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. 2023 Feb 21:12:e83291.
doi: 10.7554/eLife.83291.

Directed differentiation of human iPSCs to functional ovarian granulosa-like cells via transcription factor overexpression

Merrick D Pierson Smela #  1   2 , Christian C Kramme #  1   2 , Patrick R J Fortuna  1   2 , Jessica L Adams  1   2 , Rui Su  1   2 , Edward Dong  1   2 , Mutsumi Kobayashi  3 , Garyk Brixi  1   2   4   5 , Venkata Srikar Kavirayuni  4   5 , Emma Tysinger  4   5 , Richie E Kohman  1   2 , Toshi Shioda  3 , Pranam Chatterjee  1   2   4   5 , George M Church  1   2
Affiliations

Directed differentiation of human iPSCs to functional ovarian granulosa-like cells via transcription factor overexpression

Merrick D Pierson Smela et al. Elife. .

Erratum in

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.

Keywords: developmental biology; granulosa; human; mouse; oogenesis; ovary; regenerative medicine; stem cells; transcription factors.

Plain language summary

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.

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Conflict of interest statement

MP, MK, GB, VK, ET, RK, TS is listed as an inventor for US Provisional Application No. 63/326,640, entitled 'Methods and Compositions for Producing Granulosa-Like Cells', CK is listed as an inventor for US Provisional Application No. 63/326,640, entitled 'Methods and Compositions for Producing Granulosa-Like Cells'. CK is the VP of Cell Engineering of Gameto, Inc, PF, JA, RS, ED No competing interests declared, PC is listed an an inventor for US Provisional Application No. 63/326,640, entitled 'Methods and Compositions for Producing Granulosa-Like Cells'. PC is a co-founder and scientific advisor to Gameto, Inc, GC is listed as an inventor for US Provisional Application No. 63/326,640, entitled 'Methods and Compositions for Producing Granulosa-Like Cells'. GMC serves on the scientific advisory board of Gameto, Inc, Colossal Biosciences, and GCTx

Figures

Figure 1.
Figure 1.. 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.
Figure 2.
Figure 2.. 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—figure supplement 1.
Figure 2—figure supplement 1.. Construction of the FOXL2-T2A-tdTomato reporter human induced pluripotent stem cell (hiPSC) line.
(A) Schematic for Cas9/HDR knock-in with a donor plasmid followed by selection marker excision. Primer-binding sites for genotyping are also shown (not to scale). (B) Genotyping to verify homozygous editing. Initial screening was performed with primers 176/177. Candidate clones (denoted A, B, C, D) were further verified by additional genotyping. Note that primers 127/128 bind outside of the region used as homology arms for the donor plasmid.
Figure 2—figure supplement 2.
Figure 2—figure supplement 2.. Control of transcription factor (TF) expression plasmid copy number delivered to human induced pluripotent stem cells (hiPSCs).
A PiggyBac transposon library of barcoded doxycycline-inducible TF cDNAs was electroporated into iPSCs at varying concentrations. The amount of transposase plasmid was held constant (500 ng). After selection, gDNA was extracted and the transposon copy number was measured by quantitative polymerase chain reaction (qPCR) using primer pairs CK107/108 (amplifies RPP30 genomic control) and qMPS015 (amplifies TET3G, present in all TF expression plasmids). To calculate the copy number, a standard curve was generated using a control plasmid with a known 1:1 ratio of RPP30 and TET3G. Each point represents a biological replicate, and error bars represent 95% confidence intervals of the technical replicates. The curve in light gray is a weighted-least-squares fit of a zero-truncated Poisson mean, which represents the theoretical relationship. For pooled screening, a higher copy number improves the sensitivity to detect multiway interactions, at the cost of less specificity because the reporter-positive cells will also contain ‘free rider’ TFs that are not important. In this study, we mostly performed screening at 5 fmol (medium-copy), with two experiments at 50 fmol (high-copy).
Figure 3.
Figure 3.. 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—figure supplement 1.
Figure 3—figure supplement 1.. Evaluation of monoclonal human induced pluripotent stem cell (hiPSC) lines for yield and quality of granulosa-like cells.
(A) Lines generated by integrating the indicated transcription factor (TF) expression vectors into the F3/FOXL2-T2A-tdTomato hiPSC reporter line were evaluated by flow cytometry for FOXL2, CD82, EPCAM, and follicle-stimulating hormone receptor (FSHR). Note that line F3/N.T #5 lacks the RUNX2 expression vector (see panel C). (B) Lines generated by integrating the indicated TF expression vectors into the F66 wild-type hiPSC line were evaluated by flow cytometry for CD82 and EPCAM, as well as for estradiol production and qPCR to measure AMHR2 expression (the no-TF control was used as a reference for calculating ∆∆Ct). (C) Genotyping to detect integrated TF expression vectors in selected lines. The negative control was wild-type F66 gDNA, and the positive control was gDNA plus TF Library #1. Primer sequences are listed in Supplementary file 1.
Figure 3—figure supplement 2.
Figure 3—figure supplement 2.. Monoclonal human induced pluripotent stem cell (hiPSC) lines with integrated transcription factors (TFs) allow the efficient production of granulosa-like cells in response to doxycycline.
(A) Method of inducing granulosa-like cells from hiPSCs. Vertical black bars represent media changes. (B) Dose dependence for the production of FOXL2+CD82+EPCAM− granulosa-like cells, shown in two monoclonal lines. Granulosa-like cells are efficiently induced from both clones (but not from control cells lacking TF expression vectors) in a doxycycline-dependent manner. (C) Representative gating strategy to analyze flow cytometry data.
Figure 4.
Figure 4.. 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-aggregation.
Figure 5.
Figure 5.. Ovaroid development and germ cell maturation.
Scale bars in immunofluorescence images are 40 µm. (A) Human ovaroid (F66/N.R1.G.F #4 granulosa-like cells + hPGCLCs) sections at days 2, 4, 14, and 32 of culture, stained for FOXL2 (granulosa), OCT4 (germ cell/pluripotent), and DAZL (mature germ cell). (B) Mouse ovaroid (fetal mouse ovarian somatic cells + hPGCLCs) sections stained as in panel A. (C) Fraction of OCT4+ and DAZL+ cells relative to the total (DAPI+) over time in human ovaroids and mouse xeno-ovaroids. Counts were performed at 11 time points on images from 2 replicates of human ovaroids (F66/N.R1.G.F #4 and F66/N.R2 #1 granulosa-like cells + hPGCLCs) and 1 replicate of mouse xeno-ovaroids. (D) Human ovaroid (F66/N.R2 #1 granulosa-like cells + hPGCLCs) sections at days 4 and 8 of culture, stained for SOX17 (germ cell), TFAP2C (early germ cell), and AMHR2 (granulosa).
Figure 5—figure supplement 1.
Figure 5—figure supplement 1.. DAZL and OCT4 expression observed by immunofluorescence in day 16 ovaroids.
Some DAZL+OCT4− cells (magenta arrows) are visible, as well as DAZL+OCT4+ cells (cyan arrows). Ovaroids are also beginning to form follicle-like morphology (yellow arrows). Scale bar is 40 µm.
Figure 6.
Figure 6.. Ovaroid follicle formation.
(A) Day 35 human ovaroid (F66/N.R1.G #7 + hPGCLC) sections stained for FOXL2, OCT4, and AMHR2. Scale bars are 40 µm. Follicle-like structures are marked with yellow triangles. (B) Whole-ovaroid view of follicle-like structures in human ovaroids (F66/N.R1.G #7). Scale bars are 1 mm. (C) Section of human ovaroid (F66/N.R1.G.F #4 + hPGCLC) at day 70 of culture, stained for FOXL2, NR2F2, and AMHR2, showing multiple small follicles (yellow triangles) consisting single layers of FOXL2+AMHR2+ cells. NR2F2+ cells are interspersed between these. Scale bars are 100 µm. (D) Section of human ovaroid (F66/N.R2 #1 + hPGCLC) at day 70 of culture, stained for FOXL2, NR2F2, and AMHR2, showing an antral follicle consisting of FOXL2+AMHR2+ granulosa-like cells arranged in several layers around a central cavity. NR2F2 staining is visible outside of the follicle (marked ‘Stroma’). Scale bars are 100 µm.
Figure 7.
Figure 7.. scRNA-seq analysis of ovaroids (F66/N.R1.G.F #4 granulosa-like cells + hPGCLCs). Data from all samples (days 2, 4, 8, and 14) were combined for joint dimensionality reduction and clustering.
(A) Expression (log2 CPM) of selected granulosa (FOXL2), stroma/theca (NR2F2), and germ cell (PRDM1) markers. (B) Leiden clustering shows four main clusters; the expression (log2 CPM) of marker genes is plotted for each. (C) Mapping of cells onto a human fetal ovary reference atlas (Garcia-Alonso et al., 2022) and assignment of cell types. (D) Proportion of somatic cell types, germ cells, DAZL+ cells, and DDX4+ cells in ovaroids from each day.
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