Methods and compositions for generating granulosa-like cells

Abstract

Provided herein are methods and compositions for differentiating induced pluripotent stem cells into granulosa-like cells by overexpressing transcription factors such as NR5A1 and RUNX family proteins (e.g., RUNX1 and / or RUNX2).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 326,640, filed April 1, 2022, and U.S. Provisional Patent Application No. 63 / 444,108, filed February 8, 2023, each of which is incorporated by reference in its entirety.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (H049870758WO00-SEQ-KVC.xml; size: 7,426 bytes; and creation date: March 30, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] background Granulosa cells are specialized cells that provide critical steroids and growth hormones to the developing oocyte during maturation. Granulosa cells surround the developing oocyte and are involved in maintaining potential pregnancy by regulating hormone levels. Granulosa cell dysfunction forms the basis of many forms of human female infertility, yet efficient methods for generating granulosa cells in vitro remain elusive. Summary of the Invention [Means for solving the problem]

[0004] overview The present disclosure relates, at least in part, to methods and compositions for generating granulosa cells from pluripotent stem cells (PSCs) in vitro. The present disclosure unexpectedly demonstrates that overexpression of certain transcription factors, e.g., nuclear receptor subfamily 5 group A member 1 (NR5A1) and Runt-related transcription factor (RUNX) family members (e.g., RUNX1 and / or RUNX2), can promote the generation of granulosa cells (e.g., AMHR2) from iPSCs within as little as 5 to 7 days. + , CD82 + , FOXL2 + , and / or EPCAM- We provide experimental data demonstrating that the method is sufficient to generate cultured granulosa-like cells.

[0005] Some embodiments of the present disclosure provide a PSC comprising a modified polynucleotide comprising an open reading frame encoding a protein selected from NR5A1 and a RUNX family protein.

[0006] In some embodiments, the PSC comprises a modified polynucleotide comprising an open reading frame encoding NR5A1.

[0007] In some embodiments, the PSC comprises a modified polynucleotide comprising an open reading frame encoding a RUNX family protein.

[0008] In some embodiments, the RUNX family protein is RUNX1.

[0009] In some embodiments, the RUNX family protein is RUNX2.

[0010] In some embodiments, the PSCs express or overexpress NR5A1; RUNX1; RUNX2; NR5A1 and RUNX1; NR5A1 and RUNX2; or NR5A1, RUNX1, and RUNX2.

[0011] In some embodiments, the PSC further comprises a modified polynucleotide comprising an open reading frame encoding a transcription factor 21 (TCF21) protein.

[0012] In some embodiments, the PSCs express or overexpress TCF21.

[0013] In some embodiments, the PSC comprises a modified polynucleotide comprising an open reading frame encoding GATA binding protein 4 (GATA4).

[0014] In some embodiments, the PSCs express or overexpress GATA4.

[0015] In some embodiments, the open reading frame of the modified polynucleotide is operably linked to a heterologous promoter.

[0016] In some embodiments, the heterologous promoter is an inducible promoter.

[0017] Another aspect of the present disclosure provides a PSC comprising an overexpressed protein selected from NR5A1 and a RUNX family protein.

[0018] In some embodiments, the PSCs express or overexpress NR5A1; RUNX1; RUNX2; NR5A1 and RUNX1; NR5A1 and RUNX2; or NR5A1, RUNX1, and RUNX2.

[0019] In some embodiments, the PSCs further comprise a TCF21 protein.

[0020] In some embodiments, the PSCs express or overexpress TCF21.

[0021] In some embodiments, the PSCs further comprise a GATA4 protein.

[0022] In some embodiments, the PSCs express or overexpress GATA4.

[0023] In some embodiments, the PSCs are human PSCs.

[0024] In some embodiments, the PSCs are induced PSCs (iPSCs).

[0025] In some embodiments, the PSC comprises 1 to 20, optionally 8 to 10, copies of a modified polynucleotide comprising an open reading frame encoding a protein selected from NR5A1 and a RUNX family protein (e.g., RUNX1 and / or RUNX2).

[0026] A further aspect of the disclosure provides a composition comprising a population of PSCs as described in any one of the preceding paragraphs, or elsewhere herein.

[0027] In some embodiments, the population is at least 10,000 cells / cm 2 Includes PSCs.

[0028] Some aspects of the disclosure provide methods including culturing a population of PSCs in a medium to generate an expanded population of PSCs; and expressing a protein selected from NR5A1 and a RUNX family protein within the expanded population of PSCs to generate granulosa-like cells.

[0029] In some embodiments, the PSCs of the expanded population comprise a modified polynucleotide that includes an open reading frame encoding NR5A1.

[0030] In some embodiments, the PSCs of the expanded population comprise a modified polynucleotide comprising an open reading frame encoding a RUNX family protein.

[0031] In some embodiments, the RUNX family protein is RUNX1.

[0032] In some embodiments, the RUNX family protein is RUNX2.

[0033] In some embodiments, the PSCs of the expanded population further comprise a modified polynucleotide comprising an open reading frame encoding a TCF21 protein.

[0034] In some embodiments, the PSCs of the expanded population further comprise a modified polynucleotide comprising an open reading frame encoding a GATA4 protein.

[0035] In some embodiments, the open reading frame of the modified polynucleotide is operably linked to a heterologous promoter.

[0036] In some embodiments, the heterologous promoter is an inducible promoter.

[0037] In some embodiments, the population is 1×10 2 ~1×10 7 Includes PSCs.

[0038] In some embodiments, the population of PSCs is cultured for about 4 to 10 days. For example, the population of PSCs may be cultured for about 6 days.

[0039] In some embodiments, the granulosa-like cells are + , CD82 + , FOXL2 + , and / or EPCAM - It is.

[0040] Another aspect of the present disclosure is a method for treating a PSC comprising: (a) delivering to the PSC an engineered polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a protein selected from NR5A1 and a RUNX family protein; (b) culturing PSCs in feeder-free, serum-free medium to generate an expanded population of PSCs; and (c) culturing the expanded population of PSCs in a series of induction media containing an inducer to generate AMHR2. + , CD82 + , FOXL2 + , and / or EPCAM - generating granulosa-like cells; The present invention provides a method comprising:

[0041] In some embodiments, the method comprises delivering to the PSCs (i) a modified polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding NR5A1 and (i) a modified polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a RUNX family protein.

[0042] In some embodiments, the RUNX family protein is RUNX1.

[0043] In some embodiments, the RUNX family protein is RUNX2.

[0044] In some embodiments, the modified polynucleotide is a transposon and the delivering further comprises delivering a transposase to the PSC.

[0045] In some embodiments, the inducible promoter is a chemically inducible promoter, optionally a doxycycline inducible promoter.

[0046] In some embodiments, the feeder-free, serum-free medium of (b) comprises a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma.

[0047] In some embodiments, the solubilized basement membrane preparation comprises extracellular matrix (ECM) proteins and growth factors.

[0048] In some embodiments, the ECM protein is selected from laminin, collagen IV, heparan sulfate proteoglycan, and entactin / nidogen.

[0049] In some embodiments, the feeder-free, serum-free medium of (b) comprises a growth factor selected from recombinant human basic fibroblast growth factor (rh bFGF) and recombinant human transforming growth factor beta (rh TGFβ).

[0050] In some embodiments, the culturing in (b) is carried out for about 6 to 24 hours.

[0051] In some embodiments, the expanded population of PSCs in (c) is about 10,000 cells / cm. 2 to approximately 20,000 cells / cm 2 The cells are cultured at a density of 100 μg / ml.

[0052] In some embodiments, the culturing of (c) comprises culturing the PSCs in a first induction medium and culturing the PSCs in a second induction medium.

[0053] In some embodiments, the first induction medium comprises one or more of L-alanyl-L-glutamine, an antibiotic (e.g., penicillin and / or streptomycin), Dulbecco's Modified Eagle's Medium (DMEM) / F-12, Advanced RPMI (Roswell Park Memorial Institute) 1640 medium, a glycogen synthase kinase (GSK) 3 inhibitor, a small molecule or protein inhibitor of the BMP signaling pathway, a small molecule ROCK inhibitor, and an inducer (e.g., doxycycline).

[0054] In some embodiments, culturing the PSCs in the first induction medium is for about 36 to about 60 hours, optionally for about 48 hours.

[0055] In some embodiments, the second induction medium comprises one or more of L-alanyl-L-glutamine, an antibiotic (e.g., penicillin and / or streptomycin), Advanced RPMI 1640 medium, DMEM / F-12, and an induction agent (e.g., doxycycline).

[0056] In some embodiments, culturing the PSCs in the second induction medium is for about 96 to about 144 hours, optionally for about 120 hours.

[0057] In some embodiments, the second induction medium is removed and replaced with fresh second induction medium at about 24 hour intervals.

[0058] A further aspect of the present disclosure provides a granulosa-like cell produced by a method according to any one of the preceding claims.

[0059] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the invention will be apparent from the following drawings and detailed description of certain embodiments, and from the appended claims.

[0060] Some embodiments provide ovarian organoids comprising granulosa-like cells and human primordial germ cell-like cells (hPGCLCS) according to any one of the preceding claims.

[0061] In some embodiments, the method of any one of the preceding paragraphs further comprises combining granulosa-like cells with hPGCLCS to form ovarian organoids.

[0062] The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component illustrated in various figures is labeled by a numeral or the like. For purposes of clarity, not every component may be labeled in every figure. The drawings are as follows: [Brief description of the drawings]

[0063] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]FIG. 1 shows barcode enrichment screening for TFs that activate expression of FOXL2. TF expression plasmids were introduced into FOXL2-tdTomato reporter iPSCs. Three TF pools were evaluated: C5, containing a total of 5 fmol of all TFs (equimolar mix); B5, containing a total of 5 fmol of a subset of TFs; and B50, containing a total of 50 fmol of the same subset. After nucleofection and puromycin selection, cells were treated with doxycycline to induce TF expression in either pluripotency-supporting mTeSR™Plus medium or mesoderm induction medium. After 5 days, FOXL2+ cells were isolated by FACS and DNA was extracted. Barcode frequencies were compared between FOXL2+ cells and the initial population of cells before TF expression. [Diagram 2] Figure 2 shows the TF combinatorial screen. Various combinations of TF expression plasmids were introduced into FOXL2-tdTomato reporter iPSCs. Then, iPSCs were plated in differentiation medium (DMEM / F12, 10% KSR, 1 μg / mL doxycycline). During the first 2 days, cells were additionally treated with 3 μM CHIR99021 and 10 μM Y-27632. After 5 days of differentiation, cells were dissociated and analyzed by flow cytometry to measure the percentage of AMHR2+FOXL2+CD82+EPCAM- granulosa-like cells. A second replicate was treated with androstenedione (500 ng / mL) and FSH (0.15 IU / mL) from day 4 to day 6 of differentiation. Estradiol production was measured by ELISA. [Diagram 3] Figure 3 shows transcriptional characterization of granulosa-like cells. FOXL2+ cells generated by TF-mediated differentiation were analyzed by RNA-seq. TPM values ​​for known markers of gonadal / granulosa cells, adrenal cells, and pluripotent cells were compared between male fetal gonads, primitive and primary granulosa cells, sorted FOXL2+ cells, COV434 ovarian tumor cells, and iPSCs. Values ​​are the average of at least two biological replicates. [Figure 4]Figure 4 shows that the combination of top TFs generates high yields of granulosa-like cells. Expression plasmids for the top TFs (NR5A1, RUNX1, RUNX2, TCF21) were introduced into iPSCs and single colonies were picked to generate monoclonal lines. Clone 1F has integrated NR5A1, RUNX1, and TCF21 expression plasmids (confirmed by PCR). The day 5 differentiation protocol with doxycycline-inducible TF expression resulted in a nearly homogenous population of FOXL2+CD82+ granulosa-like cells. In comparison, spontaneous differentiation in the absence of doxycycline resulted in only a few granulosa-like cells. [Figure 5A] Hormonal signaling by granulosa-like cells. Figure 5A shows that granulosa-like cells produce estradiol in the presence of androstenedione and either FSH or forskolin (FK). Results are shown from nine monoclonal populations of granulosa-like cells (n=2 biological replicates for each of the nine clones, error bars are 95%CI), as well as COV434 and KGN human ovarian cancer cell lines (controls), HGL5 immortalized primary human granulosa cells, and primary adult mouse granulosa cells. Lines marked with asterisks are cases where FSH production was significantly (two-tailed t-test, p<.05) increased upon stimulation. Exact P values ​​are given in the source data. Monoclonal lines of iPSCs with integrated TFs (NR5A1, TCF21, GATA4, RUNX1) were generated by picking single colonies from the polyclonal populations. These lines were then subjected to a 5-day granulosa differentiation protocol. Cells were then treated with androstenedione (500 ng / mL) for 24 h in the presence or absence of FSH (0.25 IU / mL) and / or forskolin (100 μM). Estradiol production was measured by ELISA. Three of the four lines showed hormone-responsive estradiol production, while the fourth selection (found by PCR to lack RUNX1) produced high levels of estradiol constitutively. [Figure 5B]Figure 5B shows that ovaroids produce both estradiol and progesterone. Estradiol production requires androstenedione and is stimulated by FSH. Results are shown for ovaroids formed by six different monoclonal samples of granulosa-like cells (n=1 sample per ovaroid per condition) at 3 days after aggregation. [Figure 6] Figure 6 shows the protocol for inducing granulosa-like cells. hiPSCs containing integrated TF expression plasmids are cultured in mTeSR™ Plus medium on Corning® Matrigel® matrix. For induction of granulosa-like cells, hiPSCs are dissociated into single cells and plated at a density of 10,000-20,000 cells / cm2 on Corning® Matrigel® matrix in DK10 medium (DMEM / F12 with GlutaMAX™ supplement and 10% knockout serum replacement) + 3 μM CHIR99021, 10 μM Y-27632, and 1 μg / mL doxycycline to induce TF expression. After 48 hours and at 24 hour intervals thereafter, the medium is changed and replaced with fresh DK10 + 1 μg / mL doxycycline. After a total of 120 hours, the granulosa-like cells are ready for downstream experiments. [Figure 7] Figure 7 shows the fraction of OCT4+ and DAZL+ cells relative to the total (DAPI+) in human and mouse xenografts over time. Counts were performed at 11 time points for images from two replicates of human xenografts (F66 / N.R1.GF #4 and F66 / N.R2 #1 granulosa-like cells+hPGCLCs) and one replicate of mouse xenografts. [Figure 8A]Figure 8A shows scRNA-seq analysis of ovarianoids (F66 / N.R1.GF#4 granulosa-like cells + hPGCLCs). Data from all samples (days 2, 4, 8, and 14) were combined for joint dimensionality reduction and clustering. Figure 8B shows expression (log2 CPM) of selected granulosa (FOXL2), stromal / thecal (NR2F2), and germ cell (PRDM1) markers. [Figure 8B] FIG. 8B shows Leiden clustering showing four major clusters; expression of marker genes (log2 CPM) is plotted for each. [Figure 8C] FIG. 8C shows mapping of cells onto the human fetal ovary reference atlas and cell type assignment. [Figure 8D] FIG. 8D shows the percentages of somatic cell types, germ cells, DAZL+ cells, and DDX4+ cells in the ovaloids obtained on each day. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0064] Detailed Description Ovarian granulosa cells are important in many aspects of female reproduction, including oocyte development and support of hormone signaling during the menstrual cycle. Current in vitro models, including human or mouse primary granulosa cells and granulosa cell tumor lines, are inadequate to test these processes (Havelock et al. 2004). For example, the COV434 ovarian tumor line, which is commonly used as a model for granulosa cells (Zhang 2000), lacks the transcriptional and phenotypic characteristics of true granulosa cells. Previously reported protocols for differentiating induced pluripotent stem cells (iPSCs) into granulosa-like cells are either low-yielding (Lan et al. 2013; Lipskind et al. 2018) or exclusively applicable to mouse cells (Yoshino et al. 2021). Aspects of the present disclosure use direct transcription factor overexpression to mediate differentiation of iPSCs, resulting in the production of AMHR2-like cells within about 4 to about 10 days. + (anti-Mullerian hormone receptor type 2), CD82 +(Surface Antigen Classification 82), FOXL2 + (Forkhead Box L2), and / or EPCAM - (epithelial cell adhesion molecule) granulosa-like cells. It should be understood that the term "granulosa-like cells" encompasses cells that express granulosa-specific markers, e.g., AMHR2, CD82, and / or FOXL2, and / or do not express EPCAM, and that exhibit other characteristics of naturally occurring granulosa.

[0065] Aspects of the present disclosure relate to pluripotent stem cells (PSCs) comprising a modified polynucleotide comprising an open reading frame encoding a protein selected from nuclear receptor subfamily 5 group A member 1 (NR5A1) and a Runt-related transcription factor (RUNX) family protein. In some embodiments, the modified polynucleotide comprises an open reading frame encoding NR5A1. In some embodiments, the modified polynucleotide comprises an open reading frame encoding a RUNX family protein. In some embodiments, the RUNX family protein is Runt-related transcription factor 1 (RUNX1). In some embodiments, the RUNX family protein is Runt-related transcription factor 2 (RUNX2).

[0066] Granulosa-like cells Some aspects of the present disclosure provide granulosa-like cells and methods for producing such cells. Granulosa cells or follicular cells are somatic cells of the sex cord that are closely associated with the developing female gamete (oocyte / egg) in the mammalian ovary. In the primordial ovarian follicle and later in follicular development (folliculogenesis), granulosa cells progress to form a multi-layered cumulus that surrounds the oocyte in the preovulatory or antral follicle (Grafian follicle). The primary function of granulosa cells includes the production of sex steroids and a myriad of growth factors that are believed to interact with the oocyte during its development. Sex steroid production begins with follicle-stimulating hormone (FSH) from the anterior pituitary gland, which stimulates granulosa cells to convert androgens (derived from theca cells) to estradiol by aromatase during the follicular phase of the menstrual cycle. After ovulation, granulosa cells transform into granulosa lutein cells that produce progesterone. Progesterone induces the production of thick cervical mucus that can maintain a potential pregnancy and inhibits sperm entry into the uterus.

[0067] There are two types of granulosa cells: cumulus cells (CC) and mural granulosa cells (MGC). Cumulus cells surround the oocyte. They provide nutrients to the oocyte and affect oocyte development in a paracrine manner. Mural granulosa cells cover the follicle wall and surround a fluid-filled cavity. Oocytes secrete factors that determine the functional differences between CC and MGG. CCs primarily support oocyte growth and development, while MGCs primarily perform endocrine functions and support follicle growth. Cumulus cells contribute to oocyte development and show higher expression of SLC38A3, a transporter of amino acids, as well as Aldoa, Eno1, Ldh1, Pfkp, Pkm2, and Tpi1, enzymes involved in glycolysis. MGCs are more steroidogenically active and have higher levels of mRNA expression of steroidogenic enzymes such as cytochrome P450. MGCs produce increased amounts of estrogen, which triggers the LH surge. After the LH surge, the cumulus cells undergo cumulus expansion, during which they proliferate at a rate 10 times higher than MGCs in response to FSH. During expansion, CCs also produce the mucus matrix required for ovulation.

[0068] Granulosa cells express several different biomarkers that can be used to distinguish granulosa and granulosa-like cells from other cell types. For example, granulosa cells typically express the anti-Mullerian hormone receptor type 2 (AMHR2) (AMHR2 + ), CD82 molecule (CD82 + ), Fork Head Box L2 (FOXL2 + ) and epithelial cell adhesion molecule (EPCAM - ). Thus, in some embodiments, the granulosa-like cells generated by the methods provided herein are negative for AMHR2. + , CD82 + , FOXL2 + , and / or EPCAM - Granulosa-like cells (i.e., cells that express AMHR2, CD82, and / or FOXL2 proteins, but do not express EPCAM at detectable levels).

[0069] There are other characteristics of granulosa-like cells that distinguish them from non-granulosa-like cells, including, but not limited to, the expression of adhesion proteins such as adherens (B-catenin, a,catenin, N-cadherin, nectins 1-3), as well as combinations of tight junctions (JAM-A, cingulin), desmosomes (dsg2, Dsc2) and linkers (afadin, ZO-1,2 and ZONAB). Granulosa-like cells are further distinguished by the biosynthesis of a combination of estradiol, progesterone and AMH.

[0070] pluripotent stem cells The granulosa-like cells presented herein are differentiated from pluripotent stem cells, which have the ability to self-renew by dividing and to express in the three primary germ cell layers (e.g., ectoderm, endoderm, and mesoderm) of early embryos and in all cells of adults, but not in extraembryonic tissues such as placenta (Shi et al. 2017).

[0071] Non-limiting examples of pluripotent stem cells include induced pluripotent cells (iPSCs), "true" embryonic stem cells (ESCs) derived from embryos, embryonic stem cells produced by somatic cell nuclear transfer (ntESCs), and embryonic stem cells derived from non-fertilized eggs (parthenogenetic embryonic stem cells, or pESCs). In some embodiments, the pluripotent cells are human pluripotent cells.

[0072] In some embodiments, the pluripotent stem cells are embryonic stem cells, such as human embryonic stem cells. "Embryonic stem cells" is a general term for pluripotent stem cells that are generated using embryos or eggs, rather than genetically reprogrammed cells from the body. As used herein, "ESCs" encompasses true ESCs, ntESCs, and pESCs.

[0073] In other embodiments, the pluripotent stem cells are induced pluripotent stem cells, such as human induced pluripotent stem cells. iPSCs may be derived from skin or blood cells that have been reprogrammed back to an embryonic-like pluripotent state, allowing for the expression of an unlimited source of any type of human cell.

[0074] Some aspects of the disclosure provide PSCs comprising an expressed or overexpressed protein selected from NR5A1 and a RUNX family protein (e.g., RUNX1 and / or RUNX2). In some embodiments, the PSCs further comprise a TCF21 protein. In some embodiments, the protein is expressed at a level at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, or at least 100% higher than a control level. In some embodiments, the PSCs further comprise a GATA4 protein. In some embodiments, the protein is expressed at a level at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, or at least 100% higher than a control level. In some embodiments, the control level is an endogenous level of the protein, for example in a naturally occurring pluripotent stem cell. In some embodiments, the PSCs comprise NR5A1. In some embodiments, the PSCs express or overexpress NR5A1. In some embodiments, the PSC comprises a RUNX family protein (e.g., RUNX1 and / or RUNX2). In some embodiments, the PSC expresses or overexpresses a RUNX family protein (e.g., RUNX1 and / or RUNX2). In some embodiments, the PSC comprises RUNX1. In some embodiments, the PSC expresses or overexpresses RUNX1. In some embodiments, the PSC comprises RUNX2. In some embodiments, the PSC expresses or overexpresses RUNX2. In some embodiments, the PSC comprises TCF21. In some embodiments, the PSC expresses or overexpresses TCF21. In some embodiments, the PSC comprises GATA4. In some embodiments, the PSC expresses or overexpresses GATA4.

[0075] Data provided herein show that combinatorial expression of NR5A1 and a RUNX family protein (e.g., RUNX1 and / or RUNX2) results in a 2-15-fold increase in the efficiency of granulosa cell-like production over a control, optionally where the control is the efficiency of granulosa cell-like production in PSCs expressing only NR5A1 or one of the RUNX family proteins (e.g., RUNX1 and / or RUNX2). In some embodiments, the PSCs comprise NR5A1 and a RUNX family protein (e.g., RUNX1 and / or RUNX2). In some embodiments, the PSCs express or overexpress NR5A1 and a RUNX family protein (e.g., RUNX1 and / or RUNX2). In some embodiments, the PSCs comprise NR5A1 and RUNX1. In some embodiments, the PSCs express or overexpress NR5A1 and RUNX1. In some embodiments, the PSCs comprise NR5A1 and RUNX2. In some embodiments, the PSC expresses or overexpresses NR5A1 and RUNX2. In some embodiments, the PSC further comprises TCF21. In some embodiments, the PSC further expresses or overexpresses TCF21. In some embodiments, the PSC further comprises GATA4. In some embodiments, the PSC further expresses or overexpresses GATA4.

[0076] Transcription factors Granulosa-like cells provided herein, in some embodiments, are differentiated from pluripotent stem cells by expressing one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) transcription factors (i.e., proteins that control the rate of transcription). Differentiation is a process by which uncommitted or partially committed cells commit to a specialized cell fate. Aspects of the present disclosure relate to the differentiation of uncommitted pluripotent stem cells to a granulosa-like cell fate.

[0077] In some embodiments, the transcription factor is selected from NR5A1 and a RUNX family protein (e.g., RUNX1 and / or RUNX2). In some embodiments, the pluripotent stem cells, e.g., hPSCs or hiPSCs, are modified to express or overexpress NR5A1. In some embodiments, the pluripotent stem cells, e.g., hPSCs or hiPSCs, are modified to express or overexpress a RUNX family protein (e.g., RUNX1 and / or RUNX2). In some embodiments, the pluripotent stem cells, e.g., hPSCs or hiPSCs, are modified to express or overexpress RUNX1. In some embodiments, the pluripotent stem cells, e.g., hPSCs or hiPSCs, are modified to express or overexpress RUNX2. In some embodiments, the pluripotent stem cells, e.g., hPSCs or hiPSCs, are modified to express or overexpress TCF21. In some embodiments, the pluripotent stem cells, e.g., hPSCs or hiPSCs, are modified to express or overexpress GATA4. In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are modified to express or overexpress NR5A1 and a RUNX family protein (e.g., RUNX1 and / or RUNX2). In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are modified to express or overexpress NR5A1 and RUNX1. In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are modified to express or overexpress NR5A1 and RUNX2. In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are modified to further express or overexpress TCF21. In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are modified to further express or overexpress GATA4.

[0078] A cell "expresses" a particular protein if the level of the protein within the cell is detectable (e.g., using a known protein assay). A cell "overexpresses" a particular protein (e.g., a modified polynucleotide encoding the protein) if the level of the protein is higher (e.g., at least 5%, at least 10%, or at least 20% higher) than the level of the protein expressed from an endogenous, naturally-occurring polynucleotide encoding the protein.

[0079] Modified Polynucleotides and Polypeptides The pluripotent stem cells of the present disclosure, in some embodiments, comprise modified polynucleotides. Modified polynucleotides are non-naturally occurring nucleic acids (e.g., at least two nucleotides covalently linked together, in some cases including phosphodiester bonds, referred to as a phosphodiester backbone). Modified polynucleotides include recombinant and synthetic nucleic acids. Recombinant nucleic acids are molecules that are constructed by linking nucleic acids (e.g., isolated nucleic acids, synthetic nucleic acids, or combinations thereof) from two different organisms (e.g., human and mouse). Synthetic nucleic acids are molecules that have been amplified or synthesized chemically or by other means. Synthetic nucleic acids include those that have been chemically modified or otherwise modified, but that can base pair (bind) with naturally occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include molecules that are obtained by replicating any of the above.

[0080] Modified polynucleotides can include DNA (e.g., genomic DNA, cDNA, or a combination of genomic DNA and cDNA), RNA, or hybrid molecules, e.g., nucleic acids containing any combination of deoxyribonucleotides and ribonucleotides (e.g., artificial or natural) and any combination of two or more bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine.

[0081] In some embodiments, a polynucleotide is complementary DNA (cDNA), which is synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by reverse transcriptase.

[0082] The modified polynucleotides of the present disclosure can be generated using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, the nucleic acids are generated using GIBSON ASSEMBLY® cloning (see, e.g., Gibson, DGet al. Nature Methods, 343-345, 2009; and Gibson, DGet al. Nature Methods, 901-903, 2010, each of which is incorporated herein by reference). GIBSON ASSEMBLY® typically employs three enzyme activities in a single tube reaction: a 5' exonuclease, a 3' extension activity of a DNA polymerase, and a DNA ligase activity. The 5' exonuclease activity chews back the 5' terminal sequence, exposing the complementary sequence and preparing it for annealing. Polymerase activity then fills the gaps on the annealing domain. DNA ligase then seals the nicks and covalently joins the DNA fragments together. The overlapping sequences of the adjacent fragments are much longer than those used in Golden Gate assembly, thus resulting in a higher percentage of correct assembly. Other methods of generating modified polynucleotides may be used in accordance with the present disclosure.

[0083] In some embodiments, the modified polynucleotide comprises a promoter operably linked to the open reading frame. A promoter is a nucleotide sequence at which RNA polymerase binds to an initial transcript (e.g., ATG). A promoter is typically located immediately upstream (at its 5' end) from the transcription start site. In some embodiments, the promoter is a heterologous promoter. A heterologous promoter is not naturally associated with the open reading frame to which it is operably linked.

[0084] In some embodiments, the promoter is an inducible promoter. Inducible promoters can be regulated in vivo, for example, by chemical agents, temperature, or light. Inducible promoters allow for temporal and / or spatial control of gene expression, for example. Inducible promoters for use according to the present disclosure include any inducible promoter described herein or known to one of skill in the art. Examples of inducible promoters include, but are not limited to, chemically / biochemically regulated promoters and physically regulated promoters, such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, including tetracycline repressor protein (tetR), tetracycline operator sequence (tetO), and tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., rat glucocorticoid receptor, human steroid-regulated promoter ... Examples of suitable promoters include promoters based on the human estrogen receptor, moth ecdysone receptor, and promoters from the steroid / retinoid / thyroid 25 receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (a protein that binds and sequesters metal ions) genes from yeast, mouse, and human), pathogen-regulated promoters (e.g., induced by salicylic acid, ethylene, or benzothiadiazole (BTH)), temperature / heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light-responsive promoters from plant cells). In some embodiments, the inducible promoter is a tetracycline-inducible promoter. In some embodiments, the inducible promoter is a doxycycline-inducible promoter. In other embodiments, the promoter is a constitutive promoter (in vivo active, non-regulated).

[0085] An open reading frame is a contiguous stretch of codons that begins with a start codon (e.g., ATG) and ends with a stop codon (e.g., TAA, TAG, or TGA) and encodes a polypeptide, e.g., a protein. An open reading frame is operably linked to a promoter if the promoter controls transcription of the open reading frame.

[0086] Vectors used to deliver modified polynucleotides include minicircles, plasmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes. Transposon-based systems, such as the piggyBac™ system (see, e.g., Chen et al. Nature Communications. 2020; 11(1): 3446), are also contemplated herein.

[0087] The pluripotent stem cells, in some embodiments, comprise a modified polynucleotide comprising an open reading frame encoding a protein selected from NR5A1 and a RUNX family protein (e.g., RUNX1 and / or RUNX2). In some embodiments, the modified polynucleotide comprises an open reading frame encoding NR5A1. In some embodiments, the modified polynucleotide comprises an open reading frame encoding a RUNX family protein (e.g., RUNX1 and / or RUNX2). In some embodiments, the modified polynucleotide comprises an open reading frame encoding RUNX1. In some embodiments, the modified polynucleotide comprises an open reading frame encoding RUNX2. In some embodiments, the modified polynucleotide comprises an open reading frame encoding TCF21. In some embodiments, the modified polynucleotide comprises an open reading frame encoding GATA4.

[0088] In some embodiments, the pluripotent stem cells comprise a modified polynucleotide comprising an open reading frame encoding NR5A1 and a modified polynucleotide comprising an open reading frame encoding a RUNX family protein (e.g., RUNX1 and / or RUNX2). In some embodiments, the pluripotent stem cells comprise a modified polynucleotide comprising an open reading frame encoding NR5A1 and a modified polynucleotide comprising an open reading frame encoding RUNX1. In some embodiments, the pluripotent stem cells comprise a modified polynucleotide comprising an open reading frame encoding NR5A1 and a modified polynucleotide comprising an open reading frame encoding RUNX2. In some embodiments, the pluripotent stem cells further comprise a modified polynucleotide comprising an open reading frame encoding TCF21. In some embodiments, the pluripotent stem cells further comprise a modified polynucleotide comprising an open reading frame encoding GATA4.

[0089] In some embodiments, a modified polynucleotide comprising an open reading frame encoding nuclear receptor subfamily 5 group A member 1 (NR5A1) (e.g., UniprotKB Accession No. Q13285) is [ka] It encodes a protein comprising the sequence:

[0090] In some embodiments, a modified polynucleotide comprising an open reading frame encoding Runt-related transcription factor 1 (RUNX1) (e.g., UniprotKB Accession No. Q01196) is [ka] It encodes a protein comprising the sequence:

[0091] In some embodiments, a modified polynucleotide comprising an open reading frame encoding Runt-related transcription factor 2 (RUNX2) (e.g., UniprotKB Accession No. Q13950) is [ka] It encodes a protein comprising the sequence:

[0092] In some embodiments, a modified polynucleotide comprising an open reading frame encoding transcription factor 21 (TCF21) (e.g., UniprotKB Accession No. O43680) is [ka] It encodes a protein comprising the sequence:

[0093] A modified polynucleotide comprising an open reading frame encoding GATA binding protein 4 (GATA4) (e.g., UniprotKB Accession No. P43694) is, in some embodiments, [ka] It encodes a protein comprising the sequence:

[0094] The number of copies of the modified polynucleotide delivered to the PSC can vary. In some embodiments, the PSC comprises 1-20 copies of the modified polynucleotide. For example, the PSC may comprise 1-15, 1-10, 2-10, 2-15, 2-10, 5-20, 5-15, or 5-10 copies of the modified polynucleotide. In some embodiments, the PSC comprises 8-10 copies of the modified polynucleotide. More than 20 copies are also contemplated herein.

[0095] Methods for generating granulosa-like cells Methods of generating granulosa-like cells provided herein, in some aspects, include culturing a population of pluripotent stem cells (PSCs) in a medium to generate an expanded population of PSCs; and expressing a protein selected from NR5A1 and a RUNX family protein (e.g., RUNX1 and / or RUNX2) in the expanded population of PSCs to generate granulosa-like cells. In some embodiments, the method further includes expressing a TCF21 protein in the expanded population of PSCs. In some embodiments, the method further includes expressing a GATA4 protein in the expanded population of PSCs.

[0096] In some embodiments, the PSCs of the expanded population comprise a modified polynucleotide that comprises an open reading frame encoding NR5A1. In some embodiments, the PSCs of the expanded population comprise a modified polynucleotide that comprises an open reading frame encoding a RUNX family protein (e.g., RUNX1 and / or RUNX2). In some embodiments, the PSCs of the expanded population comprise a modified polynucleotide that comprises an open reading frame encoding RUNX1. In some embodiments, the PSCs of the expanded population comprise a modified polynucleotide that comprises an open reading frame encoding RUNX2. In some embodiments, the PSCs of the expanded population comprise a modified polynucleotide that comprises an open reading frame encoding TCF21. In some embodiments, the PSCs of the expanded population comprise a modified polynucleotide that comprises an open reading frame encoding GATA4.

[0097] In some embodiments, the open reading frame of the modified polynucleotide is operably linked to a heterologous promoter.

[0098] In some embodiments, the heterologous promoter is an inducible promoter, non-limiting examples of which are provided elsewhere herein.

[0099] The starting population is approximately 1 × 102 ~1×10 10 pieces, about 1×10 2 ~1×10 9 pieces, about 1×10 2 ~1×10 8 Pieces, or about 1 x 10 2 ~1×10 7 In some embodiments, the population comprises about 1 x 10 PSCs. 3 ~1×10 8 Pieces or about 1 x 10 3 ~1×10 7 In some embodiments, the population comprises about 1 x 10 PSCs. 4 ~1×10 7 Pieces or about 1 x 10 5 ~1×10 6 In some embodiments, the population comprises about 1 x 10 PSCs. 1 PSCs, approximately 1 x 10 2 PSCs, approximately 1 x 10 3 PSCs, approximately 1 x 10 4 PSCs, approximately 1 x 10 5 PSCs, approximately 1 x 10 6 PSCs, approximately 1 x 10 7 PSCs, approximately 1 x 10 8 PSCs, approximately 1 x 10 9 PSCs, or approximately 1 x 10 10 Includes PSCs.

[0100] In some embodiments, the population of PSCs is cultured for about 4 to about 10 days, about 4 to about 9 days, about 4 to about 8 days, about 4 to about 7 days, about 4 to about 6 days, about 5 to about 10 days, about 5 to about 9 days, about 5 to about 8 days, about 5 to about 7 days, or about 5 to about 6 days. In some embodiments, the population of PSCs is cultured for about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.

[0101] Some methods of the disclosure include: (a) delivering to PSCs an engineered polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a protein selected from NR5A1 and a RUNX family protein (e.g., RUNX1 and / or RUNX2); (b) culturing the PSCs in a feeder-free, serum-free medium to generate an expanded population of PSCs; and (c) culturing the expanded population of PSCs in a series of induction media comprising an inducer to generate an expanded population of PSCs. + , CD82 + , FOXL2 + , and / or EPCAM - and generating granulosa-like cells. In some embodiments, the series of induction media includes a first, a second, a third, and a fourth induction medium. In some embodiments, a modified polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a TCF21 protein is also delivered to the PCSs (e.g., in step (a)). In some embodiments, a modified polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a GATA4 protein is also delivered to the PCSs (e.g., in step (a)).

[0102] In some embodiments, the PSCs are cultured in a feeder-free serum-free medium for about 6 hours to about 24 hours. For example, the PSCs may be cultured in a feeder-free serum-free medium for about 6 hours to about 12 hours. In some embodiments, the PSCs are cultured in a feeder-free serum-free medium for about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours.

[0103] In some embodiments, the expanded population of PSCs comprises at least 5×10 3 For example, an expanded population may contain (e.g., at the time of induction) at least 1 x 10 PSCs.4 Pieces, at least 1 × 10 5 Pieces, at least 1 × 10 6 or at least 1 × 10 7 In some embodiments, the expanded population of PSCs may comprise about 5×10 3 Approximately 1 × 10 PSCs 7 Includes PSCs.

[0104] In some embodiments, the expanded population of PSCs comprises about 10,000 cells / cm. 2 to approximately 30,000 cells / cm 2 In some embodiments, the expanded population of PSCs are cultured at a density of about 10,000 cells / cm. 2 to approximately 25,000 cells / cm 2 In some embodiments, the expanded population of PSCs are cultured at a density of about 10,000 cells / cm. 2 to approximately 20,000 cells / cm 2 In some embodiments, the expanded population of PSCs are cultured at a density of about 10,000 cells / cm. 2 to approximately 15,000 cells / cm 2 In some embodiments, the expanded population of PSCs are cultured at a density of about 15,000 cells / cm. 2 to approximately 30,000 cells / cm 2 In some embodiments, the expanded population of PSCs are cultured at a density of about 15,000 cells / cm. 2 to approximately 25,000 cells / cm 2 In some embodiments, the expanded population of PSCs are cultured at a density of about 15,000 cells / cm. 2 to approximately 20,000 cells / cm 2 In some embodiments, the PSCs of the expanded population are cultured at a density of at least 10,000 / cm. 2 , at least 15,000 / cm 2 , at least 20,000 / cm 2 , at least 25,000 / cm 2 , or at least 30,000 / cm 2 The cells are cultured at a density of 100 μg / ml.

[0105] In some embodiments, the PSCs of the expanded population are cultured for 10 days or less, 9 days or less, 8 days or less, 7 days or less, 6 days or less, 5 days or less, or 4 days or less. For example, the PSCs of the expanded population may be cultured for about 4 to about 10 days, about 4 to about 9 days, about 4 to about 8 days, about 4 to about 7 days, about 4 to about 6 days, about 5 to about 10 days, about 5 to about 9 days, about 5 to about 8 days, about 5 to about 7 days, or about 5 to about 6 days. In some embodiments, the PSCs of the expanded population are cultured for about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.

[0106] In some embodiments, the expanded population of PSCs are cultured in the first induction medium for about 36 hours to about 60 hours. For example, the PSCs may be cultured in the first induction medium for about 36 hours to about 54 hours, about 36 hours to about 48 hours, about 42 hours to about 60 hours, about 42 hours to about 54 hours, about 42 hours to about 48 hours, about 48 hours to about 60 hours, or about 48 hours to about 54 hours. In some embodiments, the PSCs are cultured in the first induction medium for about 36 hours, about 42 hours, about 48 hours, about 54 hours, or about 60 hours.

[0107] In some embodiments, the expanded population of PSCs are cultured in the second induction medium for about 96 hours to about 144 hours. For example, the PSCs may be cultured in the second induction medium for about 96 hours to about 132 hours, about 96 hours to about 120 hours, about 96 hours to about 108 hours, about 108 hours to about 144 hours, about 108 hours to about 132 hours, about 108 hours to about 120 hours, about 120 hours to about 144 hours, or about 120 hours to about 132 hours. In some embodiments, the PSCs are cultured in the second induction medium for about 96 hours, about 108 hours, about 120 hours, about 132 hours, or about 144 hours.

[0108] Cultivation in the second induction medium, in some embodiments, includes several (one or more) medium changes. For example, the second induction medium may be removed and replaced with new (fresh) second designated medium (about every) 12 hours, 24 hours, 36 hours, or 48 hours. In some embodiments, the second induction medium is changed (about) every 24 hours.

[0109] Transfection method The modified polynucleotides of the present disclosure may be delivered to the PSCs using any one or more transfection methods, including chemical transfection, viral transduction, and electroporation.

[0110] In some embodiments, the modified polynucleotide is delivered on a vector. A vector is any vehicle, such as a virus or a plasmid, used to introduce a desired polynucleotide into a host cell, such as a PSC. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is not a naturally occurring viral vector. Viral vectors may be derived from adeno-associated virus (AAV), adenovirus, herpes simplex virus, lentiviral, retrovirus, varicella, variola virus, hepatitis B, cytomegalovirus, JC polyomavirus, BK polyomavirus, monkeypox virus, Herpes Zoster, Epstein-Barr virus, human herpes virus 7, Kaposi's sarcoma-associated herpesvirus, or human parvovirus B19. Other viral vectors are encompassed by this disclosure.

[0111] In some embodiments, the viral vector is an AAV vector. AAV is a small non-enveloped virus that is approximately 5 kb long and packages a single-stranded linear DNA genome that is intended for use as a gene transfer vehicle (Samulski, RJ et al., Annu Rev Virol. 2014; 1(1): 427-51). The coding region of AAV is flanked by inverted terminal repeats (ITRs), which act as origins for DNA replication and serve as primary packaging signals (McLaughlin, SK et al. Virol. 1988; 62(6): 1963-73; Hauswirth, WW et al. 1977; 78(2): 488-99). Thus, AAV vectors typically include ITR sequences. Both positive and negative strands are equally efficient and packaged into infectious virions (Zhong, L et al. Mol Ther. 2008; 16(2): 290-5; Zhou, X et al. Mol Ther. 2008; 16(3): 494-9; Samulski, RJ et al. Virol. 1987; 61(10): 3096-101). Furthermore, a small deletion in one of the two ITRs allows packaging of self-complementary vectors, whose genomes self-anneal after virus uncoating. This results in more efficient transduction of cells, but halves the coding capacity (McCarty, DM et al. Mol Ther. 2008; 16(10): 1648-56; McCarty, DM et al. Gene Ther. 2001; 8(16): 1248-54).

[0112] In some embodiments, polynucleotides are delivered to cells using a transposon / transposase system. For example, the piggyBac™ transposon system may be used. The piggyBac™ transposon is a mobile genetic element that efficiently transposes between vectors and chromosomes via a "cut and paste" mechanism (Woodard et al. 2015). During transposition, the piggyBac™ transposase recognizes transposon-specific inverted terminal repeats (ITRs) located on both ends of the transposon vector, efficiently displacing its contents from their original site and integrating them into the TTAA chromosomal site. The piggyBac™ transposon system facilitates efficient integration of polynucleotides into the cell genome.

[0113] Thus, in some embodiments, the methods comprise delivering a transposon comprising the modified polynucleotide to the PSCs and further comprising also delivering a transposase.

[0114] In some embodiments, modified polynucleotide is delivered to cell by electroporation.Electroporation is a physical transfection method that uses electric pulse to create temporary pores in cell membrane, through which modified polynucleotide can pass into cell.See, for example, Chicaybam L et al.Front.Bioeng.Biotechnol.,23 January 2017.

[0115] After transfection, the modified polynucleotide may be integrated into the genome of the PSC. In some embodiments, the modified polynucleotide may further comprise an antibiotic resistance gene to confer resistance to an antibiotic used in the antibiotic drug selection process. In this way, a "pure" population of cells containing the integrated modified polynucleotide may be obtained. In some embodiments, the population of cells containing the integrated modified polynucleotide is selected using antibiotic drug selection. Antibiotic drug selection is a process in which a population of cells is treated with an antibiotic such that only cells that are viable in the presence of the antibiotic remain in the population. Non-limiting examples of antibiotics that may be used for antibiotic drug selection include puromycin, blasticidin, geneticin, hygromycin, mycophenolic acid, zeocin, carbenicillin, kanemycin, ampicillin, and actinomycin.

[0116] Culture medium The methods presented herein, in some embodiments, include culturing PSCs in a feeder-free, serum-free medium. The medium may be, for example, a solubilized basement membrane preparation (e.g., Corning® Matrigel® Matrix) (approximately 75 μl / cm 2 ) extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma. 2 to about 150 μl / cm 2 In some embodiments, the solubilized basement membrane preparation comprises one or more extracellular matrix (ECM) proteins and one or more growth factors. For example, the ECM proteins may be selected from laminin, collagen IV, heparan sulfate proteoglycan, and entactin / nidogen.

[0117] In some embodiments, the medium further comprises one or more growth factors selected from, for example, recombinant human basic fibroblast growth factor (rh bFGF) (e.g., 80 ng / ml to 120 ng / ml) and recombinant human transforming growth factor beta (rh TGFβ) (e.g., 20 pM to 25 pM). In some embodiments, the medium further comprises rh bFGF and rh TGFβ. In some embodiments, the medium comprises mTeSR™ Plus medium (STEMCELL Technologies).

[0118] In some embodiments, the first induction medium contains L-alanyl-L-glutamine (e.g., 1.8 mM to 2.2 mM), antibiotics (e.g., penicillin and / or streptomycin) (e.g., 45 U / ml to 50 U / ml), Dulbecco's Modified Eagle Medium (DMEM) / F-12 (e.g., 15 mM HEPES, no glutamine), Advanced The first induction medium may include one or more (e.g., 2, 3, 4, or more) of RPMI (Roswell Park Memorial Institute) 1640 medium (with non-essential amino acids and sodium pyruvate), a glycogen synthase kinase (GSK) 3 inhibitor (e.g., 3 μM to 10 μM), a protein inhibitor of the BMP signaling pathway (e.g., noggin, 2 ng / mL to 20 ng / mL) or a small molecule inhibitor (e.g., dorsomorphin, e.g., 100 nM to 400 nM), a small molecule ROCK inhibitor (e.g., 9 μM to 11 μM), and an inducer (e.g., doxycycline (e.g., 50 ng / ml to 2000 ng / ml)). For example, the first induction medium may include DK10 medium, CHIR99021, Y-27632 (a small molecule ROCK inhibitor), and doxycycline.

[0119] In some embodiments, the second induction medium comprises one or more (e.g., 2, 3, 4, or more) of L-alanyl-L-glutamine, an antibiotic (e.g., penicillin and / or streptomycin), Advanced RPMI 1640 medium (with non-essential amino acids and sodium pyruvate), DMEM / F-12, and an inducer (e.g., doxycycline). For example, the second induction medium may comprise DK10 medium and doxycycline.

[0120] Advanced RPMI 1640 (Thermo Fisher Scientific) is made with glucose, non-essential amino acids, sodium pyruvate (without L-glutamine and HEPES), vitamins, inorganic salts, proteins (e.g., AlbuMAX® II, human transferrin, sand insulin recombinant full chain), and trace elements.

[0121] As used herein, DK10 medium contains KO-SR, glutamine, penicillin, streptomycin, and Dulbecco's modified Eagle's medium (DMEM) / F-12.

[0122] As used herein, Knockout™ Serum Replacement (KO-SR) is a serum-free formulation used as an alternative to fetal bovine serum.

[0123] GlutaMAX™ supplement contains L-alanyl-L-glutamine, a dipeptide replacement with L-glutamine.

[0124] CHIR99021 inhibits GSK3β (IC 50 = 6.7 nM) and GSK3α (IC 50 CHIR99021 is an aminopyrimidine derivative that is an extremely potent glycogen synthase kinase (GSK) 3 inhibitor, inhibiting both IL-1 and IL-2 (=10 nM). GSK3 is a serine / threonine kinase that is a major inhibitor of the WNT pathway; therefore, CHIR99021 functions as a WNT activator.

[0125] Noggin is a protein that binds to and inactivates proteins in the BMP family. Dorsomorphin is a small molecule inhibitor of the type I BMP receptor serine / threonine kinase. Both of these substances are considered inhibitors of the BMP signaling pathway.

[0126] Therapeutic Compositions and Methods of Use The present disclosure provides, in some embodiments, a therapeutic composition comprising the granulosa-like cells produced herein. In some embodiments, the composition further comprises a pharma- ceutically acceptable excipient. The composition, in some embodiments, is cryopreserved.

[0127] Such compositions may be administered to a subject, e.g., a human subject, using any suitable route of administration. Suitable routes of administration include parenteral routes, such as, for example, intravenous, intrathecal, parenchymal, or intraventricular routes. Suitable routes of administration include parenteral routes, such as, for example, intravenous, intrathecal, parenchymal, or intraventricular injection.

[0128] In some embodiments, the subject is a human subject. In patients suffering from primary ovarian insufficiency or menopause, impaired granulosa cell function may benefit from the use of these cells. Thus, in some embodiments, the subject has been diagnosed with ovarian insufficiency, and the granulosa cells provided herein are used to treat the ovarian insufficiency. In other embodiments, the subject is undergoing menopause, and the granulosa cells provided herein are used to treat the menopause (e.g., to alleviate symptoms associated therewith).

[0129] In addition, such compositions can be used to improve the quality and in vitro maturation of oocytes and / or embryos, for example, during the in vitro fertilization (IVF) process and / or during related assisted reproductive technology (ART) procedures, such as egg freezing. Such administration methods include, for example, co-culture of granulosa-like cells with immature and mature oocytes in vitro. In some embodiments, the subject is a human subject. Such subjects will have experienced oocyte freezing or fertilization through IVF and may suffer from, for example, infertility, age-related oocyte immaturity, polycystic ovary syndrome (PCOS) and / or ovarian hyperstimulation syndrome (OHSS), which leaves many oocytes immature and unusable. In vitro application of granulosa-like cells can improve the quality and utility of these oocytes.

[0130] The composition can be administered to a subject in a therapeutically effective amount. The term "therapeutically effective amount" refers to the number of granule membranes required to produce a therapeutic effect on a subject, either alone or in combination with at least one other active agent. The effective amount varies depending on the route of administration, excipient use, and co-use with other active agents, as understood by those skilled in the art. The amount to be administered depends on the subject to be treated, such as the strength of the individual's immune system or genetic predisposition. A suitable dosage range can be easily determined by those skilled in the art and may be on the order of micrograms of the polypeptide of the present disclosure. The dosage of the formulations disclosed herein may depend on the route of administration and varies depending on the size of the subject.

[0131] It is believed that one skilled in the art can utilize the present invention to its full extent based on the above description. The following specific embodiments are therefore to be construed as merely illustrative, and in no way limiting of the remainder of the disclosure. All publications cited in this application are incorporated by reference for the purposes or subject matter referenced in this disclosure.

[0132] Additional Embodiments The present disclosure also relates to additional embodiments described in the following numbered paragraphs: 1. A pluripotent stem cell (PSC) comprising a modified polynucleotide comprising an open reading frame encoding a protein selected from NR5A1 and a RUNX family protein. 2. The PSC of paragraph 1, comprising a modified polynucleotide comprising an open reading frame encoding NR5A1. 3. The PSC of paragraph 1 or 2, comprising a modified polynucleotide comprising an open reading frame encoding a RUNX family protein. 4. The PSC of paragraph 3, wherein the RUNX family protein is RUNX1. 5. The PSC of paragraph 3 or 4, wherein the RUNX family protein is RUNX2. 6. A PSC of any one of the preceding paragraphs, which expresses or overexpresses NR5A1; RUNX1; RUNX2; NR5A1 and RUNX1; NR5A1 and RUNX2; or NR5A1, RUNX1, and RUNX2. 7. The PSC of any one of the preceding paragraphs, further comprising a modified polynucleotide comprising an open reading frame encoding a TCF21 protein. 8. The PSC of paragraph 7, which expresses or overexpresses TCF21. 9. The PSC of any one of the preceding paragraphs, further comprising a modified polynucleotide comprising an open reading frame encoding a GATA4 protein. 10. A PSC according to paragraph 7, which expresses or overexpresses GATA4. 11. The PSC of any one of the preceding paragraphs, wherein the open reading frame of the modified polynucleotide is operably linked to a heterologous promoter. 12. The PSC of paragraph 11, wherein the heterologous promoter is an inducible promoter. 13. A pluripotent stem cell (PSC) comprising a protein selected from NR5A1 and RUNX family proteins, wherein the protein is overexpressed. 14. A PSC according to paragraph 15, which expresses or overexpresses NR5A1; RUNX1; RUNX2; NR5A1 and RUNX1; NR5A1 and RUNX2; or NR5A1, RUNX1, and RUNX2. 15. The PSC of paragraph 14, further comprising a TCF21 protein. 16. A PSC according to paragraph 15, which expresses or overexpresses TCF21. 17. A PSC according to any one of paragraphs 14 to 16, further comprising a GATA4 protein. 18. A PSC according to any one of paragraphs 14 to 17, which expresses or overexpresses GATA4. 19. The PSC of any one of the preceding paragraphs, which is a human PSC. 20. The PSC of any one of the preceding paragraphs, which is an induced PSC (iPSC). 21. The PSC of any one of the preceding paragraphs, comprising 1 to 20, optionally 8 to 10 copies of a modified polynucleotide comprising an open reading frame encoding a protein selected from NR5A1 and a RUNX family protein (e.g., RUNX1 and / or RUNX2). 22. A composition comprising a population of PSCs as described in any one of the preceding paragraphs or elsewhere herein. 23. Population is at least 10,000 / cm 2 The composition of paragraph 22, comprising the PSC 24. A method comprising: culturing a population of pluripotent stem cells (PSCs) in a medium to generate an expanded population of PSCs; and expressing a protein selected from NR5A1 and a RUNX family protein in the expanded population of PSCs to generate granulosa-like cells. 25. The method of paragraph 24, wherein the PSCs of the expanded population comprise a modified polynucleotide that includes an open reading frame encoding NR5A1. 26. The method of paragraph 24 or 25, wherein the expanded population of PSCs comprises a modified polynucleotide comprising an open reading frame encoding a RUNX family protein. 27. The method of paragraph 26, wherein the RUNX family protein is RUNX1. 28. The method of paragraph 26 or 27, wherein the RUNX family protein is RUNX2. 29. The method of any one of paragraphs 24 to 28, wherein the expanded population of PSCs further comprises a modified polynucleotide comprising an open reading frame encoding a TCF21 protein. 30. The method of any one of paragraphs 24 to 29, wherein the expanded population of PSCs further comprises a modified polynucleotide comprising an open reading frame encoding a GATA4 protein. 31. The method of any one of the preceding paragraphs, wherein the open reading frame of the modified polynucleotide is operably linked to a heterologous promoter. 32. The method of any one of the preceding paragraphs, wherein the heterologous promoter is an inducible promoter. 33. The group is 1 x 10 2 pieces~1×10 7 Any one of the methods in the preceding paragraphs, including PSC 34. The method of any one of the preceding paragraphs, wherein the population of PSCs is cultured for about 4 to 10 days. 35. The method of paragraph 34, wherein the population of PSCs is cultured for about 6 days. 36. Granulosa-like cells, AMHR2 + , CD82 + , FOXL2 + , and / or EPCAM - Any one of the methods of the preceding paragraphs 37. Granulosa-like cells, AMHR2 + , CD82 + , FOXL2 + , and EPCAM - The method of paragraph 36 38. (a) delivering to a pluripotent stem cell (PSC) a modified polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a protein selected from NR5A1 and a RUNX family protein; (b) culturing the PSCs in feeder-free, serum-free medium to generate an expanded population of PSCs; (c) Expanded populations of PSCs were cultured in a series of induction media containing inducers, and AMHR2 + , CD82 + , FOXL2 + , and / or EPCAM - generating granulosa-like cells; Methods including 39. The method of paragraph 38, comprising delivering to the PSC a modified polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding NR5A1, and a modified polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a RUNX family protein. 40. The method of paragraph 38 or 39, wherein the RUNX family protein is RUNX1. 41. The method of any one of paragraphs 38 to 40, wherein the RUNX family protein is RUNX2. 42. The method of any one of paragraphs 38 to 41, wherein the modified polynucleotide is a transposon and the delivery further comprises delivering a transposase to the PSC. 43. The method of any one of paragraphs 38 to 42, wherein the inducible promoter is a chemically inducible promoter, optionally a doxycycline inducible promoter. 44. The method of any one of paragraphs 38 to 43, wherein the feeder-free serum-free medium of (b) comprises a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma. 45. The method of paragraph 44, wherein the solubilized basement membrane preparation comprises extracellular matrix (ECM) proteins and growth factors. 46. ​​The method of paragraph 45, wherein the ECM protein is selected from laminin, collagen IV, heparan sulfate proteoglycan, and entactin / nidogen. 47. The method of any one of paragraphs 38 to 46, wherein the feeder-free, serum-free medium of (b) comprises a growth factor selected from recombinant human basic fibroblast growth factor (rh bFGF) and recombinant human transforming growth factor β (rh TGFβ). 48. The method of any one of paragraphs 38 to 47, wherein the incubation in (b) is carried out for about 6 to 24 hours. 49.(c) The expanded population of PSCs is approximately 10,000 cells / cm 2 to approximately 20,000 cells / cm 2 49. The method of any one of paragraphs 38 to 48, wherein the cells are cultured at a density of 50. The method of any one of paragraphs 38 to 49, wherein the culturing of (c) comprises culturing the PSCs in a first induction medium and culturing the PSCs in a second induction medium. 51. The method of paragraph 50, wherein the first induction medium comprises one or more of L-alanyl-L-glutamine, an antibiotic (e.g., penicillin and / or streptomycin), Dulbecco's Modified Eagle Medium (DMEM) / F-12, Advanced RPMI (Roswell Park Memorial Institute) 1640 medium, a glycogen synthase kinase (GSK) 3 inhibitor, a small molecule or protein inhibitor of the BMP signaling pathway, a small molecule ROCK inhibitor, and an inducer (e.g., doxycycline). 52. The method of paragraph 50 or 51, wherein culturing the PSCs in the first induction medium is performed for about 36 hours to about 60 hours, optionally for about 48 hours. 53. The method of any one of paragraphs 50 to 52, wherein the second induction medium comprises one or more of L-alanyl-L-glutamine, an antibiotic (e.g., penicillin and / or streptomycin), Advanced RPMI 1640 medium, DMEM / F-12, and an induction agent (e.g., doxycycline). 54. The method of any one of paragraphs 50 to 53, wherein culturing the PSCs in the second induction medium is performed for about 96 hours to about 144 hours, optionally for about 120 hours. 55. The method of paragraph 54, wherein the second induction medium is removed and replaced with fresh second induction medium at about 24 hour intervals. 56. Granulosa-like cells produced by the method of any one of the preceding paragraphs. 57. An ovarian organoid comprising granulosa-like cells and human primordial germ cell-like cells (hPGCLCS) according to any one of the preceding paragraphs. 58. The method of any one of the preceding paragraphs, further comprising combining granulosa-like cells with hPGCLCS to form ovarian organoids. EXAMPLES

[0133] Example 1. Induced differentiation of hiPSCs into granulosa-like cells by transcription factor expression Ovarian granulosa cells are important for many aspects of female reproduction, including oocyte development and supporting hormone signaling during the menstrual cycle. Current in vitro models, including human or mouse primary granulosa cells and granulosa cell tumor lines, are inadequate to study these processes. For example, the COV434 ovarian tumor line, commonly used as a model for granulosa cells, was found to lack the transcriptional and phenotypic characteristics of true granulosa cells. Previously reported protocols for differentiating iPSCs into granulosa-like cells are either low-yielding or only applicable to mouse cells. To address this challenge, we developed a robust, scalable method to generate granulosa-like cells by transcription factor (TF)-mediated differentiation of iPSCs.

[0134] First, we identified a list of candidate TFs to screen for granulosa induction. To generate this list, we identified 22 TFs that are differentially expressed in granulosa cells compared to hESCs and early mesoderm using previously published datasets for these cell types. We also included five TFs identified by previous developmental biology studies as important in mouse ovarian development. Finally, we identified nine additional TFs that are predicted to be upstream of others on the list based on co-expression data and gene regulatory network analysis that considers binding motifs.

[0135] Next, we engineered hiPSC lines with a tdTomato reporter for the granulosa-specific protein FOXL2. In addition, we generated a barcoded cDNA plasmid library for doxycycline-inducible expression of these TFs. These plasmids were then integrated into FOXL2-tdTomato reporter iPSCs, and TF expression was induced under various conditions, resulting in the expression of FOXL2. + By capturing barcodes from cells, we determined the set of TFs that were enriched within the granulosa-like population (Figure 1).

[0136] Based on these results, we screened combinations of TFs of interest to observe which TFs were most able to induce a granulosa-like phenotype (Figure 2). We found that NR5A1 expression was essential for efficient estradiol production, and that the combination of NR5A1 and RUNX family members (either RUNX1 or RUNX2) was optimal for inducing the expression of FOXL2 and surface markers of granulosa. Furthermore, FOXL2 + It was verified that granulosa-like cells express transcriptional markers of granulosa and gonadal cells, but not markers from the related adrenal lineage (Figure 3).

[0137] Monoclonal hiPSC lines were then generated with the integrated top TFs. After selection of lines with efficient induction of granulosa-like cells (Figure 4), their hormonal signaling was verified by measuring estradiol production in response to follicle-stimulating hormone (FSH) or forskolin, which increase the levels of the second messenger cAMP. Three lines containing integrated expression plasmids for NR5A1, TCF21, and RUNX1 were found to produce estradiol in response to either FSH or forskolin, as expected for granulosa cells (Figure 5). The fourth line, without the RUNX transgene, produced high levels of estradiol constitutively, suggesting that RUNX expression is required for proper regulation of estradiol production.

[0138] The entire protocol for generating granulosa-like cells is depicted in Figure 6. hiPSCs containing integrated TF expression plasmids are cultured in mTeSR™ Plus medium on Corning® Matrigel® matrix. For derivation of granulosa-like cells, hiPSCs are dissociated into single cells and cultured at approximately 10,000-20,000 cells / cm in DK10 medium (DMEM / F12 with GlutaMAX™ supplement and 10% knockout serum replacement) + approximately 3-4 μM CHIR99021, approximately 8-12 μM Y-27632, and approximately 0.5-3 μg / mL doxycycline on Corning® Matrigel® matrix or collagen I. 2 Cells were plated at a density of 100x for 1 h and TF expression was induced. After 48 h and at 24 h intervals thereafter, the medium was changed / replaced with fresh DK10 medium + approximately 0.5-3 μg / mL doxycycline. After a total of 120 h, granulosa-like cells were ready for downstream experiments.

[0139] In an alternative protocol, iPSCs are cultured on Matrigel or laminin coated plates and grown to 20-40% confluency in mTeSR Plus medium. For induction of granulosa-like cells, mTeSR Plus is removed, iPSCs are washed with phosphate-buffered saline (PBS), and Stage 1 medium containing Advanced RPMI with GlutaMAX™ supplement, CHIR99021 (approximately 5-8 μM), and either Dorsomorphin (approximately 100-400 nM) or Noggin (approximately 4-8 ng / mL) is added. After 48 hours, Stage 1 medium is removed and replaced with fresh Stage 1 medium. After an additional 48 hours, Stage 1 medium is removed and replaced with Stage 2 medium containing Advanced RPMI with GlutaMAX™ supplement and approximately 0.5-3 μg / mL doxycycline. Stage 2 medium is changed every 24 hours. Granulosa-like cells can be harvested in Stage 2 medium after approximately 3-4 days total (approximately 7-8 days total).

[0140] The protocol presented here has several advantages over previously reported methods. It rapidly produces granulosa-like cells within about 5-8 days, and the resulting cells are pure enough not to require enrichment by FACS (Figure 4). Because of the monolayer format, it is easily scalable (50 million cells are generated immediately, and the format is compatible with higher throughput assays). Most importantly, granulosa-like cells exhibit key characteristics of granulosa cells transcriptionally (Figure 3) and phenotypically (Figure 5), making them suitable as a model for ovarian biology.

[0141] Example 2. Granulosa-like cells respond to FSH and carry out steroid biosynthesis The ability of granulosa-like cells to carry out one of the main endocrine functions of granulosa cells (i.e., production of estradiol) was evaluated. 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), which produces estrone, which is then reduced to estradiol in granulosa cells by enzymes in the HSD17B family, typically HSD17B1. This pathway of estrogen synthesis in vivo is stimulated by FSH. Granulosa-like cells were treated with androstenedione in the presence or absence of FSH or forskolin (which directly increases the level of the FSHR second messenger cAMP). As controls, COV434 and KGN ovarian tumor cells, which produce estradiol from androstenedione, as well as immortalized human primary granulosa cells (HGL5) and adult mouse primary ovarian somatic cells were used. Granulosa-like cells produce estradiol from androstenedione, and in seven of the nine monoclonal lines tested, this steroidogenic activity was significantly increased upon stimulation with FSH or forskolin (Figure 5A). One of the granulosa lines (F3 / NT#5) produced high levels of estradiol under all conditions, and unlike the others, this line did not incorporate either the RUNX1 or RUNX2 expression vector (data not shown).

[0142] The levels of estradiol produced by the three FSH-responsive lines were similar to those produced by KGN human granulosa tumor cells, and also showed responsiveness to FSH and forskolin (Figure 5A). In contrast, COV434 cells showed no FSHR expression in the RNA-seq data (Figure 3), were unresponsive to FSH alone, and produced estradiol only in the presence of forskolin. HGL5 immortalized human granulosa cells did not produce estradiol under any conditions. Adult mouse primary ovarian somatic cells produced similar amounts of estradiol as hiPSC-derived granulosa-like cells (Figure 5A); however, the mouse cells showed no responsiveness to FSH or forskolin, presumably because they had already been exposed to FSH in vivo. We also examined whether granulosa-like cells maintained their steroidogenic activity during co-culture with hPGCLCs in ovarian organoids. Hormone levels were measured in ovarian supernatants in the presence or absence of androstenedione and FSH. In addition to estradiol, progesterone was also measured; granulosa cells produce progesterone in vivo after ovulation and formation of the corpus luteum. We observed production of both hormones in five of six samples (Figure 5B). Estradiol was only produced in the presence of androstenedione supplementation, and levels increased with FSH treatment. Progesterone was produced under all conditions, but was highest in the absence of androstenedione.

[0143] Example 3. Granulosa-like cells support germ cell development within the ovaloid The present method for inducing and culturing human PGC-like cells (hPGCLCs) generates cells corresponding to immature pre-migratory PGCs that lack expression of gonadal PGC markers such as DAZL. During fetal development, PGCs mature through interactions with gonadal somatic cells, where DAZL plays a key role in downregulating pluripotency factors and committing to gametogenesis. This process has recently been reconstituted in vitro using mouse fetal ovarian somatic cells, allowing the development of hPGCLCs to an oogonia-like stage. In vitro-derived human granulosa-like cells may perform a similar role and eliminate interspecies developmental mismatches. Therefore, granulosa-like cells were combined with hPGCLCs to form ovarian organoids, termed "ovarianoids."

[0144] To generate ovarian lamina, these two cell types were aggregated in low-binding U-bottom wells and then transferred to transwell cultures at the air-liquid interface. As a comparison, fetal mouse ovarian somatic cells were isolated and aggregated with hPGCLCs. OCT4 + Immunofluorescence revealed expression of the maturation marker DAZL in a subset of hPGCLCs starting from day 4 in coculture with hiPSC-derived granulosa-like cells (data not shown). In contrast, robust DAZL expression in coculture with mouse cells was not observed until day 32 (data not shown), with fainter expression observed by day 26. Similarly, in a previous study using the same hPGCLC line and anti-DAZL antibody, DAZL expression was observed exclusively after 77 days in coculture with mouse fetal testicular somatic cells. DAZL + The fraction of cells reached its maximum at day 14 in human ovarians and day 38 in mouse ovarians (Figure 7). + The fraction of cells decreased after day 8. In mouse ovarian lesions, OCT4 + The fraction of cells decreased over time. On day 16, in human ovarian tumors, DAZL + OCT4 + In addition to cells, DAZL + OCT4 -Cells were also clearly observed (in situ images not shown), and from day 38 onwards, OCT4 + DAZL over cells + There were many cells (Figure 7). + Downregulation of OCT4 in oogonia occurs in vivo during the second trimester of human fetal ovarian development, but we did not observe the transition of DAZL to exclusively cytoplasmic localization that has been reported to occur at this stage. Expression of TFAP2C, an early PGC marker, decreased during oballoid culture and was almost entirely absent by day 8. In contrast, SOX17 expression was still detectable at day 8, and expression of OCT4 and DAZL continued through day 54. Although this system allowed rapid development of hPGCLCs to the gonadal stage, the number of germ cells in both hiPSC-derived and mouse-derived oballoids decreased over long-term culture (Figure 7), indicating that they were either dying or differentiating into other lineages. Unlike mouse-derived obaloids, hiPSC-derived obaloids cultured on transwells gradually flattened, expanded, and were largely collapsed by day 38.

[0145] Nevertheless, in these long-term experiments, cubic AMHR2 + FOXL2 +We observed the formation of empty follicle-like structures composed of granulosa-like cells (in situ images not shown), suggesting that TFs can drive follicle formation even in the absence of oocytes. The formation of follicle-like structures was first observed on day 16, and by day 26, these structures had grown to a maximum diameter of 1–2 mm. On day 70, the ovarian follicular cells were giving rise to follicles of various sizes, mainly small unilamellar but also antral follicles. Cells outside the follicles were stained positive for NR2F2, a marker of ovarian stromal and theca cells. To further examine gene expression of hPGCLCs and somatic cells in this system, we performed scRNA-seq to follow gene expression on dissociated ovarian follicular cells from days 2, 4, 8, and 14 of culture, as well as on cluster cells. As expected, the largest cluster (cluster 0) contained cells expressing granulosa markers such as FOXL2, WNT4, and CD82 (Figures 8 and 8B). Cells expressing markers of secondary / antral granulosa cells such as FSHR and CYP19A1 were also found within this cluster, but these were less numerous. A smaller cluster (cluster 1) expressing the ovarian stromal marker NR2F2 was also found. NR2F2 is expressed by both stromal and thecal cells, but cells in cluster 1 did not express 17α-hydroxylase (CYP17A1), indicating that they cannot produce androgens and are not thecal cells.

[0146] Clusters of hPGCLCs expressing marker genes such as CD38, KIT, PRDM1, TFAP2C, PRDM14, NANOG, and POU5F1 were also found. Notably, the X-chromosomal lncRNAs XIST, TSIX, and XACT were all more highly expressed in hPGCLCs compared to the other clusters (on average, 80-fold, 20-fold, and 2900-fold, respectively) (Figure 8B), suggesting that hPGCLCs have initiated the process of X reactivation, which is associated with the high expression of both XIST and XACT in hPGCs. Additionally, the X-chromosomal HPRT1 gene, known to be more highly expressed in cells with two active X chromosomes, was upregulated by approximately threefold.

[0147] We next compared the in vitro generated ovarian development to a reference atlas of human fetal ovarian development. Using Scanpy ingest, samples were integrated into the atlas and each cell was annotated by the closest cell type from the in vivo data (Figure 8C). The ovarian was composed primarily of granulosa, gonadal mesenchyme, and pregranulosa lineages (Figure 8D), with a small fraction of coelomic epithelium. The fraction of granulosa cells increased from day 2 to day 8, potentially representing the maturation of the somatic cell population. As expected, neural, immune, smooth muscle, and erythroid cells present within the fetal ovary were completely absent from the ovarian. Epithelial, endothelial, and perivascular cells were detected but at very low frequencies (<1%), likely representing a low rate of off-target differentiation.

[0148] The fraction of cells expressing gonadal germ cell markers DAZL and DDX4, along with the total fraction of germ cells, was further examined over the course of the experiment (Figure 8D). The germ cell population was defined based on an integrated atlas of fetal ovaries. This population increased from day 2 to day 4, but then decreased. In comparison, DAZL + Cells and DDX4 + The fraction of cells also increased from day 2 to day 4, but remained nearly constant from day 4 to day 14 (Figure 8D). Differential gene expression analysis and gene ontology enrichment were performed using the DAZL - DAZL on cells + A quantitative analysis was performed on ovarian cells. Upregulated genes (log2fc>2, n=221) were most highly enriched over periods associated with general developmental processes, but also included adhesion and migration (e.g., "amoeboid cell migration"), and periods associated with reproductive system development. Downregulated genes (log2fc<-2, n=6451) were strongly associated with metabolic processes and mitosis. These data support the notion that DAZL in ovarian cells is a potent regulator of ovarian development. + This suggests that the cells down-regulate their metabolism and proliferation, consistent with the known role of DAZL in suppressing PGC proliferation.

[0149] method cell culture Two parental hiPSC lines were used in this study: ATCC-BXS0116 female hiPSCs, designated F3 line, and F66 line, an autologous hiPSC line derived from NIA Aging Cell Repository fibroblast cell line AG07141 using Epi5 footprint-free episomal reprogramming. The karyotypes of the parental and modified reporter lines were verified by Thermo Fisher's Cell ID (SNP-based authentication) + Karyostat, and pluripotency was assessed by Thermo Fisher Pluritest. All lines were confirmed to be normal.

[0150] hiPSCs were cultured in mTESR Plus medium (Stemcell Technologies) on standard polystyrene plates coated with hESC-qualified Matrigel (Corning). Medium was changed daily. For experiments requiring single cell dissociation, passaging was performed using 0.5 mM EDTA or TRYPLE. After each passaging, hiPSCs were treated with 10 μM Y-27632 (Ambeed) for 24 hours. COV434 cells were cultured in DMEM + 10% FBS + 1x GlutaMax (Gibco). KGN cells (RIKEN, RCB1154) were cultured in DMEM / F12 + 10% FBS + 1x GlutaMax (Gibco). HGL5 cells (ABM cat.T0650) were cultured in Prigrow IV medium (ABM) with 10% FBS. Passaging was performed using TRYPLE (Gibco). hPGCLCs were cultured in S-CM medium and passaged using Accutase (Stemcell Technologies). Mycoplasma testing was performed by PCR every 3 months; all cells tested negative.

[0151] Electroporation Electroporation was performed using a Lonza Nucleofector with a 96-well shuttle with 200,000 cells in 20 μL of P3 buffer. Pulse setting CA-137 was used for all electroporations. Selection with the appropriate drug was started 48 hours after electroporation and continued for 5 days. For the drugs used in this study, this time was sufficient to obtain a final cell population with high purity.

[0152] Reporter constructs Homology arms for FOXL2 were amplified from genomic DNA by PCR. A targeting plasmid (Figure 2-Appendix Figure 1A) containing an in-frame C-terminal T2A-tdTomato reporter and a Rox-PGK-PuroTK-Rox selection cassette was constructed by Gibson assembly. The plasmid backbone further carried an MC1-DTA marker to select against random integration. sgRNA oligos targeting the C-terminal region of FOXL2 were cloned into pX330 (Addgene#42230). For reporter line generation, 1 μg of donor plasmid and 1 μg of sgRNA plasmid were co-electroporated into hiPSCs and subsequently plated in one well of a 6-well plate. After selection with puromycin (400 ng / mL), colonies were manually picked with a P20 pipette. The generated hiPSC lines were genotyped by PCR for the presence of wild type and reporter alleles. Homozygous clones were further verified by PCR amplification of the entire FOXL2 locus (Figure 2 - Appendix Fig. 1B) and Sanger sequencing.

[0153] To excise the selection cassette, hiPSCs were electroporated with pCAGGS-Dre (1 μg). Selection was performed with ganciclovir (4 μM) and colonies were picked as above. Excision of the selection cassette was verified by genotyping. The primers used in this study are listed in Appendix file 1.

[0154] Construction of TF plasmids TF cDNAs were obtained from TFome39 or ORFeome76 as Gateway entry clones. They were cloned into a barcoded, Dox-inducible expression vector (Addgene#175503) using MegaGate cloning48. Final expression constructs were verified by Sanger sequencing, which also served to determine the barcode sequence for each TF. For library pooling, two unique barcodes were used per TF. Libraries were pooled using equimolar amounts of each plasmid (measured using QuBit).

[0155] TF screening for granulosa differentiation Pooled libraries of barcoded TF plasmids were electroporated into FOXL2-tdTomato reporter hiPSCs, typically with 5 fmol of library and 500 ng of PiggyBac transposase expression plasmid (Systems Bio). These conditions were chosen to obtain an average copy number of approximately 5 cells / cell (Figure 2 - Appendix Figure 2). Some experiments were also performed at 50 fmol to explore the effect of higher copy numbers. For the screening data shown in Figure 2, two libraries were used: library #1 contains 35 TFs and library #2 contains 18 TFs. Library #1 was used exclusively at 5 fmol, while library #2 was used at both 5 fmol and 50 fmol.

[0156] After selection with puromycin (400 ng / mL), hiPSCs were treated with doxycycline (1 μg / mL) in mTESR Plus medium. In further experiments, hiPSCs were first differentiated into mesoderm according to a previously published protocol77 before doxycycline treatment. In both sets of experiments, doxycycline treatment was continued for 5 days, after which cells were dissociated in TRYPLE and reporter-positive cells were isolated by FACS. Genomic DNA was extracted (QIAamp DNA Micro kit) from reporter-positive and -negative cells as well as from the initial population before doxycycline treatment.

[0157] Barcodes were amplified by PCR (KAPA polymerase) using 10 ng of input gDNA per reaction and typically 22 PCR cycles (denaturation at 95°C for 15 s, annealing / extension at 58°C for 20 s). ProNex beads were used to purify the PCR products and two PCRs (NEB Q5 polymerase, 6 cycles of denaturation at 98°C for 5 s, annealing at 61°C for 20 s, extension at 72°C for 5 s, final extension at 72°C for 2 min) were performed to add Illumina indexes. (Primers are shown in Appendix file 1). These amplicons were purified again using ProNex beads. Samples were normalized, pooled, and barcodes were sequenced on an Illumina MiSeq with 10% PhiX spike-in. To call barcodes, reads were aligned to a set of known barcode sequences. Selected FOXL2 + Fold changes were calculated by comparing barcode frequencies in cells with the frequencies in the starting population.

[0158] Flow cytometry / cell sorting Cells were dissociated by treatment with TRYPLE for 5 min and quenched with 4 volumes of ice-cold DMEM+10% FBS. The suspension was passed through a 70 μm cell strainer. Cells were pelleted (200 g, 5 min) and resuspended in staining buffer (PBS+3% FBS+antibody, approximately 100 μL per million cells). Staining was allowed to continue for 30 min on ice in the dark. The suspension was diluted with 9 volumes of PBS+3% FBS. Cells were pelleted (200 g, 5 min) and resuspended in PBS+3% FBS+100 ng / mL DAPI. The suspension was kept on ice in the dark until analysis. Flow cytometry was performed on a BD LSRFortessa and sorting was performed on a Sony SH800 with a 100 μm tip.

[0159] Antibody capture beads (BD Biosciences, RRID AB_10051478) or hiPSCs expressing tdTomato were used as compensation controls. Antibodies used are listed in the Key Resources Table. Data analysis was performed using the Cytoflow Python package (version 1.0.0, github.com / cytoflow / cytoflow).

[0160] Protocol for granulosa differentiation iPSCs were dissociated with TRYPLE and plated at 12,500 cells / cm on Matrigel-coated polystyrene plates in DK10 medium (DMEM-F12, 15 mM HEPES, 1x GlutaMax, 10% KSR) with Y-27632 (10 μM), CHIR99021 (3 μM), and doxycycline (1 μg / mL). 2 Cells were plated at a cell density of 100-200 μg / mL. For 24-well plates, the medium volume / well was 0.5 mL; for 6-well plates, it was 2 mL. 48 h after plating, the medium was changed to DK10 + doxycycline (1 μg / mL), followed by medium changes every 24 h. Cells were harvested on day 5 unless otherwise indicated. In differentiation of non-TF controls for RNA-seq, the protocol was the same except that the cells did not contain the TF expression plasmid.

[0161] Additional protocols for inducing granulosa-like cells hiPSCs containing integrated TF expression plasmids were cultured in mTeSR™ Plus medium on Corning® Matrigel® matrix. For granulosa-like cell induction, hiPSCs were dissociated into single cells using TRYPLE and cultured at approximately 10,000-20,000 / cm in DK10 medium (DMEM / F12 with GlutaMAX™ supplement and 10% knockout serum replacement) + approximately 3-5 μM CHIR99021, approximately 8-12 μM Y-27632, and approximately 0.5-3 μg / mL doxycycline on Corning® Matrigel® matrix or collagen I-coated plates. 2To induce TF expression, cells were seeded at a density of 100x for 24 h. After 48 h, and at 24 h intervals thereafter, the medium was changed / replaced with fresh DK10 + ~0.5-3 μg / mL doxycycline. After a total of ~120 h, the granulosa-like cells were ready for downstream experiments. · hiPSCs containing integrated TF expression plasmids were cultured in mTeSR Plus medium on Matrigel or laminin. Once the cultures had reached 20-40% confluency, the medium was removed, the iPSCs were washed with phosphate-buffered saline (PBS), and Stage 1 medium was added containing Advanced RPMI with GlutaMAX™ supplement, CHIR99021 (~6-10 μM), and either Dorsomorphin (~100-400 nM) or Noggin (~5-8 ng / mL). After 48 hours, the Stage 1 medium was removed and replaced with fresh Stage 1 medium. After another 48 hours, the Stage 1 medium was removed and replaced with Stage 2 medium containing Advanced RPMI with GlutaMAX™ supplement and ~0.5-3 μg / mL doxycycline. Stage 2 medium was changed every 24 hours. After ~3-4 days (~7-8 days total) in Stage 2 medium, the granulosa-like cells were ready for downstream experiments.

[0162] RNA-seq Total RNA was purified using the Arcturus PicoPure kit (Thermo Fisher Scientific) to identify FOXL2 + Total RNA was extracted from granulosa-like cells or from COV434 cells and hiPSCs using Monarch's total RNA miniprep kit (NEB). For experiments involving TF overexpression, TF expression plasmids were introduced into hiPSCs as described above (50 fmol / 200,000 cells). After selection with puromycin, TF expression was induced using doxycycline (1000 ng / mL).

[0163] Two biological replicates were performed for each sample (iPSCs, hiPSCs + individual TFs, sorted FOXL2 +, non-TF differentiation, KGN, COV434). Libraries were prepared using the NEBNext Ultra II Directional kit according to the manufacturer's protocol and sequenced on an Illumina NextSeq 500 (2 × 75 bp paired-end reads). TPM data shown in Figure 3 were generated using kallisto78 and reads were pseudo-aligned to the reference human transcriptome (Ensembl GRCh38 v96). Differential expression analysis was performed using DESeq2. For each sample, gene ontology enrichment was calculated for genes significantly upregulated (log2fc>3, padj<0.05) and downregulated (log2fc<-3, padj<0.05) relative to hiPSCs using PantherDB60.

[0164] TROM analysis The Transcriptome Overlap Measure (TROM) method was used to identify associated genes that capture the molecular signatures of biological samples, and biological samples were then compared by testing the overlap of their associated genes. TROM scores were calculated as -log10 (Bonferroni-corrected P-value of association) on a scale of 0 to 300. The magnitude of the TROM positively correlated with the similarity between two independent samples, with a standard threshold of 12 as a generally accepted indicator of significant similarity.

[0165] Ovaloid formation between hPGCLCs and granulosa-like cells F2 female hPGCLCs (see main resource table) were maintained in long-term culture. Briefly, hPGCLCs were cultured in STO-conditioned medium (GMEM+13% KSR and 1× NEAA, sodium pyruvate, and GlutaMax, all from Gibco) supplemented with SCF (100 ng / mL, Peprotech), ascorbic acid (50 μg / mL, Gibco), and 2-mercaptoethanol (25 μM, Gibco) on Matrigel. hPGCLCs were harvested by Accutase. To form ovaloids, granulosa-like cells were harvested by TRYPLE, counted, and mixed with F2 hPGCLCs. For hormone assays in Figure 3, we used granulosa-like cells from F3 / N.R1#6, F66 / N.R1.GF#4, F66 / N.R1.G#7, F66 / N.R2#1, F66 / N.R2#5, and F66 / N.R2.G#3. For immunofluorescence experiments in Figures 4 and 5, we used F66 / N.R1.GF#4 and F66 / N.R2#1. For scRNA-seq in Figure 6, we used F66 / N.R1.GF#4.

[0166] For each oballoid, 100,000 granulosa-like cells and 10,000 hPGCLCs were added to each well of a 96-well U-bottom low-binding plate (Corning #7007) in 200 μL of GK15 medium (GMEM, 15% KSR (with 1× GlutaMax), sodium pyruvate, and non-essential amino acids) supplemented with 10 mM Y-27632, 0.1 mM 2-mercaptoethanol, 1 μg / mL doxycycline, 100 ng / mL SCF, and 50 μg / mL primocin. Plates were centrifuged (100 g, 2 min) and incubated (37° C., 5% CO2) for 2 days. The oballoids were then transferred to transwells (collagen-coated PTFE, 3 μm pore size, 24 mm diameter, Corning #3492) for air-liquid interface culture in aMEM, 10% KSR, 55 μM 2-mercaptoethanol, 500 ng / mL doxycycline, and 50 μg / mL primocin. Typically, 5-6 oballoids were cultured on each transwell of 6 wells. The medium (1.5 mL) was changed every 2 days.

[0167] Ovaloid formation by hPGCLCs and mouse fetal ovarian somatic cells Fetal ovarian somatic cells were isolated from E12.5 female embryos of CD-1 mice (Charles River) as described by Yamashiro et al.65. For each ovarian foetal cells, 50,000 fetal ovarian somatic cells and 5,000 F2 hPGCLCs were combined. Ovaloids were cultured as described above. All mouse experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Harvard Medical School.

[0168] Immunofluorescence The oballoids were washed with PBS and fixed in 1% PFA overnight at 4°C. After another PBS wash, the oballoids were detached from the transwells. In preparation for cryosectioning, the oballoids were transferred to 10% sucrose in PBS. After 24 hours at 4°C, the 10% sucrose solution was removed and replaced with 20% sucrose in PBS. After another 24 hours at 4°C, the oballoids were embedded in OCT compound and stored at -80°C until sectioning.

[0169] Ovaloids were sectioned to 10 μm using a Leica CM3050S cryostat. Sections were transferred to Superfrost Plus slides, which were washed with PBS to remove the OCT compound. Slides were washed with PBST (0.1% Triton X-100 in PBS) and sections were circled with a Pap pen. Slides were blocked with blocking buffer (1% bovine serum albumin and 5% normal donkey serum in PBST [Jackson ImmunoResearch, #017-000-121, lot #152961]) for 30 minutes at room temperature. Blocking buffer was removed and replaced with a solution of primary antibody in blocking buffer, and slides were incubated overnight at 4°C. Antibody solution was removed and slides were washed three times for 5 minutes with PBST. Slides were incubated with secondary antibody and DAPI solution in blocking buffer in the dark for 1 hour at room temperature, then washed twice for 5 minutes with PBST and once with PBS. After staining, samples were mounted in Prolong Gold medium and covered with a coverslip. Imaging was performed on a Leica SP5 confocal microscope. Antibodies used are listed in the Key Resources table. Images were adjusted for brightness (exclusively for brightness) in ImageJ (version 2.9.0 / 1.53t) and cell counts in the case of Figure 4C were performed manually by an investigator blinded to the species of the oballoids.

[0170] Single-cell RNA sequencing Ovaloids (6 ovaloids per sample, 2 samples per time point) were dissociated using the Miltenyi Embryoid Body Dissociation Kit (Miltenyi #130-096-348). Cells were passed through a 40 μm filter, fixed using Parse Biosciences' fixation kit, and stored at -80°C until all time points were collected. Libraries were prepared using Parse Biosciences WT Mega v1 kit, generating an average of 450 bp libraries. Ovaloids were initiated in 8 of 96 samples; the remaining kit performance was used for other experiments. Libraries were sequenced on an Illumina NovaSeq 2 x 150 bp S4 flow cell with 6 bp libraries as single index and 5% PhiX spike-in. Data was separated into library fastq files and matrix counts were generated using Parse Bioscience's analysis pipeline (version 0.9.6). Downstream data processing, such as double filtering, dimensionality reduction, and clustering, was performed using Scanpy (version 1.8.2).70 For cell type assignment, the fetal ovarian dataset from the Human Germ Cell Atlas69 was used as a reference for scanpy import.

[0171] Collection of mouse primary ovarian somatic cells Female BALB / c mice (10-12 weeks old) were confirmed to be in proestrus by visual inspection. Mice were sacrificed by CO2 exposure followed by cervical dislocation, and ovaries were removed by dissection. Ovaries were placed in HEPES-buffered DMEM / F12 with 0.1% bovine serum albumin (2 ovaries per 1.5 mL tube in 500 μL of medium) and mechanically disrupted by pricking with forceps. The cell suspension was strained through a 40 μm strainer to remove oocytes and clumps, and then cultured for hormone assays.

[0172] Steroid hormone assay On day 4 of granulosa differentiation, androstenedione (500ng / mL) was added to the medium. FSH (0.25IU / mL, BioVision#4781-50 lot 5F07L47810) or forskolin (100μM, Sigma-Aldrich) was also added as indicated. Total medium volume was 0.5mL per well of a 24-well plate. We performed these assays for each of the lines listed in Figure 3-Source Data 2. 75,000 cells were seeded per well using human cell lines (COV434, KGN, or HGL5) or mouse primary ovarian somatic cells as controls. After 24 hours, the medium was analyzed for estradiol content by ELISA (DRG International, EIA-2693). Concentrations were calculated by a 4-parameter logistic curve fit using data from standards provided in the kit. Samples outside the range of the calibration curve were diluted and rerun.

[0173] To measure hormone production in ovaloids, ovaloids were assembled as described above. Androstenedione (500 ng / mL) and / or FSH (0.25 IU / mL) were added to the assembly medium (total volume of 200 μL per ovaloid). After 3 days of culture, the medium was removed and analyzed by ELISA for estradiol (DRG International, EIA-2693) and progesterone (DRG International, EIA-1561). Hormone concentrations were calculated as described above.

[0174] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter cited, which in some cases may include the entirety of the document.

[0175] The indefinite articles "a" and "an," as used herein in the specification and the claims, unless expressly stated to the contrary, should be understood to mean "at least one."

[0176] It should also be understood that in any method claimed herein that includes two or more steps or actions, unless expressly stated to the contrary, the order of the one or more method steps or actions is not necessarily limited to the order in which the method steps or actions are recited.

[0177] In the claims and above specification, all transitional phrases, such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, are to be understood to be open-ended, i.e., to mean including, but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" must be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0178] The terms "about" and "substantially" preceding a numerical value mean ±10% of the recited numerical value.

[0179] Where various values ​​are presented, each value between, as well as including, the upper and lower ends of the range is specifically contemplated and described herein.

Claims

1. Pluripotent stem cells (PSCs) comprising modified polynucleotides including an open reading frame encoding NR5A1 and a protein selected from the RUNX family of proteins.

2. The PSC according to claim 1, wherein the RUNX family protein is RUNX1 or RUNX2.

3. The PSC according to claim 1, which expresses or overexpresses NR5A1; RUNX1; RUNX2; NR5A1 and RUNX1; NR5A1 and RUNX2; or NR5A1, RUNX1, and RUNX2.

4. The PSC according to claim 1, further comprising a modified polynucleotide containing an open reading frame encoding the TCF21 protein and / or a modified polynucleotide containing an open reading frame encoding the GATA4 protein.

5. The PSC according to claim 4, which expresses or overexpresses TCF21 and / or GATA4.

6. The PSC according to claim 1, wherein the open reading frame of the modified polynucleotide is operably linked to a heterogeneous promoter or an inducible promoter.

7. Pluripotent stem cells (PSCs) comprising NR5A1 and a protein selected from the RUNX family of proteins, wherein the protein is overexpressed.

8. The PSC according to claim 7, expressing or overexpressing NR5A1; RUNX1; RUNX2; NR5A1 and RUNX1; NR5A1 and RUNX2; or NR5A1, RUNX1, and RUNX2.

9. The PSC according to claim 8, further comprising TCF21 protein and / or GATA4 protein.

10. The PSC according to claim 9, which expresses or overexpresses TCF21 and / or GATA4.

11. The PSC according to claim 7, which is a human PSC or an induced PSC (iPSC).

12. The PSC according to claim 7, comprising 1 to 20 or 8 to 10 copies of a modified polynucleotide containing an open reading frame encoding the protein selected from NR5A1 and the RUNX family of proteins.

13. A composition comprising a population of PSCs as described in claim 1, or a population of PSCs as described in claim 1 in an amount of at least 10,000 / cm².

14. A method comprising: culturing a population of pluripotent stem cells (PSCs) in a culture medium to generate an expanded population of PSCs; and expressing a protein selected from NR5A1 and RUNX family proteins within the expanded population of PSCs to generate granulosa-like cells.

15. The method according to claim 14, wherein the PSC of the expanded population comprises a modified polynucleotide comprising an open reading frame encoding NR5A1 and / or a modified polynucleotide comprising an open reading frame encoding a RUNX family protein.

16. The method according to claim 15, wherein the RUNX family protein is RUNX1 or RUNX2.

17. The method according to claim 15, wherein the PSC of the expanded population further comprises a modified polynucleotide containing an open reading frame encoding the TCF21 protein and / or a modified polynucleotide containing an open reading frame encoding the GATA4 protein.

18. The method according to claim 15, wherein the open reading frame of the modified polynucleotide is operably linked to a heterologous promoter or an inducible promoter.

19. The method according to claim 14, wherein the population comprises 1 × 10² to 1 × 10⁷ PSCs, and / or the population of PSCs is cultured for about 4 to 10 days or about 6 days.

20. The aforementioned granulosa-like cells are AMHR2 ＋ , AP82 ＋ , FOXL2 ＋ , and / or EPCAM － The method according to claim 14.

21. (a) Delivering a modified polynucleotide containing an inducible promoter operably linked to an open reading frame encoding a protein selected from NR5A1 and the RUNX family of proteins to pluripotent stem cells (PSCs); (b) Culturing the PSCs in a feeder-free, serum-free medium to produce an expanded population of PSCs; (c) The expanded population of PSCs is cultured in a series of induction media containing an inducer, and AMHR2 ＋ , AP82 ＋ , FOXL2 ＋ , and / or EPCAM － To generate granulosa-like cells, A method that includes this.

22. The method according to claim 21, comprising delivering (i) a modified polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding NR5A1 and (ii) a modified polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a RUNX family protein to a PSC.

23. The method according to claim 22, wherein the RUNX family protein is RUNX1 or RUNX2.

24. The method according to claim 21, wherein the modified polynucleotide is a transposon, and the delivery further comprises delivering a transposase to the PSC.

25. The method according to claim 21, wherein the inducible promoter is a chemically inducible promoter or a doxycycline-inducible promoter.

26. (b) The feeder-free serum-free medium is Solubilized basement membrane specimens extracted from Angelbreth-Holm-Swarm (EHS) mouse sarcoma, and / or Growth factors selected from recombinant human basic fibroblast growth factor (rh bFGF) and recombinant human transforming growth factor β (rh TGFβ). The method according to claim 21, including the method described in claim 21.

27. The method according to claim 26, wherein the solubilized basement membrane specimen comprises extracellular matrix (ECM) proteins and growth factors.

28. The method according to claim 27, wherein the ECM protein is selected from laminin, collagen IV, heparan sulfate proteoglycan, and entactin / nidogen.

29. The method according to claim 21, wherein the culture in (b) is carried out for about 6 to 24 hours.

30. The method according to claim 21, wherein the culture in (c) comprises (i) culturing the PSCs at a density of about 10,000 cells / cm² to about 20,000 cells / cm², and / or (ii) culturing the PSCs in a first induction medium and culturing the PSCs in a second induction medium.

31. The first induction medium comprises one or more of the following: L-alanyl-L-glutamine, an antibiotic, Dulbecco's Modified Eagle Medium (DMEM) / F-12, Advanced RPMI (Roswell Park Memorial Laboratory) 1640 medium, a glycogen synthase kinase (GSK) 3 inhibitor, a small molecule or protein inhibitor of the BMP signaling pathway, a small molecule ROCK inhibitor, and / or an inducer. The method according to claim 23, wherein the second induction medium comprises one or more of L-alanyl-L-glutamine, an antibiotic, Advanced RPMI 1640 medium, DMEM / F-12, and an inducer.

32. The method according to claim 30, wherein the culture of the PSC in the first induction medium is carried out for approximately 36 hours to approximately 60 hours, and optionally for approximately 48 hours.

33. The method according to claim 30, wherein the culture of the PSC in the second induction medium is carried out for about 96 hours to about 144 hours, or about 120 hours.

34. The method according to claim 33, wherein the second induction medium is removed at intervals of approximately 24 hours and replaced with fresh second induction medium.

35. Granulosa-like cells produced by the method described in claim 21.

36. An ovarian organoid comprising granulosa-like cells and human primordial germ cell-like cells (hPGCLCS) as described in claim 35.

37. The method according to claim 21, further comprising combining the granulosa-like cells with hPGCLCS to form ovarian organoids.

38. The PSC according to claim 3, wherein the PSC expresses or overexpresses NR5A1 and RUNX2.

39. The PSC according to claim 3, wherein the PSC expresses or overexpresses NR5A1, RUNX2, and GATA4.

40. The method according to claim 14, wherein the PSC expresses or overexpresses NR5A1 and RUNX2.

41. The method according to claim 14, wherein the PSC expresses or overexpresses NR5A1, RUNX2, and GATA4.

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