in vitro ovaroids. This study provides a novel resource for transcriptomic signatures of ovarian somatic cells derived in vitro. https://doi.org/10.7554/eLife.83291.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Ovaries are responsible for forming the eggs humans and other mammals need to reproduce. Once mature, the egg cell is released into the fallopian tube where it can be potentially fertilized by a sperm. Despite their crucial role, how eggs are made in the ovary is poorly understood. This is because ovaries are hard to access, making it difficult to conduct experiments on them. To overcome this, researchers have built artificial ovaries in the laboratory using stem cells from the embryos of mice which can develop into all cell types in the adult body. By culturing these embryonic stem cells under special conditions, researchers can convert them in to the two main cell types of the developing ovary: germ cells which go on to form eggs, and granulosa cells which help eggs grow and mature. The resulting lab-grown ovary can make eggs that produce live mice when fertilized. This approach has also been applied to human induced pluripotent stem cells (iPSCs), adult human cells which have been reprogrammed to a stem-like state. While this has produced human germ cells, generating human granulosa cells has been more challenging. Here, Pierson Smela, Kramme et al. show that activating a specific set of transcription factors (proteins that switch genes on or off) in iPSCs can make them transition to granulosa cells. First, the team tested random combinations of 35 transcription factors which, based on previous literature and genetic data, were likely to play a role in the formation of granulosa cells. This led to the identification of a small number of factors that caused the human iPSCs to develop features and carry out roles seen in mature granulosa cells; this includes producing an important reproductive hormone and supporting the maturation of germ cells. Pierson Smela, Kramme et al. found that growing these granulosa-like cells together with germ cells (also generated via iPSCs) resulted in structures similar to ovarian follicles which help eggs develop. These findings could help researchers build stable systems for studying how granulosa cells behave in human ovaries. This could lead to new insights about reproductive health. Introduction Oogenesis is the central process of female reproduction, yet less is understood about this process in humans relative to other model organisms. This is in part due to difficulties in obtaining samples of human fetal ovaries, and the lack of a suitable model system to study human ovarian development in vitro. The potential power of such a system is illustrated by a recent study in mice, which differentiated mouse embryonic stem cells into primordial germ cell-like cells (PGCLCs) and ovarian-like cells, and combined these cell types. Reconstituted follicles were capable of producing oocytes and live offspring (Yoshino et al., 2021). Using mouse fetal ovarian somatic cells, a similar system was published that allowed the development of human PGCLCs to the oogonia stage (Yamashiro et al., 2018). However, these mixed-species systems are inadequate for fully modeling human ovarian development. Clearly, the construction of fully human ovarian follicles from pluripotent stem cells could enable new advances in the study of female reproduction, epigenetics, and human development. Ovarian development requires paracrine and juxtacrine interaction between cells from germline and somatic lineages (Høyer et al., 2005; Albertini et al., 2001; Rodrigues et al., 2021; Su et al., 2004; Le Bouffant et al., 2010; Otsuka and Shimasaki, 2002; Hu et al., 2015). During embryonic development, the first cells committed to the germline appear at roughly 2–3 weeks post-fertilization (wpf), when primordial germ cells (PGCs) are specified from the posterior epiblast in response to bone morphogenetic protein (BMP) signaling (Tang et al., 2016; Lawson et al., 1999; Kee et al., 2006; Hayashi et al., 2011). After specification, PGCs migrate to the developing gonad and undergo further development into oogonia (in female) or spermatogonia (in male) (Richardson and Lehmann, 2010; Grimaldi and Raz, 2020). The expression of the RNA-binding proteins DAZL and DDX4 (also known as VASA) begins in gonadal PGCs (Hu et al., 2015; Anderson et al., 2007; Nicholls et al., 2019), with DAZL identified as a key factor induced by the gonadal environment that commits PGCs to the germline (Nicholls et al., 2019), restricting pluripotency and inhibiting differentiation to somatic lineages (Chen et al., 2014). Notably, gonadal PGCs from Carnegie stage 23 primate embryos express mature markers such as DAZL and DDX4, and can complete spermatogenesis upon transplantation into a recipient testis (Clark et al., 2017), whereas earlier-stage PGCLCs cannot (Sosa et al., 2018). Previous studies have provided efficient protocols for the differentiation of hiPSCs to DAZL-negative premigratory PGCLCs (Irie et al., 2015; Sasaki et al., 2015; Mitsunaga et al., 2017; Sebastiano et al., 2021; Kee et al., 2006). However, the only previously reported method that allows human primordial germ cell-like cells (hPGCLCs) to develop to later DAZL-positive stages in vitro is to aggregate them with mouse fetal ovarian (Yamashiro et al., 2018) or testicular (Hwang et al., 2020; Kobayashi et al., 2022) cells, which mimic the environment of the early gonad. Another study tested aggregation of hPGCLCs with rat neonatal testis cells (Mall et al., 2020), but only showed that the hPGCLCs could survive and did not assess maturation. The somatic cells of the developing ovary provide crucial support for oogenesis. These cells originate from the WT1-positive intermediate mesoderm (Sasaki et al., 2021; Stévant et al., 2019), and initially exist in a bipotential state, capable of supporting either male or female development. In humans, sex-specific pathways begin to diverge at around 7 wpf (Hartshorne et al., 2009). In male mammals, the Y-chromosomal transcription factor (TF) SRY activates a gene regulatory network promoting testis formation and repressing ovary formation (Koopman et al., 1991). In the absence of SRY, the TF FOXL2 represses the testis factors, allowing ovarian development to proceed (Nicol et al., 2018; Ottolenghi et al., 2005). The ovarian somatic cells further differentiate into several lineages, including the granulosa cells which surround and support developing oocytes. Proper oogenesis depends on a complex interplay of paracrine signaling between somatic and germline cells (Su et al., 2004; Le Bouffant et al., 2010; Otsuka and Shimasaki, 2002; Peng et al., 2013). Granulosa cells additionally participate in endocrine signaling, producing estradiol and progesterone which regulate diverse reproductive functions (Findlay et al., 2019). Although a few studies have attempted differentiating human pluripotent stem cells to granulosa-like cells through treatment with recombinant signaling proteins, none has resulted in an efficient method, nor has any previous study evaluated their ability to support germ cell development. For example, a study using spontaneous differentiation of hiPSCs produced a small fraction of AMHR2-positive granulosa-like cells which expressed some granulosa marker genes and produced estradiol (Lipskind et al., 2018). However, the efficiency of this spontaneous differentiation was low and not numerically reported. A related study used recombinant signaling proteins in a 12-day multistep protocol to direct the differentiation of human embryonic stem cells (hESCs) toward granulosa-like cells (Lan et al., 2013); again, the yield was low (12–36%), and although the cells upregulated some marker genes, the expression levels were much lower than observed in primary granulosa cells. Manipulation of TFs allows reprogramming of cell identity and offers a rapid and efficient way to generate granulosa-like cells. Perhaps the best-known example of cellular reprogramming using TFs is the generation of iPSCs from somatic cells (Takahashi and Yamanaka, 2006). TF expression can also guide the differentiation of iPSCs into various somatic lineages, including neurons, fibroblasts, oligodendrocytes, and vascular endothelium (Ng et al., 2021). Furthermore, a recent study demonstrated that overexpression of the TFs NR5A1 and GATA4 is sufficient to reprogram human fibroblasts into Sertoli-like cells (Liang et al., 2019), the male developmental counterparts of granulosa-like cells. Therefore, we set out to determine a set of TFs that could be used for an efficient, scalable method of generating granulosa-like cells from hiPSCs. Our overall experimental workflow is shown in Figure 1. Figure 1 Download asset Open asset Experimental workflow of the study. First, barcoded transcription factor (TF) expression vectors were integrated into FOXL2-T2A-tdTomato reporter human induced pluripotent stem cells (hiPSCs). After induction of TF expression, cells positive for tdTomato and granulosa-related surface markers were sorted, and the barcodes were sequenced. The top TFs based on barcode enrichment were selected for further characterization by combinatorial screening and bulk RNA-seq. Next, monoclonal hiPSC lines were generated that inducibly express the top TFs (see Figure 3—source data 2) and generate granulosa-like cells with high efficiency (Figure 3—figure supplement 2). Granulosa-like cells from these lines were further evaluated for estradiol production in response to follicle-stimulating hormone (FSH). Finally, they were aggregated with human primordial germ cell-like cells (hPGCLCs) to form ovaroids. These ovaroids produced estradiol and progesterone, formed follicle-like structures, and supported hPGCLC maturation as measured by immunofluorescence microscopy and scRNA-seq. Results In silico prediction of granulosa cell-regulating TFs We began by predicting candidate TFs that could direct the differentiation of hiPSCs to granulosa-like cells. We first selected 21 TFs that were differentially expressed in granulosa cells compared to hESCs and early mesoderm, using previously published datasets for these cell types (Irie et al., 2015; Zhang et al., 2018). We also included five TFs based on previous developmental biology studies, mainly showing that these TFs were important for mouse ovarian development (Manuylov et al., 2008; Niu and Spradling, 2020; Voronina et al., 2007; Richards, 2001; Nicol et al., 2019). Finally, we identified nine additional TFs that we predicted to be upstream of the others on the list, based on a gene regulatory network analysis taking into account co-expression data as well as binding motifs (Kramme et al., 2021a). The list of TFs (35 in total) is in Figure 2—source data 2. Next, we assembled a PiggyBac library by cloning cDNAs into a barcoded destination plasmid (Kramme et al., 2021b). The TFs were expressed under the control of a doxycycline-inducible promoter, which we characterized in previous work (Kramme et al., 2021a). Overexpression screening identifies TFs that drive granulosa-like cell formation To enable identification of granulosa-like cells, we used CRISPR/Cas9-mediated homology-directed repair to engineer an hiPSC line with a homozygous knock-in of a T2A-tdTomato reporter at the C-terminus of FOXL2 (Figure 2—figure supplement 1). We chose FOXL2 due to its specific expression in granulosa cells (Ottolenghi et al., 2005; Cocquet et al., 2002). Next, the pooled cDNA library was co-electroporated with a PiggyBac transposase expression plasmid, and a population of cells with integrated transposons was selected by treatment with puromycin. In these experiments, we tested several different library compositions and DNA concentrations (see Methods, Figure 2—source data 2, and Figure 2—figure supplement 2). For screening, we induced TF expression with doxycycline, and sorted reporter-positive cells after 5 days of treatment. In addition to screening TF expression in the pluripotency-supporting mTeSR Plus medium, we also expressed TFs following differentiation of hiPSCs to early-stage mesoderm by treatment with the GSK3 inhibitor CHIR99021. The TF expression resulted in a small fraction of FOXL2+ cells, from which we extracted gDNA and sequenced barcodes. In both conditions, we observed barcodes for NR5A1 to be strongly enriched in FOXL2+ cells relative to negative cells, as well as relative to the barcodes in the pre-induced hiPSC population (Figure 2A). Other TFs showed more modest barcode enrichment (RUNX2, GATA4, and TCF21), or enrichment in only one condition (KLF2 and NR2F2). Interestingly, barcodes for FOXL2 were strongly depleted (Figure 2A), suggesting negative feedback whereby exogenous FOXL2 directly or indirectly suppresses the expression of the endogenous FOXL2 reporter allele. Figure 2 with 2 supplements see all Download asset Open asset Identification of transcription factors (TFs) whose overexpression generates granulosa-like cells. (A) Pooled screening of barcoded TF cDNA libraries (see Methods for details) identifies TFs enriched in FOXL2-T2A-tdTomato+ cells. Library #1 is the full library of 35 TFs, and library #2 is a library containing a subset of 18 TFs. Empty values correspond to TFs that were absent in library #2. (B) Combinatorial screening identifies minimal TF combinations for inducing granulosa-like cells. TF combinations were integrated into human induced pluripotent stem cells (hiPSCs); a ‘1’ in the left-hand box signifies the presence of the TF in the combination corresponding to that row. For each combination, the polyclonal hiPSC population was differentiated with TF induction (see Methods). For the last 24 hr of differentiation, cells were additionally treated with FSH and androstenedione. Estradiol production and granulosa markers were measured by ELISA and flow cytometry after a total of 5 days. NR5A1 expression induced high levels of estradiol synthesis, but the combination of NR5A1 with RUNX1 or RUNX2 was required to give the best results for granulosa markers. ‘All markers’ signifies FOXL2+CD82+AMHR2+EPCAM−. Figure 2—source data 1 Barcode counts for the enrichment analysis. https://cdn.elifesciences.org/articles/83291/elife-83291-fig2-data1-v1.zip Download elife-83291-fig2-data1-v1.zip Figure 2—source data 2 List of transcription factors (TFs) included in libraries 1 and 2. https://cdn.elifesciences.org/articles/83291/elife-83291-fig2-data2-v1.docx Download elife-83291-fig2-data2-v1.docx Using the top TFs identified in barcode screening (NR5A1, RUNX1/RUNX2, TCF21, and GATA4) we then further optimized the conditions for generation of FOXL2+ cells. We included RUNX1 in this list along with RUNX2 because the two TFs are structurally and functionally similar, and RUNX1 is known to play an important role for granulosa cell maintenance in the mouse (Nicol et al., 2019). We integrated these TFs into hiPSCs and established monoclonal lines, which we screened by flow cytometry after TF induction. We monitored FOXL2-tdTomato as well as the surface markers CD82, follicle-stimulating hormone receptor (FSHR), and EpCAM. CD82 is absent in hiPSCs, but highly expressed in granulosa cells beginning at the primordial follicle stage (Zhang et al., 2018). FSHR is specific for late-stage (secondary/antral) granulosa cells and Sertoli cells (the male equivalent). By contrast, EpCAM is expressed in hiPSCs and epithelial cells, but not granulosa cells (Zhang et al., 2018). Out of 23 lines tested, 4 showed >50% FOXL2-tdTomato+CD82+EpCAM− cells after 5 days of differentiation (Figure 3—figure supplement 1A). We observed low levels of FSHR expression, suggesting that we were mainly generating cells corresponding to early (primordial/primary follicle) granulosa cells, given that FSHR only becomes strongly expressed at later stages of ovarian follicle development (Oktay et al., 1997). Further optimization by adding the GSK3 inhibitor CHIR99021 for the first 2 days (Supplementary file 2) resulted in a near-homogeneous FOXL2-tdTomato+CD82+EPCAM− population for the top cell lines (Figure 3—figure supplement 2), with this effect being doxycycline responsive. Genotyping of two top lines (Figure 3—figure supplement 1C) revealed that both had NR5A1 expression cassettes integrated, with the first line also having RUNX1 and the second line RUNX2. Subsequently, we set out to determine which TFs were sufficient for induction of granulosa-like cells. We tested combinations of hit TFs from our screening (NR5A1, RUNX2, TCF21, GATA4, KLF2, and NR2F2) as well as TFs reported to be important for ovarian function (RUNX1, WT1 [−KTS isoform], and FOXL2). We tested expression of each TF individually, as well as combinations of other TFs and our top hit NR5A1. In addition to flow cytometry, we also measured production of estradiol after treatment of the cells with FSH and androstenedione. We observed that combinations containing NR5A1 and RUNX1 or RUNX2 upregulated FOXL2-tdTomato expression, as well as granulosa surface markers AMHR2 and CD82 (Figure 2B). Estradiol production was strongly induced by NR5A1, and weakly by GATA4 and KLF2. RUNX1 and RUNX2 expression somewhat decreased estradiol production, suggesting a regulatory role of these factors. Overall, these results indicate that NR5A1 and either RUNX1 or RUNX2 are sufficient to induce a granulosa-like phenotype. TF-mediated differentiation drives granulosa-like cell formation based on gene expression signatures We next examined the gene expression of our granulosa-like cells. We compared the bulk transcriptomes of hiPSCs, COV434 and KGN ovarian tumor cells, and sorted FOXL2+CD82+ granulosa-like cells from day 5 of a polyclonal differentiation with expression of our previously identified top TFs (NR5A1, TCF21, GATA4, and RUNX1; see Figure 2). As an additional control, we included hiPSCs differentiated under the same conditions but without TF induction. In the absence of TF expression, the hiPSCs differentiated into cells expressing mesoderm markers (full gene expression data are provided in the Source Data for Figure 3). At day 5 of differentiation, we observed strong upregulation relative to hiPSCs of genes associated with both lateral mesoderm (HAND1 and BMP5) as well as paraxial mesoderm (PAX3), which potentially indicates the presence of a heterogeneous population. Upregulated genes were enriched for gene ontology terms related to heart, blood vessel, muscle, and skeletal development (Supplementary file 3). Figure 3 with 2 supplements see all Download asset Open asset Transcriptomic analysis of transcription factor (TF)-induced granulosa-like cells. (A) Gene expression of selected markers in granulosa-like cells. Log2(TPM) values for gondal/granulosa, adrenal, and pluripotent marker genes were compared between 7 wpf male and female fetal gonad somatic cells, primary and primordial granulosa cells, TF-induced FOXL2+ cells, KGN cells, COV434 cells, and human induced pluripotent stem cells (hiPSCs). (B) Transcriptome overlap measure (TROM) comparison of TF-induced FOXL2+ cells, COV434 cells, and hiPSCs with published in vivo data from different time points in ovarian development. (C) Regulatory effects of granulosa-related TFs. RNA-seq was performed after 2 days of TF overexpression in hiPSCs (TFs shown in magenta). A differential gene expression (DEG) analysis was performed for all samples relative to the hiPSC control (n = 2 biological replicates each). Black arrows represent significant (false discovery rate <0.05) upregulation, with the width proportional to the log2-fold change. Interactions are shown between TFs (magenta) and granulosa marker genes (yellow), as well as the stromal/theca marker NR2F2 (red) and the pre-granulosa marker LGR5 (green). (D–I) Volcano plots showing DEGs in the TF overexpression experiments. Colors are as in panel C; other DEGs not listed in panel C are shown in blue. Not all DEGs could be labeled due to space limits, but they are listed in the Source Data for this figure. Figure 3—source data 1 Gene expression data from bulk RNA-seq. https://cdn.elifesciences.org/articles/83291/elife-83291-fig3-data1-v1.zip Download elife-83291-fig3-data1-v1.zip Figure 3—source data 2 List of monoclonal human induced pluripotent stem cell (hiPSC) lines used for granulosa-like cell production. https://cdn.elifesciences.org/articles/83291/elife-83291-fig3-data2-v1.docx Download elife-83291-fig3-data2-v1.docx With TF induction, we observed expression of bipotential gonad and granulosa markers, notably including AMHR2, CD82, FOXL2, FSHR, IGFBP7, KRT19, STAR, and WNT4 (Figure 3A). The ovarian stromal/theca cell marker NR2F2 was also upregulated. The expression levels (transcripts per million (TPM)) were generally comparable to those observed in previously published data from granulosa cells and human fetal gonad (Zhang et al., 2018; Sybirna et al., 2020). The only major exception was WT1, which had much weaker expression than in vivo (although still greater than hiPSCs and KGN cells). We also examined the expression of pluripotent and adrenal markers to check for incomplete or off-target differentiation. We did not observe adrenal marker expression, although we did note some residual POU5F1 (encoding the OCT4 protein) expression that may indicate that our polyclonal population did not fully differentiate. COV434 cells, which were commonly considered as granulosa-like cells but recently reclassified as small cell ovarian carcinoma (Price et al., 2012; Zhang et al., 2000; Karnezis et al., 2021), did not express most granulosa markers, but did express high levels of WT1 and NR2F2. By contrast, KGN cells expressed high levels of most granulosa markers, but not WT1. We also performed a transcriptome-wide comparison of our TF-induced granulosa-like cells with three published datasets spanning weeks 6–21 of human fetal ovarian development (Tang et al., 2015; Lecluze et al., 2020; Yatsenko et al., 2019). Tang et al. sequenced RNA from male and female gonadal somatic cells at gestational week 7, whereas Yatsenko et al. and Leclucze et al. sequenced RNA from whole fetal ovaries over a range of gestational weeks. Using the transcriptome overlap measure (TROM) (Li et al., 2017), we computed similarity scores between our samples and these datasets (Figure 3B). We found a high degree of similarity (−log10(padj) = 50.5) between our granulosa-like cells and human fetal ovaries. The closest match was with gestational week 12 whole fetal ovary from the Leclucze et al. dataset (which sequenced RNA from whole ovaries at different gestational weeks) but we also observed a significant similarity (−log10(padj) of 12.7–15.8) with the other two datasets we examined. By contrast, COV434 and KGN cells showed a more modest similarity to the fetal ovary, and hiPSCs showed none at all. To further elucidate the regulatory effects of individual TFs that we identified as granulosa related (FOXL2, WT1, NR5A1, GATA4, TCF21, and RUNX1), we integrated their cDNA plasmids into hiPSCs and induced expression with doxycycline. For these experiments, we harvested RNA after 2 days of TF expression, since we were interested in short-term effects. We performed differential gene expression analysis relative to hiPSCs, and examined regulatory effects among TFs, and between TFs and marker genes (Figure 3C). Overall, we observed that FOXL2, NR5A1, GATA4, and RUNX1 had the greatest effects on granulosa marker genes. Therefore, we generated additional hiPSC lines by integrating these TFs (Figure 3—figure supplement 1). To rule out any effects of the T2A-tdTomato reporter allele potentially perturbing FOXL2 function, we generated these in a wild-type background (F66 hiPSC line). We measured surface marker expression by flow cytometry after induction, confirmed expression of the granulosa marker AMHR2 by qPCR, and additionally measured estradiol production in the presence of FSH and androstenedione (Figure 3—figure supplement 1B). We selected the clones (Figure 3—source data 2) that showed the most robust overall phenotype based on a combination of surface markers, AMHR2, expression and estradiol production. In the differential expression analysis, we additionally found that GATA4 upregulated NR2F2, a marker of ovarian stromal and theca cells (Figure 3C, G). LGR5, previously reported as a marker of pre-granulosa cells (Niu and Spradling, 2020), was upregulated by GATA4 and RUNX1 (Figure 3C, F, G). We also confirmed a previous report that FOXL2 expression upregulated the cyclin-dependent kinase inhibitor CDKN1B (Gustin et al., 2016), as well as downregulating ORC1 and MYC (Source Data for Figure 3), suggesting suppression of cellular proliferation. Finally, we conducted a gene ontology enrichment analysis of upregulated and downregulated genes for each TF (Mi et al., 2021). Significantly enriched terms were mainly related to generic developmental processes (e.g., ‘tissue development’, see Supplementary file 3) although terms related to gonad development were also significantly enriched for NR5A1 upregulated genes. Granulosa-like cells respond to FSH and perform steroidogenesis We next validated the ability of our granulosa-like cells to carry out one of the key endocrine functions of granulosa cells: the production of estradiol. In the ovary, theca cells convert cholesterol to androstenedione, which is the substrate for estradiol production in granulosa cells. The rate-limiting step is oxidative decarboxylation by CYP19A1 (aromatase), producing estrone, which is subsequently reduced to estradiol by enzymes in the HSD17B family, typically HSD17B1 in granulosa cells (Hakkarainen et al., 2015). In vivo, this pathway of estrogen synthesis is stimulated by FSH (Sasson et al., 2004; Welsh et al., 1984). We treated our granulosa-like cells with androstenedione, in the presence or absence of FSH or forskolin (which directly increases levels of the FSHR second messenger cAMP). As controls, we used COV434 and KGN ovarian tumor cells, which were previously reported to produce estradiol from androstenedione (Price et al., 2012; Zhang et al., 2000; Nishi et al., 2001), as well as immortalized primary human granulosa cells (HGL5) and primary adult mouse ovarian somatic cells. Our granulosa-like cells produced estradiol from androstenedione, and in seven out of the nine monoclonal lines we tested, this steroidogenic activity significantly increased upon stimulation with FSH or forskolin (Figure 4A, see Source Data for statistical tests). One of our granulosa lines (F3/N.T #5) produced high levels of estradiol in all conditions, and this line, unlike the others, had neither RUNX1 nor RUNX2 expression vectors integrated (Figure 3—figure supplement 1C). Figure 4 Download asset Open asset Hormonal signaling by granulosa-like cells. (A) Granulosa-like cells produce estradiol in the presence of androstenedione and either FSH or forskolin (FK). Results are shown from nine monoclonal populations (see Figure 3—source data 2) of granulosa-like cells (n = 2 biological replicates for each of 9 clones, error bars are 95% CI), as well as the COV434 and KGN human ovarian cancer cell lines, HGL5 immortalized primary human granulosa cells, and primary adult mouse granulosa cells. Asterisks mark lines where FSH production significantly (two-tailed t-test, p < 0.05) increased upon stimulation. Exact p values are given in the Source Data. (B) Ovaroids produce both estradiol and progesterone. Estradiol production requires androstenedione and is stimulated by FSH. Results are shown for ovaroids formed with six different monoclonal samples of granulosa-like cells (n = 1 sample per ovaroid per condition), at 3 days post-agg" @default.
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W4321462022 title "Decision letter: Directed differentiation of human iPSCs to functional ovarian granulosa-like cells via transcription factor overexpression" @default.
W4321462022 doi "https://doi.org/10.7554/elife.83291.sa1" @default.
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