Methods and compositions for producing particle layer cell-like cells

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 326,640, filed on April 1, 2022, and U.S. Provisional Application No. 63 / 444,108, filed on February 8, 2023, under 35 U.S.C. §119(e), each of which is incorporated herein by reference in its entirety.

[0003] References to Electronic Sequence Listings

[0004] The contents of the electronic sequence listing (H049870758WO00-SEQ-KVC.xml; size: 7,426 bytes; creation date: March 30, 2023) are incorporated herein by reference in their entirety.

[0005] background

[0006] Granulosa cells are specialized cells that, upon maturation, provide key steroids and growth hormones to the developing oocyte. Granulosa cells surround the developing oocyte and participate in maintaining a potential pregnancy by regulating hormone levels. Granulosa cell dysfunction underlies multiple forms of human female infertility, but effective methods for generating granulosa cells in vitro remain unclear.

[0007] Overview

[0008] The present disclosure relates, at least in part, to methods and compositions for generating granulosa cells in vitro from pluripotent stem cells (PSCs). The present disclosure provides experimental data that unexpectedly demonstrate 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)) is sufficient to generate granulosa cells (e.g., AMHR2+, CD82+, FOXL2+, and / or EPCAM-granulosa cell-like cells) from iPSCs in as little as 5 to 7 days.

[0009] Some aspects of the present disclosure provide a PSC comprising: an engineered polynucleotide comprising an open reading frame encoding a protein selected from the group consisting of NR5A1 and a RUNX family protein.

[0010] In some embodiments, the PSC comprises an engineered polynucleotide comprising an open reading frame encoding NR5A1.

[0011] In some embodiments, the PSC comprises an engineered polynucleotide comprising an open reading frame encoding a RUNX family protein.

[0012] In some embodiments, the RUNX family protein is RUNX1.

[0013] In some embodiments, the RUNX family protein is RUNX2.

[0014] In some embodiments, the PSC expresses or overexpresses: NR5A1; RUNX1; RUNX2; NR5A1 and RUNX1; NR5A1 and RUNX2; or NR5A1, RUNX1, and RUNX2.

[0015] In some embodiments, the PSC further comprises an engineered polynucleotide comprising an open reading frame encoding a transcription factor 21 (TCF21) protein.

[0016] In some embodiments, the PSC expresses or overexpresses TCF21.

[0017] In some embodiments, the PSC comprises an engineered polynucleotide comprising an open reading frame encoding GATA binding protein 4 (GATA4).

[0018] In some embodiments, the PSC expresses or overexpresses GATA4.

[0019] In some embodiments, the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

[0020] In some embodiments, the heterologous promoter is an inducible promoter.

[0021] Other aspects of the present disclosure provide a PSC comprising: a protein selected from NR5A1 and a RUNX family protein, wherein the protein is overexpressed.

[0022] In some embodiments, the PSC expresses or overexpresses: NR5A1; RUNX1; RUNX2; NR5A1 and RUNX1; NR5A1 and RUNX2; or NR5A1, RUNX1, and RUNX2.

[0023] In some embodiments, the PSC further comprises TCF21 protein.

[0024] In some embodiments, the PSC expresses or overexpresses TCF21.

[0025] In some embodiments, the PSC further comprises a GATA4 protein.

[0026] In some embodiments, the PSC expresses or overexpresses GATA4.

[0027] In some embodiments, the PSC is a human PSC.

[0028] In some embodiments, the PSC is an induced PSC (iPSC).

[0029] In some embodiments, the PSC comprises 1-20, optionally 8-10 copies of an engineered polynucleotide comprising an open reading frame encoding a protein selected from NR5A1 and a RUNX family protein (eg, RUNX1 and / or RUNX2).

[0030] A further aspect of the present disclosure provides a composition comprising: a population of PSCs as described in any of the preceding paragraphs or elsewhere herein.

[0031] In some embodiments, the population comprises at least 10,000 / cm 2 PSC.

[0032] Some aspects of the present disclosure provide a method comprising: culturing a population of PSCs in a culture medium to produce an expanded population of PSCs; and expressing a protein selected from NR5A1 and RUNX family proteins in the PSCs of the expanded population to produce granulosa cell-like cells.

[0033] In some embodiments, PSCs of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding NR5A1.

[0034] In some embodiments, the PSCs of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding a RUNX family protein.

[0035] In some embodiments, the RUNX family protein is RUNX1.

[0036] In some embodiments, the RUNX family protein is RUNX2.

[0037] In some embodiments, the PSCs of the expanded population further comprise an engineered polynucleotide comprising an open reading frame encoding a TCF21 protein.

[0038] In some embodiments, the PSCs of the expanded population further comprise an engineered polynucleotide comprising an open reading frame encoding a GATA4 protein.

[0039] In some embodiments, the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

[0040] In some embodiments, the heterologous promoter is an inducible promoter.

[0041] In some embodiments, the population comprises 1x10 2 -1x10 7 PSCs.

[0042] In some embodiments, the population of PSCs is cultured for about 4-10 days. For example, the population of PSCs can be cultured for about 6 days.

[0043] In some embodiments, the granulosa cell-like cells are AMHR2 + 、CD82 + , FOXL2 + and / or EPCAM - .

[0044] Other aspects of the present disclosure provide a method comprising: (a) delivering an engineered polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a protein selected from the group consisting of NR5A1 and RUNX family proteins to a PSC; (b) culturing the PSC in a feeder-free, serum-free medium to produce an expanded population of PSCs; and (c) culturing the expanded population of PSCs in a series of induction mediums comprising an inducing agent to produce AMHR2 + 、CD82 + , FOXL2 + and / or EPCAM - Granular cell-like cells.

[0045] In some embodiments, the method comprises delivering to a PSC (i) an engineered polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding NR5A1 and (ii) an engineered polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a RUNX family protein.

[0046] In some embodiments, the RUNX family protein is RUNX1.

[0047] In some embodiments, the RUNX family protein is RUNX2.

[0048] In some embodiments, the engineered polynucleotide is a transposon, and the delivering further comprises delivering a transposase to the PSC.

[0049] In some embodiments, the inducible promoter is a chemically inducible promoter, optionally a doxycycline inducible promoter.

[0050] 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.

[0051] In some embodiments, the solubilized basement membrane preparation comprises extracellular matrix (ECM) proteins and growth factors.

[0052] In some embodiments, the ECM protein is selected from laminin, collagen IV, heparin sulfate proteoglycans, and entactin / nidogen.

[0053] 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 β (rh TGFβ).

[0054] In some embodiments, the culturing of (b) continues for about 6-24 hours.

[0055] In some embodiments, the PSCs in the expanded population of (c) are approximately 10,000 cells / cm 2 Up to about 20,000 cells / cm 2 Density culture.

[0056] 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.

[0057] In some embodiments, the first induction medium comprises one or more of L-alanyl-L-glutamine, antibiotics (e.g., penicillin and / or streptomycin), Dulbecco's modified Eagle's medium (DMEM) / F-12, Advanced RPMI (Roswell Park Memorial Institute) 1640 medium, glycogen synthase kinase (GSK) 3 inhibitors, small molecule or protein inhibitors of the BMP signaling pathway, small molecule ROCK inhibitors, and inducers (e.g., doxycycline).

[0058] In some embodiments, the PSCs are cultured in the first induction medium for about 36 to about 60 hours, optionally about 48 hours.

[0059] In some embodiments, the second induction medium comprises one or more of L-alanyl-L-glutamine, antibiotics (eg, penicillin and / or streptomycin), Advanced RPMI 1640 medium, DMEM / F-12, and an inducer (eg, doxycycline).

[0060] In some embodiments, the PSCs are cultured in the second induction medium for about 96 to about 144 hours, optionally about 120 hours.

[0061] In some embodiments, the second induction medium is removed and replaced with fresh second induction medium at intervals of about 24 hours.

[0062] A further aspect of the disclosure provides a granulosa cell-like cell produced by the method of any one of the preceding claims.

[0063] The details of one or more embodiments of the invention are set forth in the following description. Other features and advantages of the invention will be apparent from the following drawings and detailed description of several embodiments and the appended claims.

[0064] Some aspects provide ovarian organoids comprising the granulosa cell-like cells of any of the preceding claims and human primordial germ cell-like cells (hPGCLCS).

[0065] In some embodiments, the method of any of the preceding paragraphs further comprises combining granulosa cell-like cells with hPGCLCS to form ovarian organoids.

[0066] BRIEF DESCRIPTION OF THE DRAWINGS

[0067] The drawings are not drawn to scale. In the drawings, each identical or nearly identical component shown in various figures is represented by a like numeral. For clarity, not every component is labeled in every figure. In the drawings:

[0068] Figure 1 Barcode enrichment screen for TFs that activate FOXL2 expression is shown. 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 mixture); 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, FOXL2 expression was detected in mTeSR cells that support pluripotency. TM Cells were treated with doxycycline to induce TF expression in either Plus medium or in mesoderm induction medium. After 5 days, FOXL2+ cells were isolated by FACS and DNA was extracted. The barcode frequencies were compared between FOXL2+ cells and the initial cell population before TF expression.

[0069] Figure 2TF combination screening is shown. Various combinations of TF expression plasmids were introduced into FOXL2-tdTomato reporter iPSCs. The iPSCs were then inoculated into differentiation medium (DMEM / F12, 10% KSR, 1μg / mL doxycycline). In the first 2 days, the cells were also treated with 3μM CHIR99021 and 10μM Y-27632. After 5 days of differentiation, the cells were dissociated and analyzed by flow cytometry to measure the proportion of AMHR2+FOXL2+CD82+EPCAM-granulosa cell-like cells. From the 4th to the 6th day of differentiation, the second replicate was treated with androstenedione (500ng / mL) and FSH (0.15IU / mL). The production of estradiol was measured by ELISA.

[0070] Figure 3 Transcriptional signatures of granulosa-like cells are shown. 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 mean of at least two biological replicates.

[0071] Figure 4 The combination of the top-ranked TFs was shown to produce granulosa cell-like cells at a high yield. Expression plasmids for the top-ranked TFs (NR5A1, RUNX1, RUNX2, TCF21) were introduced into iPSCs, and single colonies were picked to generate monoclonal lines. Clone 1F integrated NR5A1, RUNX1, and TCF21 expression plasmids (confirmed by PCR). A 5-day differentiation protocol using doxycycline-induced TF expression produced a nearly uniform population of FOXL2+CD82+ granulosa cell-like cells. In contrast, spontaneous differentiation without doxycycline produced only a small number of granulosa cell-like cells.

[0072] Figure 5A-5B Demonstrating hormone signaling in granulosa cell-like cells. Figure 5AGranulosa cell-like cells are shown to produce estradiol in the presence of androstenedione and FSH or forskolin (FK). The results show 9 monoclonal populations from granulosa cell-like cells (n=2 biological replicates for each of the 9 clones, with 95% CI error bars), as well as COV434 and KGN human ovarian cancer cell lines (control), HGL5 immortalized primary human granulosa cells, and primary adult mouse granulosa cells. Asterisks mark cell lines that produce significantly (two-tailed t-test, p<.05) increased FSH production after stimulation. The exact p value is given in the source data. iPSC monoclonal lines with integrated TFs (NR5A1, TCF21, GATA4, RUNX1) were generated by selecting single colonies from polyclonal populations. These cell lines were then subjected to a 5-day granulosa cell differentiation protocol. The cells were then treated with androstenedione (500 ng / mL) for 24 hours in the presence or absence of FSH (0.25 IU / mL) and / or forskolin (100 μM). The production of estradiol was measured by ELISA. Three of the four cell lines showed hormone-responsive estradiol production, while the fourth cell line (which was found to lack RUNX1 by PCR) constitutively produced high levels of estradiol. Figure 5B Ovarian organoids (ovaroids) are shown to produce estradiol and progesterone. Estradiol production requires androstenedione and is stimulated by FSH. Results show ovarian organoids formed from six different monoclonal samples of granulosa cell-like cells (n=1 sample per ovarian organoid per condition) 3 days after aggregation.

[0073] Figure 6 A scheme for inducing granulosa cell-like cells is shown. hiPSCs containing an integrated TF expression plasmid were mTeSR on substrate TM To induce granulosa cell-like cells, hiPSCs were dissociated into single cells and plated at 10,000-20,000 cells / cm 2 The density of the cells was added to DK10 medium (containing GlutaMAX) supplemented with 3 μM CHIR99021, 10 μM Y-27632 and 1 μg / mL doxycycline to induce TF expression. TM The cells were inoculated in DMEM / F12 supplemented with 10% Knockout serum replacement. After 48 hours, and subsequently at 24 hour intervals, the medium was changed and replaced with fresh DK10 + 1 μg / mL doxycycline. After a total of 120 hours, the granulocyte-like cells were ready for downstream experiments.

[0074] Figure 7Shown is the expression of OCT4 over time in human ovarian organoids and mouse xenograft ovarian organoids + and DAZL + Cells relative to total amount (DAPI + Images from two replicates of human ovarian organoids (F66 / N.R1.GF#4 and F66 / N.R2#1 granulosa-like cells+hPGCLCs) and one replicate of mouse xenogeneic ovarian organoids were counted at 11 time points.

[0075] Figures 8A-8D scRNA-seq analysis of ovarian organoids (F66 / N.R1.GF#4 granulosa-like cells + hPGCLCs) is shown. Data from all samples (days 2, 4, 8, and 14) were combined for joint dimensionality reduction and clustering. Fig. 8A The expression (log2 CPM) of selected granulosa cell (FOXL2), matrix / theca (NR2F2), and germ cell (PRDM1) markers is shown. Figure 8B Leiden clustering shows four major clusters; expression (log2 CPM) of marker genes is plotted for each cluster. Figure 8C Shown is the mapping of cells onto the human fetal ovary reference atlas and the assignment of cell types. Fig.8D Displays somatic cell types, germ cells, DAZL in ovarian organoids every day + Cells and DDX4 + The proportion of cells.

[0076] Detailed Description

[0077] Ovarian granulosa cells are important for many aspects of female reproduction, including supporting oocyte development and hormonal signaling during the menstrual cycle. Current in vitro models, including primary human or mouse granulosa cells and granulosa cell tumor lines, are insufficient to study these processes (Havelock et al., 2004). For example, the COV434 ovarian tumor line (Zhang, 2000), which is commonly used as a granulosa cell model, lacks the transcriptional and phenotypic characteristics of true granulosa cells. Previously reported protocols for differentiating induced pluripotent stem cells (iPSCs) into granulosa cell-like cells either have low yields (Lan et al. 2013; Lipskind et al. 2018) or are only applicable to mouse cells (Yoshino et al. 2021). Aspects of the present disclosure relate to a robust, scalable method that uses direct transcription factor overexpression to mediate iPSC differentiation to generate AMHR2 in about 4 to about 10 days. + (Anti-Müllerian hormone receptor type 2), CD82 + (Cluster of Differentiation 82), FOXL2+ (fork head frame L2) and / or EPCAM - It should be understood that the term "granulosa cell-like cells" encompass cells that express granulosa cell-specific markers (e.g., AMHR2, CD82, and / or FOXL2) and / or do not express EPCAM and exhibit other characteristics of naturally occurring granulosa cells.

[0078] Aspects of the present disclosure relate to pluripotent stem cells (PSCs) comprising: an engineered polynucleotide comprising an open reading frame encoding a protein selected from nuclear receptor subfamily 5 group A member 1 (NR5A1) and Runt-related transcription factor (RUNX) family proteins. In some embodiments, the engineered polynucleotide comprises an open reading frame encoding NR5A1. In some embodiments, the engineered 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).

[0079] Granular cell-like cells

[0080] Certain aspects of the present disclosure provide granulosa cell-like cells and methods for producing such cells. Granula cells or follicle cells are somatic cells of the reproductive cord, which are closely related to the developing female gametes (oocytes / ovum) in the mammalian ovary. In the primordial follicle and in the subsequent follicular development (folliculogenesis), granulosa cells develop into a multilayer cumulus that surrounds the oocyte before ovulation or in the antral follicle (Graafian follicle). The main functions of granulosa cells include the production of sex steroids and numerous growth factors that are believed to interact with the oocyte during oocyte development. The production of sex steroids begins with follicle stimulating hormone (FSH) from the anterior pituitary, which stimulates granulosa cells to convert androgens (from theca cells) into estradiol by aromatase during the follicular phase of the menstrual cycle. After ovulation, granulosa cells become granulosa luteal cells that produce progesterone. Progesterone can maintain potential pregnancy and lead to the production of thick cervical mucus, which inhibits sperm from entering the uterus.

[0081] 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 influence the development of the oocyte in a paracrine manner. Mural granulosa cells line the wall of the follicle and surround the fluid-filled antrum. The oocyte secretes factors that determine the functional differences between CC and MGC. CC mainly supports the growth and development of the oocyte, while MGC mainly plays an endocrine function and supports the growth of follicles. Cumulus cells contribute to the development of the oocyte and show higher expression of the amino acid transporter SLC38A3 and the enzymes Aldoa, Eno1, Ldh1, Pfkp, Pkm2 and Tpi1 responsible for glycolysis. MGCs have higher steroidogenic activity and have higher levels of mRNA expression of steroidogenic enzymes such as cytochrome P450. MGCs produce more and more estrogen, which causes a surge in LH. After the LH surge, cumulus cells undergo cumulus expansion, in which they proliferate at ten times the rate of MGCs in response to FSH. During the expansion process, the CC also produces the mucus matrix required for ovulation.

[0082] Granular layer cells express many different biomarkers that can be used to distinguish granular layer cells and granular layer cell-like cells from other cell types. For example, granular layer cells are usually positive for anti-Mullerian hormone receptor type 2 (AMHR2) (AMHR2+), CD82 molecules (CD82+), forkhead box L2 (FOXL2+), and negative for epithelial cell adhesion molecule (EPCAM-). Therefore, in some embodiments, the granular layer cell-like cells produced by the methods provided herein are AMHR2+, CD82+, FOXL2+ and / or EPCAM-granular layer cell-like cells (i.e., cells expressing AMHR2, CD82 and / or FOXL2 proteins but not expressing detectable levels of EPCAM).

[0083] Granulocyte-like cells also have other characteristics that distinguish them from non-granulocyte-like cells, including but not limited to the expression of a combination of adhesion proteins, such as adhesion proteins (B-catenin, α-catenin, N-cadherin, synaptone 1-3) as well as tight junction proteins (JAM-A, cingulin), desmosomes (dsg2, Dsc2) and connexins (afadin, ZO-1,2 and ZONAB). In addition, granulocyte-like cells can also be distinguished by the biosynthesis of a combination of estradiol, progesterone and AMH.

[0084] Pluripotent stem cells

[0085] The granulosa cell-like cells provided herein are differentiated from pluripotent stem cells. Pluripotent stem cells are cells with the following abilities: self-renewal by division and the ability to develop into the three major germ cell layers of the early embryo (e.g., ectoderm, endoderm, and mesoderm), and therefore can develop into all cells of the human body, excluding extraembryonic tissues such as the placenta (Shi et al., 2017).

[0086] Non-limiting examples of pluripotent stem cells include induced pluripotent cells (iPSCs), "true" embryonic stem cells (ESCs) derived from embryos, embryonic stem cells made by somatic cell nuclear transfer (ntESCs), and embryonic stem cells from unfertilized eggs (parthenogenetic embryonic stem cells or pESCs). In some embodiments, the pluripotent cells are human pluripotent cells.

[0087] In some embodiments, the pluripotent stem cell is an embryonic stem cell, such as a human embryonic stem cell. "Embryonic stem cell" is a general term for pluripotent stem cells made using embryos or eggs, rather than cells genetically reprogrammed from the body. As used herein, "ESC" includes true ESC, ntESC and pESC.

[0088] In other embodiments, the pluripotent stem cells are induced pluripotent stem cells, such as human induced pluripotent stem cells. iPSCs can be derived from skin or blood cells that have been reprogrammed back to an embryonic-like pluripotent state, enabling the development of an unlimited source of any type of human cell.

[0089] Some aspects of the present disclosure provide a PSC comprising: a protein selected from NR5A1 and a RUNX family protein (e.g., RUNX1 and / or RUNX2), wherein the protein is expressed or overexpressed. In some embodiments, the PSC also comprises TCF21 protein. In some embodiments, the expression level of the protein is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, or at least 100% higher than the control level. In some embodiments, the PSC also comprises GATA4 protein. In some embodiments, the expression level of the protein is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, or at least 100% higher than the control level. In some embodiments, the control level is the endogenous level of the protein, such as in naturally occurring pluripotent stem cells. In some embodiments, the PSC comprises NR5A1. In some embodiments, the PSC expresses or overexpresses 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.

[0090] The data provided herein show that the combined expression of NR5A1 and RUNX family proteins (e.g., RUNX1 and / or RUNX2) results in a 2-15-fold increase in granular layer cell-like production efficiency relative to a control, wherein optionally, the control is a granular layer cell-like production efficiency in a PSC expressing only one of NR5A1 or RUNX family proteins (e.g., RUNX1 and / or RUNX2). In some embodiments, PSC comprises NR5A1 and RUNX family proteins (e.g., RUNX1 and / or RUNX2). In some embodiments, PSC expresses or overexpresses NR5A1 and RUNX family proteins (e.g., RUNX1 and / or RUNX2). In some embodiments, PSC comprises NR5A1 and RUNX1. In some embodiments, PSC expresses or overexpresses NR5A1 and RUNX1. In some embodiments, PSC comprises NR5A1 and RUNX2. In some embodiments, PSC expresses or overexpresses NR5A1 and RUNX2. In some embodiments, PSC also 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.

[0091] Transcription Factors

[0092] In some embodiments, the granulosa cell-like cells provided herein 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 transcription rates). Differentiation is the process by which uncommitted cells or partially committed cells are directed to a specialized cell fate. Aspects of the present disclosure relate to the differentiation of uncommitted pluripotent stem cells to a granulosa cell-like cell fate.

[0093] In some embodiments, the transcription factor is selected from NR5A1 and RUNX family proteins (e.g., RUNX1 and / or RUNX2). In some embodiments, pluripotent stem cells (e.g., hPSC or hiPSC) are engineered to express or overexpress NR5A1. In some embodiments, pluripotent stem cells (e.g., hPSC or hiPSC) are engineered to express or overexpress RUNX family proteins (e.g., RUNX1 and / or RUNX2). In some embodiments, pluripotent stem cells (e.g., hPSC or hiPSC) are engineered to express or overexpress RUNX1. In some embodiments, pluripotent stem cells (e.g., hPSC or hiPSC) are engineered to express or overexpress RUNX2. In some embodiments, pluripotent stem cells, such as hPSC or hiPSC, are engineered to express or overexpress TCF21. In some embodiments, pluripotent stem cells, such as hPSC or hiPSC, are engineered to express or overexpress GATA4. In some embodiments, pluripotent stem cells, such as hPSC or hiPSC, are engineered to express or overexpress NR5A1 and RUNX family proteins (e.g., RUNX1 and / or RUNX2). In some embodiments, pluripotent stem cells, such as hPSC or hiPSC, are engineered to express or overexpress NR5A1 and RUNX1. In some embodiments, pluripotent stem cells, such as hPSC or hiPSC, are engineered to express or overexpress NR5A1 and RUNX2. In some embodiments, pluripotent stem cells (e.g., hPSC or hiPSC) are engineered to further express or overexpress TCF21. In some embodiments, pluripotent stem cells (e.g., hPSC or hiPSC) are engineered to further express or overexpress GATA4.

[0094] A cell "expresses" a particular protein if the level of the protein in the cell is detectable (e.g., using a known protein assay). A cell "overexpresses" a particular protein (e.g., an engineered polynucleotide encoding the protein) if the level of the protein is greater (e.g., at least 5% greater, at least 10% greater, or at least 20% greater) than the level of the protein expressed by an endogenous, naturally occurring polynucleotide encoding the protein.

[0095] Engineered polynucleotides and peptides

[0096] In some embodiments, the pluripotent stem cells of the present disclosure include engineered polynucleotides. Engineered polynucleotides are nucleic acids that do not exist in nature (e.g., at least two nucleotides are covalently linked together, and in some cases, contain phosphodiester bonds, referred to as phosphodiester backbones). Engineered polynucleotides include recombinant nucleic acids and synthetic nucleic acids. Recombinant nucleic acids are molecules constructed by connecting nucleic acids (e.g., isolated nucleic acids, synthetic nucleic acids, or combinations thereof) from two different organisms (e.g., humans and mice). Synthetic nucleic acids are molecules synthesized by amplification or chemical synthesis or other means. Synthetic nucleic acids include those nucleic acids that are chemically modified or otherwise modified but can be base paired (bound) with naturally occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include those molecules produced by replication of any of the above.

[0097] Engineered polynucleotides can include DNA (e.g., genomic DNA, cDNA, or a combination of genomic DNA and cDNA), RNA, or hybrid molecules, for example, wherein the nucleic acid comprises any combination of (e.g., artificial or natural) deoxyribonucleotides and ribonucleotides, and any combination of two or more bases (including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine).

[0098] In some embodiments, the polynucleotide is complementary DNA (cDNA).cDNA is synthesized from a single-stranded RNA (eg, messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by reverse transcriptase.

[0099] The engineered 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, nucleic acids are generated using GIBSON Clonal generation (see, eg, Gibson, DG et al. Nature Methods, 343-345, 2009; and Gibson, DG et al. Nature Methods, 901-903, 2010, each of which is incorporated herein by reference). GIBSON Three enzyme activities are usually used in a single tube reaction: 5' exonuclease, 3' extension activity of DNA polymerase and DNA ligase activity. The 5' exonuclease activity digests the 5' end sequence (chew back) and exposes the complementary sequence for annealing. The polymerase activity then fills the gap on the annealed domain. The DNA ligase then seals the nick and covalently connects the DNA fragments together. The overlapping sequences of adjacent fragments are much longer than those used in Golden Gate Assembly, thus resulting in a higher percentage of correct assembly. According to the present disclosure, other methods for producing engineered polynucleotides can be used.

[0100] In some embodiments, the engineered polynucleotide comprises a promoter operably connected to an open reading frame. A promoter is a nucleotide sequence (e.g., ATG) to which RNA polymerase binds to initiate transcription. A promoter is generally located directly upstream (at the 5' end) of the transcription start site. In some embodiments, a promoter is a heterologous promoter. A heterologous promoter is not naturally associated with the open reading frame to which it is operably connected.

[0101] In some embodiments, the promoter is an inducible promoter. An inducible promoter can be regulated in vivo by, for example, chemicals, temperature, or light. An inducible promoter can achieve, for example, temporal and / or spatial control of gene expression. Inducible promoters used according to the present disclosure include any inducible promoters described herein or known to those of ordinary skill in the art. Examples of inducible promoters include, but are not limited to, chemically / biochemically regulated and physically regulated promoters, such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, which include tetracycline repressor protein (tetR), tetracycline operator sequence (tetO), and tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptor, and promoters from the steroid / retinoic acid / thyroid 25 receptor superfamily), metal-regulated promoters (e.g., promoters from metallothionein (proteins that bind and sequester metal ions) genes derived from yeast, mouse, and human), pathogenicity-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 (active in vivo, not regulated).

[0102] An open reading frame is a continuous segment 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 regulates the transcription of the open reading frame.

[0103] Vectors for delivering engineered polynucleotides include minicircles, plasmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes. Transposon-based systems, such as piggyBac, are also contemplated herein. TM system (e.g. Chen et al. Nature Communications. 2020; 11(1): 3446).

[0104] In some embodiments, the pluripotent stem cell comprises an engineered polynucleotide comprising an open reading frame encoding a protein selected from NR5A1 and RUNX family proteins (e.g., RUNX1 and / or RUNX2). In some embodiments, the engineered polynucleotide comprises an open reading frame encoding NR5A1. In some embodiments, the engineered polynucleotide comprises an open reading frame encoding RUNX family proteins (e.g., RUNX1 and / or RUNX2). In some embodiments, the engineered polynucleotide comprises an open reading frame encoding RUNX1. In some embodiments, the engineered polynucleotide comprises an open reading frame encoding RUNX2. In some embodiments, the engineered polynucleotide comprises an open reading frame encoding TCF21. In some embodiments, the engineered polynucleotide comprises an open reading frame encoding GATA4.

[0105] In some embodiments, pluripotent stem cells include an engineered polynucleotide containing an open reading frame encoding NR5A1 and an engineered polynucleotide containing an open reading frame encoding RUNX family proteins (e.g., RUNX1 and / or RUNX2). In some embodiments, pluripotent stem cells include an engineered polynucleotide containing an open reading frame encoding NR5A1 and an engineered polynucleotide containing an open reading frame encoding RUNX1. In some embodiments, pluripotent stem cells include an engineered polynucleotide containing an open reading frame encoding NR5A1 and an engineered polynucleotide containing an open reading frame encoding RUNX2. In some embodiments, pluripotent stem cells also include an engineered polynucleotide containing an open reading frame encoding TCF21. In some embodiments, pluripotent stem cells also include an engineered polynucleotide containing an open reading frame encoding GATA4.

[0106] In some embodiments, the engineered polynucleotide comprising an open reading frame encoding Nuclear Receptor Subfamily 5 Group A Member 1 (NR5A1) (e.g., UniprotKB Accession No. Q13285) encodes a protein comprising the following sequence:

[0107] MDYSYDEDLDELCPVCGDKVSGYHYGLLTCESCKGFFKRTVQNNKHYTCTESQSCKIDKTQRKRCPFCRFQKCLTVGMRLEAVRADRMRGGRNKFGPMYKRDRALKQQKKAQIRAN GFKLETGPPMGVPPPPPPAPDYVLPPSLHGPEPKGLAAGPPAGPLGDFGAPALPMAVPGAHGPLAGYLYPAFPGRAIKSEYPEPYASPPQPGLPYGYPEPFSGGPNVPELILQLLQ LEPDEDQVRARILGCLQEPTKSRPDQPAAFGLLCRMADQTFISIVDWARRCMVFKELEVADQMTLLQNCWSELLVFDHIYRQVQHGKEGSILLVTGQEVELTTVATQAGSLLHSLVLRAQELVLQLLALQLDRQEFVCLKFIILFSLDLKFLNNHILVKDAQEKANAALLDYTLCHYPHCGDKFQQLLLCLVEVRALSMQAKEYLYHKHLGNEMPRNNLLIEMLQAKQT (SEQ ID NO: 1) In some embodiments, the engineered polynucleotide comprising an open reading frame encoding Runt-related transcription factor 1 (RUNX1) (e.g., UniprotKB Accession No. Q01196) encodes a protein comprising the following sequence:

[0108] MRIPVDASTSRRFTPPSTALSPGKMSEALPLGAPDAGAALAGKLRSGDRSMVEVLADHPGELVRTDSPNFLCSVLPTHWRCNKTLPIAFKVVALGDVPDGTLVTVMAGNDENYS AELRNATAAMKNQVARFNDLRFVGRSGRGKSFTLTITVFTNPPQVATYHRAIKITVDGPREPRRHRQKLDDQTKPGSLSFSERLSELEQLRRTAMRVSPHHPAPTPNPRASLNH STAFNPQPQSQMQDTRQIQPSPPWSYDQSYQYLGSIASPSVHPATPISPGRASGMTTLSAELSSRLSTAPDLTAFSDPRQFPALPSISDPRMHYPGAFTYSPTPVTSGIGIGMS AMGSATRYHTYLPPPYPGSSQAQGGPFQASSPSYHLYYGASAGSYQFSMVGGERSPPRILPPCTNASTGSALLNPSLPNQSDVVEAEGSHSNSPTNMAPSARLEEAVWRPY(SEQ ID NO:2)

[0109] In some embodiments, an engineered polynucleotide comprising an open reading frame encoding Runt-related transcription factor 2 (RUNX2) (e.g., UniprotKB Accession No. Q13950) encodes a protein comprising the following sequence:

[0110] MASNSLFSTVTPCQQNFFWDPSTSRRFSPPSSSLQPGKMSDVSPVVAAQQQQQQQQQQQQQQQQQQQQQEAAAAAAAAAAAAAAAAAVPRLRPPHDNRTMVEIIADHPAELVRTDSPNFLCSVLPSHWR CNKTLPVAFKVVALGEVPDGTVVTVMAGNDENYSAELRNASAVMKNQVARFNDLRFVGRSGRGKSFTLTITVFTNPPQVATYHRAIKVTVDGPREPRRHRQKLDDSKPSLFSDRLSDLGRIPHPSMRVGVP PQNPRPSLNSAPSPFNPQGQSQITDPRQAQSSPPWSYDQSYPSYLSQMTSPSIHSTTPLSSTRGTGLPAITDVPRRISDDDTATSDFCLWPSTLSKKSQAGASELGPFSDPRQFPSISSLTESRFSNPRMH YPATFTYTPPVTSGMSLGMSATTHYHTYLPPPYPGSSQSQSGPFQTSSTPYLYYGTSSGSYQFPMVPGGDRSPSRMLPPCTTTSNGSTLLNPNLPNQNDGVDADGSHSSSPTVLNSSGRMDESVWRPY(SEQ ID NO:3)

[0111] In some embodiments, the engineered polynucleotide comprising an open reading frame encoding transcription factor 21 (TCF21) (e.g., UniprotKB Accession No. 043680) encodes a protein comprising the following sequence:

[0112] MSTGSLSDVEDLQEVEMLECDGLKMDSNKEFVTSNESTEESSNCENGSPQKGRGGLGKRRKAPTKKSPLSGVSQEGKQVQRNAANARERARMRVLSKAFSRLKTTLPWVPPDTKLSKLDTLRLASSYIAHLRQILANDKYENGYIHPVNLTWPFMVAGKPESDLKEVVTASRLCGTTAS(SEQ ID NO: 4)

[0113] In some embodiments, the engineered polynucleotide comprising an open reading frame encoding GATA binding protein 4 (GATA4) (e.g., UniprotKB Accession No. P43694) encodes a protein comprising the following sequence:

[0114] MYQSLAMAANHGPPPGAYEAGGPGAFMHGAGAASSPVYVPTPRVPSSVLGLSYLQGGGAGSASGGASGGSSGGAASGAGPGTQQGSPGWSQAGADGAAYTPPPVSPRFSFP GTTGSLAAAAAAAAAREAAAYSSGGGAAGAGLAGREQYGRAGFAGSYSSPYPAYMADVGASWAAAAAASAGPFDSPVLHSLPGRANPAARHPNLDMFDDFSEGRECVNCGAM STPLWRRDGTGHYLCNACGLYHKMNGINRPLIKPQRRLSASRRVGLSCANCQTTTTTLWRRNAEGEPVCNACGLYMKLHGVPRPLAMRKEGIQTRKRKPKNLNKSKTPAAP SGSESLPPASGASSNSSNATTSSSEEMRPIKTEPGLSSHYGHSSSVSQTFSVSAMSGHGPSIHPVLSALKLSPQGYASPVSQSPQTSSKQDSWNSLVLADSHGDIITA(SEQ IDNO: 5)

[0115] The number of copies of the engineered polynucleotide delivered to the PSC may vary. In some embodiments, the PSC comprises 1-20 copies of the engineered 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 engineered polynucleotide. In some embodiments, the PSC comprises 8-10 copies of the engineered polynucleotide. More than 20 copies are also contemplated herein.

[0116] Methods for generating granulosa cell-like cells

[0117] The method for producing granular layer cell-like cells provided herein includes in some aspects culturing a pluripotent stem cell (PSC) population in a culture medium to produce an amplified population of PSC; and expressing a protein selected from NR5A1 and RUNX family proteins (e.g., RUNX1 and / or RUNX2) in the PSC of the amplified population to produce granular layer cell-like cells. In some embodiments, the method also includes expressing TCF21 protein in the PSC of the amplified population. In some embodiments, the method also includes expressing GATA4 protein in the PSC of the amplified population.

[0118] In some embodiments, the PSC of the amplified population comprises an engineered polynucleotide containing an open reading frame encoding NR5A1. In some embodiments, the PSC of the amplified population comprises an engineered polynucleotide containing an open reading frame encoding a RUNX family protein (e.g., RUNX1 and / or RUNX2). In some embodiments, the PSC of the amplified population comprises an engineered polynucleotide containing an open reading frame encoding RUNX1. In some embodiments, the PSC of the amplified population comprises an engineered polynucleotide containing an open reading frame encoding RUNX2. In some embodiments, the PSC of the amplified population comprises an engineered polynucleotide containing an open reading frame encoding TCF21. In some embodiments, the PSC of the amplified population comprises an engineered polynucleotide containing an open reading frame encoding GATA4.

[0119] In some embodiments, the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

[0120] In some embodiments, the heterologous promoter is an inducible promoter, non-limiting examples of which are provided elsewhere herein.

[0121] The population (starting population) contains about 1x10 2 -1x10 10 , about 1x10 2 -1x10 9 , about 1x10 2 -1x10 8 or about 1x10 2 -1x10 7 In some embodiments, the population comprises about 1x10 3

[0122] -1x10 8 or about 1x10 3 -1x10 7 In some embodiments, the population comprises about 1x10 4

[0123] -1x10 7 or about 1x10 5 -1x10 6 In some embodiments, the population comprises about 1x10 1 PSC, about 1x10 2 PSC, about 1x10 3 PSC, about 1x10 4 PSC, about 1x10 5 PSC, about 1x10 6 PSC, about 1x107 PSC, about 1x10 8 PSC, about 1x10 9 PSC or about 1x10 10 PSCs.

[0124] 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.

[0125] Some methods of the present disclosure provide methods, which include (a) delivering an engineered polynucleotide comprising an inducible promoter operably connected to an open reading frame encoding a protein selected from NR5A1 and RUNX family proteins (e.g., RUNX1 and / or RUNX2) to PSC; (b) culturing PSC in a feeder-free, serum-free medium to produce an amplified population of PSC; and (c) culturing the PSC of the amplified population in a series of induction mediums comprising an inducing agent to produce AMHR2+, CD82+, FOXL2+ and / or EPCAM- granulosa cell-like cells. In some embodiments, a series of induction mediums include a first induction medium, a second induction medium, a third induction medium, and a fourth induction medium. In some embodiments, an engineered polynucleotide comprising an inducible promoter operably connected to an open reading frame encoding TCF21 protein is also delivered to PSC (e.g., in step (a)). In some embodiments, an engineered polynucleotide comprising an inducible promoter operably connected to an open reading frame encoding GATA4 protein is also delivered to PSC (e.g., in step (a)).

[0126] In some embodiments, the PSC is cultured in a feeder-free, serum-free medium for about 6 to about 24 hours. For example, the PSC can be cultured in a feeder-free, serum-free medium for about 6 to about 12 hours. In some embodiments, the PSC is 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.

[0127] In some embodiments, the expanded population of PSCs comprises at least 5×10 3 For example, the expanded population (e.g., upon induction) can contain at least 1x104 , at least 1x10 5 , at least 1x10 6 or at least 1x10 7 In some embodiments, the expanded population of PSCs comprises about 5x10 3 PSC is about 1x10 7 PSCs.

[0128] In some embodiments, the PSCs in the expanded population are approximately 10,000 cells / cm 2 to approximately 30,000 cells / cm 2 In some embodiments, the PSCs of the expanded population are cultured at a density of about 10,000 cells / cm 2 to approximately 25,000 cells / cm 2 In some embodiments, the PSCs of the expanded population are cultured at a density of about 10,000 cells / cm 2 Up to about 20,000 cells / cm 2 In some embodiments, the PSCs of the expanded population are cultured at a density of about 10,000 cells / cm 2 to approximately 15,000 cells / cm 2 In some embodiments, the PSCs of the expanded population are cultured at a density of about 15,000 cells / cm 2 to about 30,000 cells / cm 2 In some embodiments, the PSCs of the expanded population are cultured at a density of about 15,000 cells / cm 2 to approximately 25,000 cells / cm 2 In some embodiments, the PSCs of the expanded population are cultured at a density of about 15,000 cells / cm 2 Up to 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 Density culture.

[0129] In some embodiments, the PSCs of the expanded population are cultured for no more than 10 days, no more than 9 days, no more than 8 days, no more than 7 days, no more than 6 days, no more than 5 days, or no more than 4 days. For example, the PSCs of the expanded population can 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.

[0130] In some embodiments, the PSCs of the expanded population are cultured in the first induction medium for about 36 to about 60 hours. For example, the PSCs can be cultured in the first induction medium for about 36 to about 54 hours, about 36 to about 48 hours, about 42 to about 60 hours, about 42 to about 54 hours, about 42 to about 48 hours, about 48 to about 60 hours, or about 48 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.

[0131] In some embodiments, the PSCs of the expanded population are cultured in the second induction medium for about 96 to about 144 hours. For example, the PSCs may be cultured in the second induction medium for about 96 to about 132 hours, about 96 to about 120 hours, about 96 to about 108 hours, about 108 to about 144 hours, about 108 to about 132 hours, about 108 to about 120 hours, about 120 to about 144 hours, or about 120 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.

[0132] In some embodiments, culturing in the second induction medium comprises several (one or more) medium changes. For example, the second induction medium can be removed and replaced with a new (fresh) second indicator medium every (about) 12 hours, every 24 hours, every 36 hours, or every 48 hours. In some embodiments, the second induction medium is replaced every (about) 24 hours.

[0133] Transfection method

[0134] The engineered polynucleotides of the present disclosure can be delivered to PSCs using one or more transfection methods, including chemical transfection methods, viral transduction methods, and electroporation.

[0135] In some embodiments, the engineered polynucleotide is delivered on a vector. A vector is any vehicle, such as a virus or plasmid, for transferring the desired polynucleotide to a host cell (e.g., PSC). In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is not a naturally occurring viral vector. The viral vector can be from adeno-associated virus (AAV), adenovirus, herpes simplex virus, lentivirus, retrovirus, varicella, smallpox virus, hepatitis B, cytomegalovirus, JC polyomavirus, BK polyomavirus, monkeypox virus, herpes zoster virus, Epstein-Barr virus, human herpes virus 7, Kaposi's sarcoma-associated herpes virus, or human parvovirus B19. The present disclosure also encompasses other viral vectors.

[0136] In some embodiments, the viral vector is an AAV vector. AAV is a small, non-enveloped virus that packages a single-stranded linear DNA genome of about 5 kb and has been engineered for use as a gene transfer vector (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 serve as the starting point of DNA replication and act as the main packaging signal (McLaughlin, SK et al. Virol. 1988; 62 (6): 1963-73; Hauswirth, WW et al. 1977; 78 (2): 488-99). Therefore, AAV vectors generally include ITR sequences. Both positive and negative strands are packaged equally well into virions and are able to infect (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). In addition, a small deletion in one of the two ITRs allows packaging of self-complementary vectors in which the genome anneals to itself after viral uncoating. This results in more efficient transduction of cells, but the coding capacity is reduced by half (McCarty, DM et al. Mol Ther. 2008; 16 (10): 1648-56; McCarty, DM et al. Gene Ther. 2001; 8 (16): 1248-54).

[0137] In some embodiments, a transposon / transposase system is used to deliver a polynucleotide to a cell. For example, piggyBac TM Transposon system. piggyBac TM Transposons are mobile genetic elements that efficiently transpose between vectors and chromosomes via a “cut and paste” mechanism (Woodard et al., 2015).TM The transposase recognizes the transposon-specific inverted terminal repeats (ITRs) located at both ends of the transposon vector and efficiently moves the contents from the original site and integrates them into the TTAA chromosomal locus. piggyBac TM The transposon system facilitates the efficient integration of polynucleotides into the cellular genome.

[0138] Thus, in some embodiments, the method further comprises delivering a transposon comprising the engineered polynucleotide to the PSC, and also delivering a transposase.

[0139] In some embodiments, the engineered polynucleotide is delivered to the cell using electroporation. Electroporation is a physical transfection method that uses electric pulses to create temporary holes in the cell membrane through which the engineered polynucleotide can enter the cell. See, e.g., Chicaybam L et al. Front. Bioeng. Biotechnol., 23 January 2017.

[0140] After transfection, the engineered polynucleotide can be integrated into the genome of the PSC. In some embodiments, the engineered polynucleotide may also include an antibiotic resistance gene to confer resistance to the antibiotic used in the antibiotic drug selection process. In this way, a "pure" cell population containing the integrated engineered polynucleotide can be obtained. In some embodiments, antibiotic drug selection is used to select a cell population containing the integrated engineered polynucleotide. Antibiotic drug selection is the process of treating a cell population with an antibiotic so that only cells that can survive in the presence of the antibiotic will remain in the population. Non-limiting examples of antibiotics that can be used for antibiotic drug selection include: puromycin, blasticidin, geneticin, hygromycin, mycophenolic acid, bleomycin, carbenicillin, kanamycin, ampicillin and actinomycin.

[0141] Culture medium

[0142] In some embodiments, the methods provided herein include culturing PSCs in a feeder-free, serum-free medium. The medium may include, for example, a dissolved basement membrane preparation extracted from an Engelbreth-Holm-Swarm (EHS) mouse sarcoma (e.g. Matrix) (at about 75 μl / cm 2 About 150 μl / cm 2 In some embodiments, the dissolved basement membrane preparation comprises one or more extracellular matrix (ECM) proteins and one or more growth factors. For example, the ECM protein can be selected from laminin, collagen IV, heparin sulfate proteoglycans, and paranectin / nidogen.

[0143] In some embodiments, the culture medium further comprises one or more growth factors, for example, selected from recombinant human basic fibroblast growth factor (rh bFGF) (e.g., 80 ng / ml to 120 ng / ml) and recombinant human transforming growth factor β (rh TGFβ) (e.g., 20 pM to 25 pM). In some embodiments, the culture medium further comprises rh bFGF and rh TGFβ. In some embodiments, the culture medium comprises mTeSR TM Plus medium (STEMCELL Technologies).

[0144] In some embodiments, the first induction medium comprises one or more (e.g., 2, 3, 4 or more) of the following, the first induction medium comprises 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's medium (DMEM) / F-12 (e.g., 15 mM HEPES, without glutamine), Advanced RPMI (Roswell Park Memorial In some embodiments, the first induction medium may include one or more of DK10 medium, CHIR99021, Y-27632 (a small molecule ROCK inhibitor), and doxycycline.

[0145] In some embodiments, the second induction medium comprises one or more (e.g., 2, 3, 4 or more) of L-alanyl-L-glutamine, antibiotics (e.g., penicillin and / or streptomycin), Advanced RPMI 1640 medium (containing 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.

[0146] Advanced RPMI 1640 (Thermo Fisher Scientific) consists of glucose, nonessential amino acids, sodium pyruvate (without L-glutamine and HEPES), vitamins, inorganic salts, proteins (e.g. II, human transferrin and recombinant insulin full chain) and trace elements.

[0147] The DK10 medium used herein contains KO-SR, glutamine, penicillin, streptomycin, and Dulbecco's Modified Eagle's Medium (DMEM) / F-12.

[0148] KnockOut TM KO-SR is a serum-free preparation used to replace fetal bovine serum.

[0149] GlutaMAX TM The supplement contains L-alanyl-L-glutamine, which is a dipeptide substitute for L-glutamine.

[0150] CHIR99021 is an aminopyrimidine derivative that is a very potent glycogen synthase kinase (GSK) 3 inhibitor, inhibiting GSK3β (IC 50 =6.7 nM) and GSK3α (IC 50 =10 nM). GSK3 is a serine / threonine kinase that is a key inhibitor of the WNT pathway; therefore, CHIR99021 acts as a WNT activator.

[0151] Noggin is a protein that binds and inactivates proteins in the BMP family. Dorsomorphin is a small molecule inhibitor of type I BMP receptor serine / threonine kinases. Both substances are considered inhibitors of the BMP signaling pathway.

[0152] Therapeutic compositions and methods of use

[0153] In some embodiments, the present disclosure provides a therapeutic composition comprising the granulosa cell-like cells produced herein. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the composition is cryopreserved.

[0154] Such compositions can be administered to a subject, such as a human subject, using any suitable route of administration. Suitable routes of administration include, for example, parenteral routes, such as intravenous, intrathecal, parenchymal, or intraventricular routes. Suitable routes of administration include, for example, parenteral routes, such as intravenous, intrathecal, parenchymal, or intraventricular injection.

[0155] In some embodiments, the subject is a human subject. Patients with primary ovarian failure or menopause in which granulosa cell function is impaired can benefit from the use of these cells. Therefore, in some embodiments, the subject has been diagnosed with ovarian failure, and the granulosa cells provided herein are used to treat ovarian failure. In other embodiments, the subject is experiencing menopause, and the granulosa cells provided herein are used to treat menopause (e.g., to alleviate symptoms associated with menopause).

[0156] In addition, such compositions can be used to improve the quality and in vitro maturation of oocytes and / or embryos, such as during in vitro fertilization (IVF) and / or during related assisted reproductive technology (ART) procedures (such as egg freezing). Such methods of application include, for example, co-culturing granulosa cell-like cells with immature and mature oocytes in vitro. In some embodiments, the subject is a human subject. For example, such subjects will receive freezing of oocytes or fertilization by IVF, and may suffer from infertility, age-related oocyte immaturity, polycystic ovary syndrome (PCOS) and / or ovarian hyperstimulation syndrome (OHSS), which can cause many oocytes to be immature and unusable. In vitro application of granulosa cell-like cells can improve the quality and availability of these oocytes.

[0157] The composition can be administered to a subject in a therapeutically effective amount. The term "therapeutically effective amount" refers to the amount of granulosa cells required to produce a therapeutic effect on a subject alone or in combination with at least one other active agent. As recognized by those skilled in the art, the effective amount varies depending on the route of administration, excipient use, and co-use with other active agents. The amount to be administered depends on the subject to be treated, including, for example, the strength of the individual's immune system or genetic predisposition. A suitable dosage range is readily determined by those skilled in the art, and it can be a microgram-level polypeptide of the present disclosure. The dosage of the formulation disclosed herein may depend on the route of administration and vary according to the size of the subject.

[0158] It is believed that those skilled in the art can make full use of the present invention according to the above description. Therefore, the following specific embodiments should be interpreted as illustrative only, and do not limit the remainder of the present disclosure in any way. All publications cited in this application are incorporated by reference for the purpose or theme cited in the present disclosure.

[0159] Additional Implementation

[0160] The present disclosure also relates to additional embodiments listed in the following numbered paragraphs:

[0161] 1. A pluripotent stem cell (PSC), comprising: an engineered polynucleotide containing an open reading frame encoding a protein selected from the group consisting of NR5A1 and RUNX family proteins.

[0162] 2. The PSC of paragraph 1, wherein the PSC comprises an engineered polynucleotide comprising an open reading frame encoding NR5A1.

[0163] 3. The PSC of paragraph 1 or 2, wherein the PSC comprises an engineered polynucleotide comprising an open reading frame encoding a RUNX family protein.

[0164] 4. The PSC of paragraph 3, wherein the RUNX family protein is RUNX1.

[0165] 5. The PSC of paragraph 3 or 4, wherein the RUNX family protein is RUNX2.

[0166] 6. The PSC of any of the preceding paragraphs, wherein the PSC expresses or overexpresses: NR5A1; RUNX1; RUNX2; NR5A1 and RUNX1; NR5A1 and RUNX2; or NR5A1, RUNX1 and RUNX2.

[0167] 7. The PSC of any of the preceding paragraphs, wherein the PSC further comprises an engineered polynucleotide comprising an open reading frame encoding a TCF21 protein.

[0168] 8. The PSC of paragraph 7, wherein the PSC expresses or overexpresses TCF21.

[0169] 9. The PSC of any of the preceding paragraphs, wherein the PSC further comprises an engineered polynucleotide comprising an open reading frame encoding a GATA4 protein.

[0170] 10. The PSC of paragraph 7, wherein the PSC expresses or overexpresses GATA4.

[0171] 11. The PSC of any of the preceding paragraphs, wherein the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

[0172] 12. The PSC of paragraph 11, wherein the heterologous promoter is an inducible promoter.

[0173] 13. A pluripotent stem cell (PSC), comprising: a protein selected from NR5A1 and RUNX family proteins, wherein the protein is overexpressed.

[0174] 14. The PSC of paragraph 15, wherein the PSC expresses or overexpresses: NR5A1; RUNX1; RUNX2; NR5A1 and RUNX1; NR5A1 and RUNX2; or NR5A1, RUNX1 and RUNX2.

[0175] 15. The PSC of paragraph 14, wherein the PSC further comprises TCF21 protein.

[0176] 16. The PSC of paragraph 15, wherein the PSC expresses or overexpresses TCF21.

[0177] 17. The PSC of any one of claims 14-16, wherein the PSC further comprises a GATA4 protein.

[0178] 18. The PSC of any one of claims 14-17, wherein the PSC expresses or overexpresses GATA4.

[0179] 19. The PSC of any of the preceding paragraphs, wherein the PSC is a human PSC.

[0180] 20. The PSC of any of the preceding paragraphs, wherein the PSC is an induced PSC (iPSC).

[0181] 21. The PSC of any of the preceding paragraphs, wherein the PSC comprises 1-20, optionally 8-10 copies of an engineered polynucleotide comprising an open reading frame encoding a protein selected from NR5A1 and a RUNX family protein (eg, RUNX1 and / or RUNX2).

[0182] 22. A composition comprising: a population of PSCs as described in any of the preceding paragraphs or elsewhere herein.

[0183] 23. The composition of paragraph 22, wherein the population comprises at least 10,000 / cm 2 PSC.

[0184] 24. A method comprising: culturing a population of pluripotent stem cells (PSCs) in a culture medium to produce an expanded population of PSCs; and expressing a protein selected from NR5A1 and RUNX family proteins in the PSCs of the expanded population to produce granulosa cell-like cells.

[0185] 25. The method of paragraph 24, wherein the PSCs of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding NR5A1.

[0186] 26. The method of paragraph 24 or 25, wherein the PSCs of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding a RUNX family protein.

[0187] 27. The method of paragraph 26, wherein the RUNX family protein is RUNX1.

[0188] 28. The method of paragraph 26 or 27, wherein the RUNX family protein is RUNX2.

[0189] 29. The method of any of paragraphs 24-28, wherein the PSCs of the expanded population further comprise an engineered polynucleotide comprising an open reading frame encoding a TCF21 protein.

[0190] 30. The method of any of paragraphs 24-29, wherein the PSCs of the expanded population further comprise an engineered polynucleotide comprising an open reading frame encoding a GATA4 protein.

[0191] 31. The method of any of the preceding paragraphs, wherein the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

[0192] 32. The method of any of the preceding paragraphs, wherein the heterologous promoter is an inducible promoter.

[0193] 33. The method of any of the preceding paragraphs, wherein the population comprises 1x10 2 -1x10 7 PSCs.

[0194] 34. The method of any of the preceding paragraphs, wherein the population of PSCs is cultured for about 4-10 days.

[0195] 35. The method of paragraph 34, wherein the population of PSCs is cultured for about 6 days.

[0196] 36. The method of any of the preceding paragraphs, wherein the granulosa cell-like cells are AMHR2 + 、CD82 + , FOXL2 + and / or EPCAM - .

[0197] 37. The method of paragraph 36, wherein the granulosa cell-like cells are AMHR2 + 、CD82 + , FOXL2 + and EPCAM - .

[0198] 38. A method comprising:

[0199] (a) delivering 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 to a pluripotent stem cell (PSC);

[0200] (b) culturing the PSCs in a feeder-free, serum-free medium to produce an expanded population of PSCs; and

[0201] (c) culturing the expanded population of PSCs in a series of induction media containing an induction agent to produce AMHR2 + 、CD82+ , FOXL2 + and / or EPCAM - Granular cell-like cells.

[0202] 39. The method of paragraph 38, comprising delivering to PSCs (i) an engineered polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding NR5A1 and (ii) an engineered polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a RUNX family protein.

[0203] 40. The method of paragraph 38 or 39, wherein the RUNX family protein is RUNX1.

[0204] 41. The method of any of paragraphs 38-40, wherein the RUNX family protein is RUNX2.

[0205] 42. The method of any of paragraphs 38-41, wherein the engineered polynucleotide is a transposon and the delivering further comprises delivering a transposase to the PSC.

[0206] 43. The method of any of paragraphs 38-42, wherein the inducible promoter is a chemically inducible promoter, optionally a doxycycline inducible promoter.

[0207] 44. The method of any of paragraphs 38-43, wherein the feeder-free, serum-free medium of (b) comprises a solubilized basement membrane preparation extracted from an Engelbreth-Holm-Swarm (EHS) mouse sarcoma.

[0208] 45. The method of paragraph 44, wherein the solubilized basement membrane preparation comprises extracellular matrix (ECM) proteins and growth factors.

[0209] 46. ​​The method of paragraph 45, wherein the ECM protein is selected from the group consisting of laminin, collagen IV, heparin sulfate proteoglycans, and paranectin / nidogen.

[0210] 47. The method of any of paragraphs 38-46, 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).

[0211] 48. The method of any of paragraphs 38-47, wherein the culturing of (b) continues for about 6-24 hours.

[0212] 49. The method of any of paragraphs 38-48, wherein the PSCs of the expanded population of (c) are at about 10,000 cells / cm 2 Up to 20,000 cells / cm 2Density culture.

[0213] 50. The method of any of paragraphs 38-49, wherein the culturing of (c) comprises culturing the PSCs in a first induction medium and culturing the PSCs in a second induction medium.

[0214] 51. The method of paragraph 50, wherein the first induction medium comprises one or more of L-alanyl-L-glutamine, antibiotics (e.g., penicillin and / or streptomycin), Dulbecco's modified Eagle's medium (DMEM) / F-12, Advanced RPMI (Roswell Park Memorial Institute) 1640 medium, glycogen synthase kinase (GSK) 3 inhibitors, small molecule or protein inhibitors of the BMP signaling pathway, small molecule ROCK inhibitors, and inducers (e.g., doxycycline).

[0215] 52. The method of paragraph 50 or 51, wherein the PSCs are cultured in the first induction medium for about 36 to about 60 hours, optionally about 48 hours.

[0216] 53. The method of any one of paragraphs 50-52, wherein the second induction medium comprises one or more of L-alanyl-L-glutamine, antibiotics (e.g., penicillin and / or streptomycin), Advanced RPMI 1640 medium, DMEM / F-12, and an inducer (e.g., doxycycline).

[0217] 54. The method of any of paragraphs 50-53, wherein the PSCs are cultured in the second induction medium for about 96 to about 144 hours, optionally for about 120 hours.

[0218] 55. The method of paragraph 54, wherein the second induction medium is removed and replaced with fresh second induction medium at intervals of about 24 hours.

[0219] 56. A granulosa cell-like cell produced by the method of any of the preceding paragraphs.

[0220] 57. An ovarian organoid comprising the granulosa cell-like cells of any of the preceding paragraphs and human primordial germ cell-like cells (hPGCLCS).

[0221] 58. The method of any of the preceding paragraphs, further comprising combining granulosa cell-like cells with hPGCLCS to form ovarian organoids. Example

[0222] Example 1. Directed differentiation of hiPSCs into granulosa cell-like cells by transcription factor expression

[0223] Ovarian granulosa cells are important for many aspects of female reproduction, including supporting oocyte development and hormonal signaling during the menstrual cycle. Current in vitro models, including primary human or mouse granulosa cells and granulosa cell tumor lines, are inadequate for studying these processes. For example, studies have found that the COV434 ovarian tumor line, which is commonly used as a granulosa cell model, lacks transcriptional and phenotypic characteristics of authentic granulosa cells. Previously reported protocols for differentiating iPSCs into granulosa-like cells either have low yields or are applicable only to mouse cells. To address this challenge, a robust, scalable method to generate granulosa-like cells through transcription factor (TF)-mediated differentiation of iPSCs was developed.

[0224] First, a list of candidate TFs for screening granulosa cell induction was identified. To generate this list, 22 TFs differentially expressed in granulosa cells compared to hESCs and early mesoderm were identified using previously published datasets for these cell types. Five TFs were also included that were identified by previous developmental biology studies as being important in mouse ovarian development. Finally, nine additional TFs predicted to be upstream of other TFs in the list were identified based on gene regulatory network analysis that took into account co-expression data and binding motifs.

[0225] Next, we engineered hiPSC lines using the tdTomato reporter for the granulosa cell-specific protein FOXL2. In addition, we generated a barcoded cDNA plasmid library for doxycycline-inducible expression of these TFs. By integrating these plasmids into FOXL2-tdTomato reporter iPSCs, inducing TF expression under various conditions, and capturing barcodes from FOXL2+ cells, we identified a set of TFs that are enriched in the granulosa cell-like population ( Figure 1 ).

[0226] Based on these results, we screened TF combinations of interest to see which TFs best induced a granulosa cell-like phenotype ( Figure 2 ). The study found that NR5A1 expression is essential for efficient estradiol production, and that a combination of NR5A1 and a RUNX family member (RUNX1 or RUNX2) is optimal for inducing FOXL2 expression and granulosa cell surface markers. We further validated that FOXL2+ granulosa cell-like cells express transcriptional markers of granulosa cells and gonadal cells, but not markers from the related adrenal lineage ( Figure 3 ).

[0227] Next, monoclonal hiPSC lines integrating the highest-ranked TFs were generated. After selecting cell lines with efficient induction of granulosa cell-like cells ( Figure 4), their hormone signaling was verified by measuring estradiol production in response to follicle-stimulating hormone (FSH) or forskolin, which increases levels of the second messenger cAMP. The study found that three cell lines containing integrated expression plasmids for NR5A1, TCF21, and RUNX1 produced estradiol in response to FSH or forskolin, as expected for granulosa cells (Figure 5). A fourth cell line without the RUNX transgene produced high levels of estradiol constitutively, indicating that RUNX expression is necessary for the proper regulation of estradiol production.

[0228] Figure 6 The overall protocol for the production of granulosa cell-like cells is described. hiPSCs containing an integrated TF expression plasmid were cultured in mTeSR on substrate TM To induce granulosa cell-like cells, hiPSCs were dissociated into single cells and plated per cm 2 The cells were cultured at a density of about 10,000 to 20,000 cells in DK10 medium (containing GlutaMAX) supplemented with about 3 to 4 μM CHIR99021, about 8 to 12 μM Y-27632, and about 0.5 to 3 μg / mL doxycycline to induce TF expression. TM The cells were inoculated in DMEM / F12 supplemented with 10% Knockout serum replacement. Matrix or collagen I. After 48 hours, and subsequently at 24 hour intervals, the medium was changed and replaced with fresh DK10 medium + approximately 0.5-3 μg / mL doxycycline. After a total of 120 hours, the granulocyte-like cells were ready for downstream experiments.

[0229] In an alternative protocol, iPSCs are cultured on Matrigel or laminin-coated plates and grown to 20-40% confluence in mTeSR Plus medium. To induce granulocyte-like cells, mTeSR Plus is removed, iPSCs are washed with phosphate-buffered saline (PBS), and stage 1 medium containing Advanced RPMI and GlutaMAX is added. TM Supplement, CHIR99021 (approximately 5-8 μM) and either dorsomorphin (approximately 100-400 nM) or noggin (approximately 4-8 ng / mL). After 48 hours, remove the Phase 1 medium and replace with fresh Phase 1 medium. After another 48 hours, remove the Phase 1 medium and replace with Phase 2 medium containing Advanced RPMI and GlutaMAX TMSupplements and approximately 0.5-3 μg / mL doxycycline. Stage 2 medium is changed every 24 hours. Granulocyte-like cells are harvested after approximately 3-4 days in stage 2 medium (approximately 7-8 days total).

[0230] The protocol presented here has several advantages over previously reported methods. It is rapid, produces granulosa cell-like cells in approximately 5-8 days, and the resulting cells are sufficiently pure ( Figure 4 ), no enrichment by FACS is required. Due to the monolayer format, it is easily scalable (50 million cells can be generated at a time, and the format is compatible with higher throughput assays). Most importantly, granulosa cell-like cells are transcriptionally active ( Figure 3 ) and phenotype ( Figure 5 ), making them suitable as a model for ovarian biology.

[0231] Example 2. Granulosa-like cells respond to FSH and produce steroids

[0232] The ability of granulosa cell-like cells to carry out one of the key endocrine functions of granulosa cells, namely the production of estradiol, was assessed. In the ovary, theca cells convert cholesterol to androstenedione, which is the substrate for estradiol production in granulosa cells. The rate-limiting step is the oxidative decarboxylation by CYP19A1 (aromatase) to produce estrone, which is subsequently reduced to estradiol by enzymes in the HSD17B family (usually HSD17B1 in granulosa cells). In vivo, this estrogen synthesis pathway is stimulated by FSH. Granulosa cell-like cells were treated with androstenedione in the presence or absence of FSH or forskolin, which directly increases the levels of the FSHR second messenger cAMP. As controls, COV434 and KGN ovarian tumor cells, which produce estradiol from androstenedione, as well as immortalized primary human granulosa cells (HGL5) and primary adult mouse ovarian somatic cells were used. Granulosa-like cells produce estradiol from androstenedione, and in seven of the nine monoclonal cell lines tested, this steroidogenic activity was significantly increased after stimulation with FSH or forskolin ( Figure 5A One of these granulosa cell lines (F3 / NT#5) produced high levels of estradiol under all conditions and, unlike the other cell lines, had neither RUNX1 nor RUNX2 expression vectors integrated (data not shown).

[0233] The three FSH-responsive cell lines produced estradiol levels similar to those produced by KGN human granulosa cell tumor cells, which also showed responses to FSH and forskolin ( Figure 5A In contrast, FSHR expression was not shown in the RNA-seq data ( Figure 3) COV434 cells 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. Primary adult mouse ovarian somatic cells produced similar amounts of estradiol as hiPSC-derived granulosa cell-like cells ( Figure 5A ); however, the mouse cells did not respond to FSH or forskolin, probably because they had already been exposed to FSH in vivo. It was also investigated whether granulosa cell-like cells maintained their steroidogenic activity during co-culture of ovarian organoids (ovaroids) with hPGCLCs. Hormone levels in the supernatant of ovarian organoids in the presence or absence of androstenedione and FSH were measured. In addition to estradiol, progesterone was measured; granulosa cells produce progesterone in vivo after ovulation and corpus luteum formation. Production of both hormones was observed in five of the six samples ( Figure 5B ). Estradiol was produced only 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.

[0234] Example 3. Granulosa-like cells support germ cell development in ovarian organoids

[0235] Current methods of inducing and culturing human PGC-like cells (hPGCLCs) produce 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 the downregulation of pluripotency factors and the commitment of gametogenesis. Recently, this process has been reproduced in vitro using mouse fetal ovarian somatic cells, which allows hPGCLCs to develop to the oogonia-like stage. In vitro derived human granulosa cell-like cells may play a similar role, with the potential to eliminate developmental mismatches between species. Therefore, granulosa cell-like cells are combined with hPGCLCs to form ovarian organoids, referred to as "ovarian organoids (ovaroid)".

[0236] To generate ovarian organoids, both cell types were aggregated in low-binding U-bottom wells and then transferred to air-liquid interface Transwell culture. As a comparison, mouse fetal ovarian somatic cells were isolated and aggregated with hPGCLCs. After 4 days of co-culture with hiPSC-derived granulosa cell-like cells, expression of the maturation marker DAZL began to be observed by immunofluorescence in the OCT4+ hPGCLC subset (data not shown). In contrast, robust DAZL expression in co-culture with mouse cells was not observed until day 32 (data not shown), with weaker expression visible on day 26. Similarly, in a previous study using the same hPGCLC line and anti-DAZL antibody, DAZL expression was only observed after 77 days of co-culture with mouse fetal testicular somatic cells. DAZL + The proportion of cells reached a maximum at day 14 in human ovarian organoids and at day 38 in mouse ovarian organoids ( Figure 7 ). In human ovarian organoids, OCT4 + The proportion of cells decreased after day 8. In mouse ovarian organoids, OCT4 + The proportion of cells also decreased over time. In human ovarian organoids, at day 16, except for DAZL + OCT4 + Extracellular, DAZL + OCT4 - cells were also evident (in situ images not shown), and after day 38, the number of DAZL+ cells was greater than the total number of OCT4+ cells ( Figure 7 ). DAZL + Downregulation of OCT4 in oogonia occurs during the second trimester of human fetal ovarian development; however, the shift to an exclusively cytoplasmic localization of DAZL that has been reported to occur at this stage was not observed. Expression of the early PGC marker TFAP2C decreased during ovarian organoid culture and was almost completely absent by day 8. In contrast, SOX17 expression was still visible at day 8, and OCT4 and DAZL expression persisted until day 54. Although this system allows for rapid development of hPGCLCs to the gonadal stage, germ cell numbers in both hiPSC-derived and mouse-derived ovarian organoids declined in long-term culture ( Figure 7 ), indicating that they were either dying or differentiating into other lineages. Unlike mouse-derived ovarian organoids, hiPSC-derived ovarian organoids cultured on Transwells gradually flattened and widened, and most had collapsed by day 38.

[0237] Nevertheless, in these long-term experiments, the formation of empty follicle-like structures composed of cuboidal AMHR2+FOXL2+ granulosa cell-like cells was observed (in situ images not shown), suggesting that TFs can drive folliculogenesis even in the absence of oocytes. The formation of follicle-like structures was first visible on day 16, and by day 26, the largest of these structures had grown to 1–2 mm in diameter. At day 70, ovarian organoids had developed follicles of various sizes, predominantly small, unilamellar follicles, but also antral follicles. Cells outside the follicles stained positive for NR2F2, a marker for ovarian stromal and theca cells. To further examine gene expression of hPGCLCs and somatic cells in this system, scRNA-seq was performed on dissociated ovarian organoids on days 2, 4, 8, and 14 of culture, and cells were clustered based on gene expression. As expected, the largest cluster (cluster 0) contained cells expressing granulosa cell markers such as FOXL2, WNT4, and CD82 (Figures 8 and 8B). Cells expressing secondary / antral granulosa cell markers such as FSHR and CYP19A1 were also found in this cluster, although these cells were much less numerous. There was also a smaller cluster (Cluster 1) expressing the ovarian stromal marker NR2F2. NR2F2 is expressed by stromal cells and theca cells, but the cells in Cluster 1 do not express 17α-hydroxylase (CYP17A1), indicating that they cannot produce androgens and are not theca cells.

[0238] hPGCLC clusters expressing marker genes such as CD38, KIT, PRDM1, TFAP2C, PRDM14, NANOG, and POU5F1 were also observed. Notably, the X-chromosome lncRNAs XIST, TSIX, and XACT were all expressed more highly in hPGCLCs compared to the other clusters (on average, approximately 80-fold, approximately 20-fold, and approximately 2900-fold, respectively) ( Figure 8B ), suggesting that hPGCLCs were initiating the X reactivation process, which in hPGCs was associated with high expression of XIST and XACT. The X chromosome HPRT1 gene, known to be more highly expressed in cells with two active X chromosomes, was also upregulated by about 3-fold.

[0239] Next, the in vitro generated ovarian organoids were compared to a reference atlas of human fetal ovarian development. Scanpy ingest was used to integrate the samples into the atlas and annotate each cell with the cell type that most closely matched the in vivo data ( Figure 8C ). Ovarian organoids are mainly composed of granulosa cells, gonadal mesenchyme, and pregranulosa cell lineages ( Fig.8D), of which a small fraction is coelomic epithelium. The proportion of granulosa cells increases from day 2 to day 8, likely representing the maturation of the somatic cell population. As expected, neural cells, immune cells, smooth muscle cells, and erythrocytes present in the fetal ovary are completely absent in ovarian organoids. Epithelial cells, endothelial cells, and perivascular cells were detected, but at very low frequencies (1% or less), likely representing a low rate of off-target differentiation.

[0240] We also examined the overall proportion of germ cells and the proportion of cells expressing the gonadal germ cell markers DAZL and DDX4 during the experiment ( Fig.8D ). The germ cell population was defined based on the integration of the fetal ovarian atlas. This population increased from day 2 to day 4 but declined thereafter. In contrast, DAZL + and DDX4 + The proportion of cells also increased from day 2 to day 4, but remained essentially unchanged from day 4 to day 14 ( Fig.8D ). DAZL+ cells were compared with DAZL - Differential gene expression analysis and gene ontology enrichment of cells. Upregulated genes (log2fc>2, n=221) were most enriched in terms related to general developmental1 processes, but also included terms related to adhesion and migration (e.g., “amoeba-like cell migration”) and reproductive system development. Downregulated genes (log2fc<-2, n=6451) were strongly associated with metabolic processes and mitotic cell division. These data suggest that DAZL in ovarian organoids + The cells were downregulating their metabolism and proliferation, which is consistent with the known role of DAZL in inhibiting PGC proliferation.

[0241] method

[0242] Cell culture

[0243] Two parental hiPSC lines were used in this study: ATCC-BXS0116 female hiPSCs (referred to as the F3 line) and the F66 line, an in-house hiPSC line derived from the NIA Senescent Cell Bank fibroblast line AG07141 using Epi5 footprint-free episomal reprogramming. The karyotypes of the parental lines as well as the engineered reporter lines were verified by Thermo Fisher Cell ID (SNP-based authentication) + Karyostat, and pluripotency was assessed by Thermo Fisher Pluritest. All cell lines were authenticated as normal.

[0244] hiPSCs were cultured in mTESRPlus medium (Stemcell Technologies) on standard polystyrene plates coated with hESC-qualified Matrigel (Corning). The medium was changed every day. Passaging was performed using 0.5mM EDTA or TRYPLE (for experiments requiring single cell dissociation). After each passage, hiPSCs were treated with 10μM 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+1XGlutaMax (Gibco). HGL5 cells (ABM cat.T0650) were cultured in Prigrow IV medium (ABM) containing 10% FBS. Passaging was performed using TRYPLE (Gibco). hPGCLCs were cultured in S-CM medium and passaged with Accutase (Stemcell Technologies). Mycoplasma testing was performed by PCR every 3 months; all cells tested negative.

[0245] Electroporation

[0246] Electroporation was performed with 200,000 cells in 20 μL P3 buffer using a Lonza Nucleofector equipped with a 96-well shuttle. All electroporations used the pulse setting CA-137. Selection with the appropriate agent began 48 hours after electroporation and continued for 5 days. For the agents used in this study, this period of time was sufficient to produce a high purity final cell population.

[0247] Reporter factor construction

[0248] Homology arms of FOXL2 were amplified from genomic DNA by PCR. A targeting plasmid containing a C-terminal T2A-tdTomato reporter in frame and a Rox-PGK-PuroTK-Rox selection cassette ( Figure 2 -Replenish Figure 1A). The plasmid backbone also has an MC1-DTA marker to select for random integration. sgRNA oligonucleotides targeting the C-terminal region of FOXL2 were cloned into pX330 (Addgene #42230). To generate reporter lines, 1 μg of donor plasmid and 1 μg of sgRNA plasmid were co-electroporated into hiPSCs, which were subsequently seeded into 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 to determine the presence of wild-type and reporter alleles. PCR amplification of the entire FOXL2 locus ( Figure 2 -Replenish Figure 1 B) Homozygous clones were further verified by PCR and Sanger sequencing.

[0249] 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 described above. Excision of the selection cassette was verified by genotyping. The primers used in this study are listed in Supplementary File 1.

[0250] TF plasmid construction

[0251] TF cDNA was obtained as Gateway entry clones from TFome39 or ORFeome76. They were cloned into barcoded Dox inducible expression vectors (Addgene #175503) using MegaGate clone 48. The final expression construct was verified by Sanger sequencing, which was also used to determine the barcode sequence of each TF. During library merging, two unique barcodes were used for each TF. Libraries were merged using equimolar amounts of each plasmid (measured using QuBit).

[0252] TF screening for granulosa cell differentiation

[0253] The pooled barcoded TF plasmid library was electroporated into FOXL2-tdTomato reporter hiPSCs, typically 5 fmol of library and 500 ng of PiggyBac transposase expression plasmid (Systems Bio). These conditions were chosen to give an average copy number of approximately 5 / cell ( Figure 2 -Replenish Figure 2 ). Some experiments were also performed at 50 fmol to explore the effect of higher copy numbers. Figure 2 In the screening data shown in , two libraries were used: library #1, containing 35 TFs, and library #2, containing 18 TFs. Library #1 was used only at 5 fmol, while library #2 was used at both 5 fmol and 50 fmol.

[0254] After selection with puromycin (400 ng / mL), hiPSCs were treated with doxycycline (1 μg / mL) in mTESR Plus medium. In additional experiments, hiPSCs were first differentiated into mesoderm according to a previously published protocol 77 and then treated with doxycycline. In two sets of experiments, doxycycline treatment lasted for 5 days, and then cells were dissociated with TRYPLE and reporter-positive cells were isolated by FACS. Genomic DNA was extracted from reporter-positive and -negative cells and the initial population before doxycycline treatment (QIAamp DNA Micro Kit).

[0255] The barcodes were amplified by PCR (KAPA polymerase) using 10 ng of input gDNA per reaction, typically for 22 cycles of PCR (95°C 15 sec denaturation, 58°C 20 sec annealing / extension). The PCR products were purified using ProNex beads and a second round of PCR (NEB Q5 polymerase, 6 cycles of 98°C 5 sec denaturation, 61°C 20 sec annealing, 72°C 5 sec extension, and a final 72°C extension for 2 min) was performed to add the Illumina index. (Primers are given in Supplementary File 1). These amplicons were purified again using ProNex beads. Samples were normalized and pooled, and barcodes were sequenced on an Illumina MiSeq with a 10% PhiX spike-in. To call barcodes, reads were aligned to a set of known barcode sequences. Fold changes were calculated by comparing the barcode frequencies in the sorted FOXL2+ cells with those in the starting population.

[0256] Flow cytometry / cell sorting

[0257] Cells were dissociated by treating with TRYPLE for 5 minutes, which was quenched with 4 volumes of ice-cold DMEM+10% FBS. The suspension was passed through a 70 μm cell strainer. The cells were precipitated (200 g, 5 minutes) and resuspended in staining buffer (PBS+3% FBS+antibody, about 100 μL per million cells). Staining was continued on ice for 30 minutes in the dark. The suspension was diluted with 9 volumes of PBS+3% FBS. The cells were precipitated (200 g, 5 minutes) and resuspended in PBS+3% FBS+100 ng / mL DAPI. The suspension was placed on ice and kept away from light until analysis. Flow cytometry was performed on a BD LSRFortessa, and sorting was performed on a Sony SH800 equipped with a 100 μm chip.

[0258] Antibody capture beads (BD Biosciences, RRID AB_10051478) or hiPSCs expressing tdTomato were used as compensation controls. The antibodies used are given in the Key Resource table. Data analysis was performed using the Cytoflow Python package (version 1.0.0, github.com / cytoflow / cytoflow).

[0259] Granulosa cell differentiation protocol

[0260] iPSCs were dissociated with TRYPLE and centrifuged at 12,500 / cm 2 The cell density was seeded on Matrigel-coated polystyrene plates in DK10 medium (DMEM-F12, 15mM HEPES, 1X GlutaMax, 10% KSR) with Y-27632 (10μM), CHIR99021 (3μM) and doxycycline (1μg / mL). For 24-well plates, the volume of medium per well was 0.5mL; for 6-well plates, it was 2mL. After 48 hours of seeding, the medium was replaced with DK10+doxycycline (1μg / mL), and the medium was subsequently replaced every 24 hours. Unless otherwise stated, cells were harvested on day 5. In the TF-free control differentiation for RNA-seq, the protocol was the same, except that the cells did not contain the TF expression plasmid.

[0261] Alternative protocols for inducing granulosa cell-like cells

[0262] ·exist mTeSR on substrate TM hiPSCs containing an integrated TF expression plasmid were cultured in DK10 medium (containing GlutaMAX Plus medium) with about 3-5 μM CHIR99021, about 8-12 μM Y-27632, and about 0.5-3 μg / mL doxycycline to induce TF expression. To induce granulosa cell-like cells, hiPSCs were dissociated into single cells using TRYPLE and cultured at a density of about 10,000-20,000 per square centimeter. TM The cells were inoculated in DMEM / F12 supplemented with 10% Knockout serum replacement. Matrix or collagen I coated plates. After 48 hours, and subsequently at 24 hour intervals, the medium was changed and replaced with fresh DK10 + approximately 0.5-3 μg / mL doxycycline. After a total of approximately 120 hours, the granulocyte-like cells were ready for downstream experiments.

[0263] hiPSCs with integrated TF expression plasmids were cultured in mTeSRPlus medium on Matrigel or laminin. When the culture reached 20-40% confluence, the medium was removed, the iPSCs were washed with phosphate-buffered saline (PBS), and stage 1 medium containing Advanced RPMI and GlutaMAX was added. TM Supplement, CHIR99021 (about 6-10 μM) and doxorubicin (about 100-400 nM) or noggin (about 5-8 ng / mL). After 48 hours, remove the Phase 1 medium and replace with fresh Phase 1 medium. After another 48 hours, remove the Phase 1 medium and replace with Phase 2 medium, which contains Advanced RPMI and GlutaMAX TM Supplements and approximately 0.5-3 μg / mL doxycycline. Phase 2 medium was changed every 24 hours. After approximately 3-4 days of culture in Phase 2 medium (approximately 7-8 days total), the granulosa cell-like cells were ready for downstream experiments.

[0264] RNA-seq

[0265] Total RNA was extracted from sorted FOXL2+ granulosa cells using the Arcturus PicoPure kit (Thermo Fisher) or from COV434 cells and hiPSCs using the Monarch Total RNA Miniprep kit (NEB). For experiments involving TF overexpression, TF expression plasmids were integrated into hiPSCs as described above (50 fmol / 200,000 cells). After selection with puromycin, TF expression was induced using doxycycline (1000 ng / mL).

[0266] Two biological replicates were collected for each sample (iPSC, hiPSC+individual TF, sorted FOXL2+, no 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 NextSeq500 (2x75bp paired-end reads). Figure 3 The TPM data shown in Figure 1 were generated by pseudo-aligning reads to the reference human transcriptome (Ensembl GRCh38 v96) using kallisto78. Differential expression analysis was performed using DESeq2. Gene ontology enrichment of significantly upregulated (log2fc>3, padj<0.05) and downregulated (log2fc<-3, padj<0.05) genes in each sample relative to hiPSC was calculated using PantherDB60.

[0267] TROM analysis

[0268] The transcriptome overlap measure (TROM) method was used to identify relevant genes that capture the molecular features of biological samples, and then biological samples were compared by testing the overlap of their relevant genes 58. TROM scores were calculated as -log10 (Bonferroni-corrected association p-value) and ranged from 0 to 300. The TROM magnitude was positively correlated with the similarity between two independent samples, with a standard threshold of 12, which is a generally accepted indicator of significant similarity.

[0269] Formation of ovarian organoids using hPGCLCs and granulosa cell-like cells

[0270] F2 female hPGCLCs (see Key Resources Table) were maintained in long-term culture. Briefly, hPGCLCs were cultured in STO-conditioned medium (GMEM with 13% KSR and 1X NEAA, sodium pyruvate, and GlutaMax, all from Gibco) on Matrigel and supplemented with SCF (100 ng / mL, Peprotech), ascorbic acid (50 μg / mL, Gibco), and 2-mercaptoethanol (25 μM, Gibco). hPGCLCs were harvested with Accutase. To form ovarian organoids, granulosa cell-like cells were harvested with TRYPLE, counted, and mixed with F2 hPGCLCs. For Figure 3 For hormone assays in , we used granulosa cell-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. Figure 4 For the immunofluorescence experiments in Figure 5, we used F66 / N.R1.GF#4 and F66 / N.R2#1. Figure 6 For scRNA-seq in , we used F66 / N.R1.GF#4.

[0271] For each ovarian organoid, 100,000 granulosa cell-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, and 1X 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. The plate was centrifuged (100 g, 2 minutes) and incubated (37°C, 5% CO2) for two days. Subsequently, the ovarian organoids were transferred to Transwells (collagen-coated PTFE, 3 μm pore size, 24 mm diameter, Corning #3492) and cultured at the air-liquid interface using aMEM, 10% KSR, 55 μM 2-mercaptoethanol, 500 ng / mL doxycycline, and 50 μg / mL primosin. Typically, 5-6 ovarian organoids were cultured in each 6-well Transwell. The culture medium (1.5 mL) was replaced every 2 days.

[0272] Generation of ovarian organoids using hPGCLCs and mouse fetal ovarian somatic cells

[0273] 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 organoid, 50,000 fetal ovarian somatic cells and 5,000 F2hPGCLCs were combined. Ovarian organoids were cultured as described above. All mouse experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Harvard Medical School.

[0274] Immunofluorescence

[0275] Ovarian organoids were washed with PBS and fixed with 1% PFA overnight at 4°C. After one more wash with PBS, ovarian organoids were detached from the Transwell. To prepare for cryosectioning, ovarian organoids 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, ovarian organoids were embedded in OCT compound and stored at -80°C until sectioning.

[0276] Ovarian organoids were cut into 10 μm slices using a Leica CM3050S cryostat. The slices were transferred to Superfrost Plus slides and washed with PBS to remove the OCT compound. The slides were washed with PBST (0.1% TritonX-100 in PBS) and the slices were circled with a pap pen. The slides were blocked with blocking buffer (1% bovine serum albumin and 5% normal donkey serum in PBST [Jackson ImmunoResearch, #017-000-121, lot number 152961]) at room temperature for 30 minutes. The blocking buffer was removed and replaced with the primary antibody solution in the blocking buffer, and the slides were incubated overnight at 4 ° C. The antibody solution was removed and the slides were washed 3x5 minutes with PBST. The slides were incubated in the dark at room temperature for 1 hour with the secondary antibody and DAPI solution in the blocking buffer, then washed twice with PBST for 5 minutes each and once with PBS. After staining, the samples were mounted in Prolong Gold medium and covered with a coverslip. Imaging was performed on a Leica SP5 confocal microscope. The antibodies used are given in the Key Resources table. The brightness of the images (and only the brightness) was adjusted in ImageJ (version 2.9.0 / 1.53t). Figure 4 Cell counts for C were performed manually by a researcher who was blinded to the species of the ovarian organoids.

[0277] Single-cell RNA sequencing

[0278] Ovarian organoids were isolated using the Miltenyi embryoid body dissociation kit (Miltenyi #130-096-348) (6 ovarian organoids per sample, 2 samples per time point). Cells were passed through a 40 μm filter, fixed using the ParseBiosciences fixation kit, and stored at -80 °C until all time points were collected. Libraries were prepared using the ParseBiosciences WT Mega v1 kit, generating an average of 450 bp libraries. Ovarian organoids accounted for 8 of the 96 samples; the remaining kit capacity was used for other experiments. Libraries were sequenced on an Illumina NovaSeq 2x150bp S4 flow cell using a single index, 6bp, library, and 5% PhiX spike-in. Data were demultiplexed into library fastq files, and count matrices were generated using Parse Bioscience's analysis pipeline (version 0.9.6). Downstream data processing, such as bimodal filtering, dimensionality reduction, and clustering 70, was performed using Scanpy (version 1.8.2). For cell type assignment, the fetal ovary dataset from the Human Germ Cell Atlas69 was used as a reference for ScanPy InGest.

[0279] Collection of primary mouse ovarian somatic cells

[0280] Female BALB / c mice (10-12 weeks old) were confirmed to be in proestrus by visual inspection. Mice were killed by CO2 exposure followed by cervical dislocation, and ovaries were removed by dissection. Ovaries were placed in HEPES-buffered DMEM / F12 containing 0.1% bovine serum albumin (2 ovaries per 1.5 mL tube, 500 μL culture medium) and mechanically disrupted by puncturing with forceps. Cell suspensions were filtered through a 40 μm filter to remove oocytes and clumps, and then cultured for hormone determination.

[0281] Steroid hormone measurement

[0282] On day 4 of granulosa cell differentiation, androstenedione (500 ng / mL) was added to the culture medium. FSH (0.25 IU / mL, BioVision #4781-50 lot 5F07L47810) or forskolin (100 μM, Sigma-Aldrich) was also added as indicated. The total culture medium volume per well of a 24-well plate was 0.5 mL. Figure 3 -These assays were performed for each line listed in Source Data 2. For controls using human cell lines (COV434, KGN or HGL5) or mouse primary ovarian somatic cells, 75,000 cells were seeded per well. After 24 hours, the estradiol content in the culture medium was analyzed by ELISA (DRG International, EIA-2693). Concentrations were calculated by 4-parameter logistic curve fitting using the standard data provided in the kit. Samples outside the range of the calibration curve were diluted and rerun.

[0283] To measure hormone production in ovarian organoids, ovarian organoids were aggregated as described above. Androstenedione (500 ng / mL) and / or FSH (0.25 IU / mL) were added to the aggregation medium (total volume of 200 μL / ovarian organoid). After 3 days of culture, the culture 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.

[0284] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which they are cited, which in some cases may be the entire document.

[0285] The indefinite articles "a" and "an" as used in this specification and claims are to be understood as meaning "at least one", unless expressly stated otherwise.

[0286] It should also be understood that in any method claimed herein that includes multiple steps or actions, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are described unless explicitly stated otherwise.

[0287] In the claims and the foregoing description, all transitional phrases, such as "comprises," "comprising," "carrying," "having," "containing," "involving," "holding," "comprising," etc., shall be understood to be open-ended, i.e., meaning including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in Section 2111.03 of the U.S. Patent Office Manual of Patent Examining Procedures.

[0288] The terms "about" and "substantially" preceding a numerical value mean ±10% of the numerical value.

[0289] Where a range of values ​​is provided, every value between and including the upper and lower limits of that range is specifically contemplated and described herein.

Claims

1. A pluripotent stem cell (PSC), comprising: an engineered polynucleotide containing an open reading frame encoding a protein selected from the group consisting of NR5A1 and RUNX family proteins.

2. The PSC of claim 1, wherein the PSC comprises an engineered polynucleotide comprising an open reading frame encoding NR5A1.

3. The PSC of claim 1, wherein the PSC comprises an engineered polynucleotide comprising an open reading frame encoding a RUNX family protein.

4. The PSC of claim 3, wherein the RUNX family protein is RUNX1.

5. The PSC of claim 3, wherein the RUNX family protein is RUNX2.

6. The PSC of claim 1, wherein the PSC expresses or overexpresses: NR5A1; RUNX1; RUNX2; NR5A1 and RUNX1; NR5A1 and RUNX2; or NR5A1, RUNX1, and RUNX2.

7. The PSC of claim 1, wherein the PSC further comprises an engineered polynucleotide comprising an open reading frame encoding a TCF21 protein.

8. The PSC of claim 7, wherein the PSC expresses or overexpresses TCF21.

9. The PSC of claim 1, wherein the PSC further comprises an engineered polynucleotide comprising an open reading frame encoding a GATA4 protein.

10. The PSC of claim 9, wherein the PSC expresses or overexpresses GATA4.

11. The PSC of claim 1, wherein the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

12. The PSC of claim 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. The PSC of claim 13, wherein the PSC expresses or overexpresses: NR5A1; RUNX1; RUNX2; NR5A1 and RUNX1; NR5A1 and RUNX2; or NR5A1, RUNX1, and RUNX2.

15. The PSC of claim 14, wherein the PSC further comprises TCF21 protein.

16. The PSC of claim 15, wherein the PSC expresses or overexpresses TCF21.

17. The PSC of claim 14, wherein the PSC further comprises a GATA4 protein.

18. The PSC of claim 15, wherein the PSC expresses or overexpresses GATA4.

19. The PSC of claim 13, wherein the PSC is a human PSC.

20. The PSC of claim 13, wherein the PSC is an induced PSC (iPSC).

21. The PSC of claim 13, wherein the PSC comprises 1-20, optionally 8-10 copies of an engineered 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 the PSCs of claim 1.

23. The composition of claim 22, wherein the population comprises at least 10,000 cells / cm 2 PSC.

24. A method comprising: culturing a population of pluripotent stem cells (PSCs) in a culture medium to produce an expanded population of PSCs; And a protein selected from NR5A1 and RUNX family proteins is expressed in PSCs of the expanded population to generate granulosa cell-like cells.

25. The method of claim 24, wherein the PSCs of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding NR5A1.

26. The method of claim 24, wherein the PSCs of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding a RUNX family protein.

27. The method of claim 26, wherein the RUNX family protein is RUNX1.

28. The method of claim 26, wherein the RUNX family protein is RUNX2.

29. The method of claim 25, wherein the PSCs of the expanded population further comprise an engineered polynucleotide comprising an open reading frame encoding a TCF21 protein.

30. The method of claim 25, wherein the PSCs of the expanded population further comprise an engineered polynucleotide comprising an open reading frame encoding a GATA4 protein.

31. The method of claim 25, wherein the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

32. The method of claim 31, wherein the heterologous promoter is an inducible promoter.

33. The method of claim 24, wherein the population comprises 1 x 10 2 -1x10 7 PSCs.

34. The method of claim 24, wherein the population of PSCs is cultured for about 4-10 days.

35. The method of claim 29, wherein the population of PSCs is cultured for about 6 days.

36. The method of claim 24, wherein the granulosa cell-like cells are AMHR2 + 、CD82 + , FOXL2 + and / or EPCAM - .

37. The method of claim 36, wherein the granulosa cell-like cells are AMHR2 + 、CD82 + , FOXL2 + and EPCAM - .

38. A method comprising: (a) delivering 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 to a pluripotent stem cell (PSC); (b) culturing the PSCs in a feeder-free, serum-free medium to produce an expanded population of PSCs; and (c) culturing the expanded population of PSCs in a series of induction media containing an induction agent to produce AMHR2 + 、CD82 + , FOXL2 + and / or EPCAM - Granular cell-like cells.

39. The method of claim 38, comprising delivering to the PSC (i) an engineered polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding NR5A1 and (ii) an engineered polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a RUNX family protein.

40. The method of claim 38, wherein the RUNX family protein is RUNX1.

41. The method of claim 38, wherein the RUNX family protein is RUNX2.

42. The method of claim 38, wherein the engineered polynucleotide is a transposon and delivering further comprises delivering a transposase to the PSC.

43. The method of claim 38, wherein the inducible promoter is a chemically inducible promoter, optionally a doxycycline inducible promoter.

44. The method of claim 38, wherein the feeder-free, serum-free medium of (b) comprises a solubilized basement membrane preparation extracted from an Engelbreth-Holm-Swarm (EHS) mouse sarcoma.

45. The method of claim 44, wherein the solubilized basement membrane preparation comprises extracellular matrix (ECM) proteins and growth factors.

46. ​​The method of claim 45, wherein the ECM protein is selected from the group consisting of laminin, collagen IV, heparin sulfate proteoglycans, and paranectin / nidogen.

47. The method of claim 38, wherein the feeder-free, serum-free medium of (b) comprises a growth factor selected from the group consisting of recombinant human basic fibroblast growth factor (rh bFGF) and recombinant human transforming growth factor beta (rh TGFβ).

48. The method of claim 38, wherein the culturing of (b) continues for about 6-24 hours.

49. The method of claim 38, wherein the PSCs of the expanded population of (c) are approximately 10,000 cells / cm 2 Up to 20,000 cells / cm 2 Density culture.

50. The method of claim 38, 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 claim 40, wherein the first induction medium comprises one or more of L-alanyl-L-glutamine, antibiotics (e.g., penicillin and / or streptomycin), Dulbecco's Modified Eagle's Medium (DMEM) / F-12, Advanced RPMI (Roswell Park Memorial Institute) 1640 medium, glycogen synthase kinase (GSK) 3 inhibitors, small molecule or protein inhibitors of the BMP signaling pathway, small molecule ROCK inhibitors, and inducers (e.g., doxycycline).

52. The method of claim 50, wherein the PSCs are cultured in the first induction medium for about 36 to about 60 hours, optionally about 48 hours.

53. The method of claim 50, wherein the second induction medium comprises one or more of L-alanyl-L-glutamine, antibiotics (e.g., penicillin and / or streptomycin), Advanced RPMI 1640 medium, DMEM / F-12, and an inducer (e.g., doxycycline).

54. The method of claim 50, wherein the PSCs are cultured in the second induction medium for about 96 to about 144 hours, optionally about 120 hours.

55. The method of claim 54, wherein the second induction medium is removed and replaced with fresh second induction medium at intervals of about 24 hours.

56. A granulosa cell-like cell produced by the method of claim 38.

57. An ovarian organoid comprising the granulosa cell-like cells of claim 56 and human primordial germ cell-like cells (hPGCLCS).

58. The method of claim 38, further comprising combining granulosa cell-like cells with hPGCLCS to form ovarian organoids.

Images