Compositions and methods for producing ovarian support cells

Abstract

Featured are methods and compositions for production of ovarian support cells. In particular, the disclosure features methods of culturing or producing ovarian support cells from pluripotent stem or progenitor cells. The ovarian support cells described herein may be used for various purposes, including the treatment of ovarian decline, infertility, and a wide array of other reproductive and gynecologic conditions, as well as for modeling particular components of the human reproductive system in order to identify and characterize new therapeutic interventions.

Description

[0001] PATENT

[0002] Attorney Docket No.: 51763-009WO2

[0003] COMPOSITIONS AND METHODS FOR PRODUCING OVARIAN SUPPORT CELLS

[0004] TECHNICAL FIELD

[0005] This disclosure relates to the fields of ovarian and uterine synthetic biology, particularly in the context of therapeutic interventions, organoid model development, and assisted reproductive technology (ART).

[0006] BACKGROUND

[0007] One in ten women struggle with infertility, often requiring assisted reproductive technology (ART), such as in vitro fertilization (IVF) or forms of hormone replacement therapy (HRT), in order to augment their likelihood of successful conception and maintaining pregnancy. A wide variety of mechanisms underlie the ovarian and uterine conditions that hinder fertility, and challenges remain in studying these mechanisms and treating these conditions. These challenges include limitations on the availability of suitable animal models, confounding variables from existing in v / 'vo data, and overly simplistic representations of in vitro cell culture models that utilize single-cell populations to replicate a complex, multicellular environment of the human female reproductive system.

[0008] In addition to the challenges described above, menopause represents another substantial threat to reproductive health for which there is a paucity of effective treatment options. Menopause can cause debilitating symptoms, and the loss of hormonal balance that accompanies menopause is linked to such peripheral health crises as Alzheimer’s disease, osteoporosis, cardiovascular disease, and stroke. Existing attempts to counteract menopause are associated with significant detriments. Hormonal replacement therapy, for example, is commonly used to treat menopausal symptoms, but has achieved mixed efficacy and is poorly regulated. Another technique that has been explored in an effort to remedy the impact of menopause is ovarian tissue cryopreservation. However, this technique is a largely unproven and invasive procedure, requiring surgery to remove ovarian tissue followed by autotransplantation later in the patient’s life. However, in addition to being an expensive, invasive, and virtually inaccessible to patients already post-menopausal, this intervention poses significant health risks, as removing ovarian tissue may reduce fertility and induce early menopause, among other potentially dangerous side effects.

[0009] Accordingly, there remains a need for new models of the female reproductive system for elucidating mechanisms of human disease, as well as new methods for improving fertility and other complications associated with ovarian or uterine diseases.

[0010] SUMMARY OF THE INVENTION

[0011] The present disclosure provides compositions and methods for producing an engineered ovarian support cell (OSO) (e.g., ovarian theca cells, among other ovarian support cells described herein). Particularly, the disclosure provides methods of producing OSCs from a progenitor cell that replicate the structural and functional features of OSCs that are found in healthy ovarian or uterine tissue.

[0012] The disclosure also provides methods for producing an implant that includes (i) a population of engineered OSCs and (ii) an extracellular matrix component, such that the implant may regulate the PATENT

[0013] Attorney Docket No.: 51763-009WO2 cyclic production of endogenous gonadal hormones upon delivery to a subject. The subject may be one, for instance, that has or is at risk of developing a form of reproductive dysfunction (e.g., menopause, premature menopause, ovarian decline, primary ovarian insufficiency (POI) , polycystic ovarian syndrome (POOS), endometriosis, uterine fibroids, gynecological cancer, interstitial cystitis, pelvic inflammatory disease (PI D) , vaginitis, cervical dysplasia, uterine fibroids, pelvic floor prolapse, and / or interstitial cystitis). By regulating a subject’s hormone production upon delivery, an implant of this disclosure may be used for treating infertility and / or any one of the foregoing conditions, among other forms of reproductive dysfunction.

[0014] Additionally, this disclosure provides methods for assembling a population of engineered OSCs into an organoid that replicates female reproductive tissues or organs. The organoids of the disclosure may be applicable for research or therapeutic purposes, such as evaluating the safety and efficacy of therapeutics for treating infertility, ovarian decline, or other reproductive disorders. Moreover, the organoids of the disclosure may be used for determining the toxicity profile of novel drugs or therapeutics, such as those that are suspected of altering or disrupting hormone secretion female reproductive tissues.

[0015] Furthermore, in addition to methods of producing in vivo implants and organoids, the disclosure also provides methods of performing in vitro maturation of oocytes with the engineered OSCs. Such methods are particularly useful for individuals that are seeking forms of ART, such as IVF, and wish to increase the number of usable oocytes from a method of oocyte retrieval following ovarian stimulation.

[0016] In one aspect, the disclosure features a method of preparing a composition including one or more ovarian support cells (OSCs), in which the method includes culturing one or more induced pluripotent stem cells (iPSCs) in vitro in a cell culture medium including an alkanethiol, a mitogen-activated protein kinase (MAPK) pathway inhibitor, a bone morphogenetic protein (BMP) pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, a fibroblast growth factor (FGF), a keto acid, a WNT pathway activator, and / or a BMP.

[0017] In another aspect, the disclosure features a method of preparing a composition including one or more OSCs, the method including:

[0018] (a) culturing one or more iPSCs in vitro;

[0019] (b) differentiating the one or more iPSCs by:

[0020] (i) inducing, in the one or more iPSCs, expression or overexpression of one or more transcription factors including NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, GATA4, or any combination thereof; and / or

[0021] (ii) culturing the one or more iPSCs in a cell culture medium including an alkanethiol, a MAPK pathway inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, and / or a BMP; and, optionally,

[0022] (c) determining that the one or more differentiated cells resulting from (b) exhibit a gene expression profile that is indicative of one or more OSCs, thereby confirming OSC identity.

[0023] In another aspect, the disclosure features a method of preparing a composition including one or more OSCs, the method including culturing one or more iPSCs in a cell culture medium including an alkanethiol, a MAPK pathway inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, and / or a BMP, wherein the iPSCs were previously induced to express or overexpress one or more transcription factors including NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, GATA4, or any combination thereof, thereby producing the one or more OSCs. PATENT

[0024] Attorney Docket No.: 51763-009WO2

[0025] In a further aspect, the disclosure features a method of producing a mature oocyte for use in an ART procedure, the method including co-culturing one or more oocytes that have been previously retrieved from a human subject with one or more OSCs that have been differentiated from one or more iPSCs, wherein the one or more iPSCs were differentiated in a cell medium including an alkanethiol, a MAPK pathway inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, a BMP, or a combination thereof.

[0026] In another aspect, the disclosure features a method of inducing oocyte maturation in vitro, the method including co-culturing one or more oocytes with one or more OSCs that have been previously differentiated from one or more iPSCs, wherein the one or more iPSCs were differentiated in a cell medium including an alkanethiol, a MAPK pathway inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, a BMP, or a combination thereof.

[0027] In an additional aspect, the disclosure features a method of preparing a composition including one or more OSCs, the method including:

[0028] (a) culturing one or more iPSCs in vitro;

[0029] (b) differentiating the one or more iPSCs by:

[0030] (i) inducing, in the one or more iPSCs, expression or overexpression of one or more transcription factors including NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, GATA4, or any combination thereof; and / or

[0031] (ii) culturing the one or more iPSCs in a cell culture medium including an alkanethiol, a MAPK pathway inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, and / or a BMP;

[0032] (c) determining that the one or more differentiated cells resulting from (b) exhibit a gene expression profile that is indicative of one or more OSCs; and

[0033] (d) preparing an ovarian or uterine implant including the one or more OSCs and an extracellular matrix (ECM) component.

[0034] In another aspect, the disclosure features a method of preparing an ovarian or uterine implant including one or more OSCs and an ECM component, wherein the method includes culturing one or more differentiated iPSCs in a cell culture medium including an alkanethiol, a MAPK pathway inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, and / or a BMP, wherein the one or more iPSCs were previously induced to express or overexpress one or more transcription factors including NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, and / or GATA4, wherein the method further includes determining that the one or more differentiated iPSCs exhibit a gene expression profile that is indicative of one or more OSCs.

[0035] In another aspect, the disclosure features a method of preparing a composition including one or more OSCs, the method including:

[0036] (a) culturing one or more iPSCs in vitro;

[0037] (b) differentiating the one or more iPSCs by:

[0038] (i) inducing, in the one or more iPSCs, expression or overexpression of one or more transcription factors including NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, GATA4, or any combination thereof; and / or

[0039] (ii) culturing the one or more iPSCs in a cell culture medium including an alkanethiol, a MAPK pathway inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, and / or a BMP; PATENT

[0040] Attorney Docket No.: 51763-009WO2

[0041] (c) determining that the one or more differentiated cells resulting from (b) exhibit a gene expression profile that is indicative of one or more OSCs; and

[0042] (d) preparing an organoid including the one or more OSCs.

[0043] In another aspect, the disclosure features a method of preparing an organoid including a population of OSCs, the method including:

[0044] (a) introducing, into one or more iPSCs, one or more nucleic acid molecules that encode NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, and / or GATA4;

[0045] (b) culturing the one or more iPSCs in a cell culture medium thereby differentiating the one or more iPSCs into a population of ovarian granulosa cells, ovarian stroma cells, ovarian lutein cells, and / or ovarian theca cells; and

[0046] (c) contacting the cells resulting from (a) with an ECM component, optionally wherein the ECM component includes one or more of collagen, an elastin, a fibronectin, a vitronectin, a laminin, a cell adhesion protein, or a plant-derived protein or protein polymer.

[0047] In another aspect, the disclosure features a method of determining whether a candidate pharmaceutical intervention is efficacious in treating a disease or condition of the human female reproductive system, the method including:

[0048] (a) contacting the candidate pharmaceutical intervention with an organoid that includes a population of OSCs and an ECM substrate, wherein the population of OSCs were previously produced by culturing one or more iPSCs in a cell culture medium including an alkanethiol, a MAPK pathway inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, and / or a BMP;

[0049] (b) determining that the organoid exhibits (i) an increase in one or more metrics of ovarian and / or uterine function relative to a measurement of the one or more metrics of ovarian and / or uterine function obtained prior to the contacting, and / or (ii) a decrease in one or more metrics of severity of the disease or condition relative to a measurement of the one or more metrics of severity of the disease or condition obtained prior to the contacting; and, optionally,

[0050] (c) releasing the candidate pharmaceutical intervention for treatment of the disease or condition in a subject in need thereof.

[0051] In another aspect, the disclosure features a method of preparing an organoid including a population of OSCs, wherein the method includes culturing one or more differentiated iPSCs in a cell culture medium including an alkanethiol, a MAPK pathway inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, and / or a BMP, wherein the one or more iPSCs were previously induced to express or overexpress one or more transcription factors including NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, and / or GATA4, wherein the method further includes determining that the one or more differentiated iPSCs exhibit a gene expression profile that is indicative of one or more OSCs.

[0052] In some embodiments, prior to performing the methods of a preceding aspect, the one or more iPSCs were previously induced to express or overexpress one or more transcription factors including NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, GATA4, or any combination thereof.

[0053] In some embodiments, the alkanethiol is beta-mercaptoethanol. In some embodiments, the betamercaptoethanol is present in the cell culture medium at a concentration between about 0.1 mM to about PATENT

[0054] Attorney Docket No.: 51763-009WO2

[0055] 1 mM (e.g., about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, or about 1 mM).

[0056] In some embodiments, the BMP pathway inhibitor is LDN-193189 (CAS No. 1062368-24-4). In some embodiments, the LDN-193189 is present in the cell culture medium at a concentration between about 5 nM to about 10 nM (e.g., about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, or about 10 nM).

[0057] In some embodiments, the MAPK pathway inhibitor is PD0325901 (CAS No. 391210-10-9). In some embodiments, the PD0325901 is present in the cell culture medium at a concentration between about 1 nM to about 10 pM (e.g., about 1 nM to about 10 nM, about 10 nM to about 50 nM, about 50 nM to about 100 nM, about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 pM, about 1 pM to about 2 pM, about 2 pM to about 3 pM, about 3 pM to about 4 pM, about 4 pM to about 5 pM, about 5 pM to about 6 pM, about 6 pM to about 7 pM, about 7 pM to about 8 pM, about 8 pM to about 9 pM, about 9 pM to about 10 pM).

[0058] In some embodiments, the nonessential amino acid is one or more nonessential amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, and tyrosine. In some embodiments, the one or more nonessential amino acids are present in the cell culture medium at a concentration between about 0.1 mM to about 1 mM (e.g., about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, or about 1 mM).

[0059] In some embodiments, the sonic hedgehog pathway activator is a smoothened agonist (CAS No. 364590-63-6). In some embodiments, the smoothened agonist is present in the cell culture medium at a concentration of about 0.1 pM to about 1 pM (e.g., about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, or about 1 pM).

[0060] In some embodiments, the keto acid is sodium pyruvate. In some embodiments, the sodium pyruvate is present in the cell culture medium at a concentration between about 1 mM to about 2 mM (e.g., about 1 mM, about 1 .5 mM, or about 2 mM).

[0061] In some embodiments, the Wnt pathway activator is CHIR99021 (CAS No. 252917-06-9). In some embodiments, CHIR99021 is present in the cell culture medium at a concentration between about 1 pM to about 7 pM (e.g., about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, or about 7 pM).

[0062] In some embodiments, the FGF is selected from the group consisting of FGF1 , FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21 , FGF22, FGF23, or a combination thereof. In some embodiments, the FGF is present in the cell culture medium at a concentration between about 1 ng / mL to about 100 ng / mL (e.g., about 1 ng / mL to about 10 ng / mL, about 10 ng / mL to about 20 ng / mL, about 20 ng / mL to about 30 ng / mL, about 30 ng / mL to about 40 ng / mL, about 40 ng / mL about 50 ng / mL, about 50 ng / mL to about 60 ng / mL, about 60 ng / mL to about 70 ng / mL, about 70 ng / mL to about 80 ng / mL, about 80 ng / mL to about 90 ng / mL, or about 90 ng / mL to about 100 ng / mL). In some embodiments, the FGF is FGF9.

[0063] In some embodiments, the BMP is selected from the group consisting of BMP1 , BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMP9, BMP10, BMP15, BMP16, or a combination PATENT

[0064] Attorney Docket No.: 51763-009WO2 thereof. In some embodiments, the BMP is present in the cell culture medium at a concentration between about 1 ng / mL to about 10 ng / mL (e.g., about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, or about 10 ng / mL). In some embodiments, the BMP is BMP4.

[0065] In some embodiments, the cell culture medium includes one or more morphogens and / or growth factors including an insulin-like growth factor (IGF), an insulin-like growth factor binding protein (IGFBP), an epidermal growth factor (EGF), and / or a transforming growth factor (TGF).

[0066] In some embodiments, the cell culture medium includes an IGF. In some embodiments, the IGF is IGF1 and / or IGF2. In some embodiments, the cell culture medium includes an IGFBP. In some embodiments, the IGFBP is selected from the group consisting of IGFBP1 , IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6, IGFBP7, or a combination thereof. In some embodiments, the cell culture medium includes an EGF. In some embodiments, the EGF is EGF and / or amphiregulin. In some embodiments, the cell culture medium includes a TGF. In some embodiments, the TGF is TGF-a and / or an isoform of TGFp, including TGF-p1 , TGF-p2, TGF-p3, or a combination thereof.

[0067] In some embodiments, the one or more iPSCs are cultured on a matrix. In some embodiments, the matrix includes alginate, laminin, collagen, vitronectin, chitosan, hyaluronic acid, Poly-D-Lactone, or a mixture thereof, optionally wherein the laminin is selected from the group consisting of laminin-111 , laminin-211 , laminin-121 , laminin-221 , laminin-332, laminin-311 , laminin-321 , laminin-411 , laminin-421 , laminin-511 , laminin-521 , laminin-213, or a combination thereof. In some embodiments, the matrix includes laminin-521 . In some embodiments, the matrix includes vitronectin.

[0068] In some embodiments, the one or more iPSCs are reprogrammed using a transposase method to carry one or more inducible transcription factors. In some embodiments, the one or more iPSCs are transformed via electroporation or liposome-mediated transformation. In some embodiments, the one or more iPSCs are transformed via viral-mediated gene transfer.

[0069] In some embodiments, the expression or overexpression of the one or more transcription factors is induced by way of a doxycycline-responsive transcription regulatory element.

[0070] In some embodiments, the one or more iPSCs are human iPSCs. In some embodiments, the one or more iPSCs were previously retrieved from a subject. In some embodiments, the subject has or is at risk of having a decline in ovarian function. In some embodiments, the decline in ovarian function is a decline in one or more of follicular development, oocyte release, and oocyte maturation.

[0071] In some embodiments of the above aspects, the gene expression profile includes one or more genes selected from NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, GATA4, or a combination thereof.

[0072] In some embodiments of the above aspects, the one or more OSCs or population of OSCs include one or more ovarian theca cells. In some embodiments, the one or more ovarian theca cells express one or more genes selected from NR2F2 and GATA4. In some embodiments, the one or more ovarian theca cells express a cytochrome P450 enzyme, 17p-hydroxysteroid dehydrogenase (HSD17B), luteinizing hormone receptor (LHR), smooth muscle actin (SMA), platelet-derived growth factor receptor beta (PDGFRp), or a combination thereof. In some embodiments, the cytochrome P450 enzyme is CYP11A1 (P450scc), CYP17A1 (P450c17), and / or CYP19A1 (aromatase).

[0073] In some embodiments, the one or more ovarian theca cells express one or more hormones. In some embodiments, the one or more ovarian theca cells express one or more hormones in the presence of or upon contact with LH. In some embodiments, the one or more hormones is progesterone and / or an PATENT

[0074] Attorney Docket No.: 51763-009WO2 androgen. In some embodiments, the androgen comprises androstenedione, testosterone, dihydrotestosterone, or a combination thereof.

[0075] In some embodiments, the one or more ovarian theca cells is co-cultured with a population of cells including one or more cell types, wherein the one or more cell types include a granulosa cell, an ovarian stroma cell, an ovarian lutein cell, one or more germ cells, one or more uterine cells, and / or an iPSC. In some embodiments, the one or more germ cells include a primordial germ cell-like cell (PGCLC), an oogonium, an oocyte, or a combination thereof. In some embodiments, the one or more uterine cells include a uterine endometrial cell, a uterine myometrial cell, a uterine perimetrial cell, or a combination thereof.

[0076] In some embodiments, the one or more ovarian theca cells is co-cultured with one or more cells that express one or more genes including:

[0077] (a) FOXL2, AMHR2, CD82, FSHR, IGFBP7, KRT19, STAR, and / or WNT4;

[0078] (b) NR2F2;

[0079] (c) KRT19, CYP19A1 , STAR, CYP17A1 , and / or PGR;

[0080] (d) NANOS3, CD, ITGA6, EpCAM, BLIMP1 , TFAP2C, and / or SOX17;

[0081] (e) DDX4, DAZL, and / or STRA8; and / or

[0082] (f) SYCP1 , ZP1 , ZP2, REC8, LHX8, and / or SOHLH1 .

[0083] In a further aspect, the disclosure features a method of selecting an OSC, wherein the method includes:

[0084] (a) contacting a pluripotent progenitor cell with one or more differentiation agents, optionally including an alkanethiol, a MAPK pathway inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, and / or a BMP in a cell culture medium, thereby differentiating the pluripotent progenitor cell into the OSC;

[0085] (b) determining the OSC from (a) exhibits a gene expression profile including expression of one or more of a cytochrome P450 enzyme, 17p-hydroxysteroid dehydrogenase (HSD17B), luteinizing hormone receptor (LHR), smooth muscle actin (SMA), and platelet-derived growth factor receptor beta (PDGFRp); and

[0086] (c) selecting the OSC for use in an ART procedure, preparation an ovarian or uterine implant including the OSC, or preparation an organoid including the OSC.

[0087] In another aspect, the disclosure features a method of selecting an OSC for use in an ART procedure, wherein the method includes:

[0088] (a) contacting a pluripotent progenitor cell with one or more differentiation agents, optionally including an alkanethiol, a MAPK pathway inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, and / or a BMP in a cell culture medium, thereby differentiating the pluripotent progenitor cell into the OSC;

[0089] (b) determining the OSC from (a) exhibits a gene expression profile including expression of one or more of a cytochrome P450 enzyme, 17p-hydroxysteroid dehydrogenase (HSD17B), luteinizing hormone receptor (LHR), smooth muscle actin (SMA), and platelet-derived growth factor receptor beta (PDGFRp); and

[0090] (c) releasing the OSC for co-culture with an oocyte.

[0091] In a further aspect, the disclosure features a method of selecting an OSC for preparation of an ovarian or uterine implant, wherein the method includes: PATENT

[0092] Attorney Docket No.: 51763-009WO2

[0093] (a) contacting a pluripotent progenitor cell with one or more differentiation agents, optionally including an alkanethiol, a MAPK pathway inhibitor, a bone morphogenetic protein BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, and / or a BMP in a cell culture medium, thereby differentiating the pluripotent progenitor cell into the OSC;

[0094] (b) determining the OSC from (a) exhibits a gene expression profile including expression of one or more of a cytochrome P450 enzyme, 17p-hydroxysteroid dehydrogenase (HSD17B), luteinizing hormone receptor (LHR), smooth muscle actin (SMA), and platelet-derived growth factor receptor beta (PDGFRp); and

[0095] (c) selecting the OSC for contact with an extracellular matrix component.

[0096] In another aspect, the disclosure features a method of selecting an OSC for preparation of an organoid, wherein the method includes:

[0097] (a) contacting a pluripotent progenitor cell with one or more differentiation agents, optionally including an alkanethiol, a MAPK pathway inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, and / or a BMP in a cell culture medium, thereby differentiating the pluripotent progenitor cell into the OSC;

[0098] (b) determining the OSC from (a) exhibits a gene expression profile including expression of one or more of a cytochrome P450 enzyme, 17p-hydroxysteroid dehydrogenase (HSD17B), luteinizing hormone receptor (LHR), smooth muscle actin (SMA), and platelet-derived growth factor receptor beta (PDGFRp); and

[0099] (c) selecting the OSC for contact with an extracellular matrix component.

[0100] In some embodiments, the pluripotent progenitor cell was previously induced to express or overexpress one or more transcription factors including NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, GATA4, or any combination thereof. In some embodiments, the pluripotent progenitor cell is an iPSC. In some embodiments, the pluripotent progenitor cell is a human cell. In some embodiments, the pluripotent progenitor cell was previously retrieved from a subject. In some embodiments, the subject has or is at risk of having a decline in ovarian function. In some embodiments, the decline in ovarian function is a decline in one or more of follicular development, oocyte release, and oocyte maturation.

[0101] In some embodiments of the above aspects, the alkanethiol is beta-mercaptoethanol. In some embodiments, the beta-mercaptoethanol is present in the cell culture medium at a concentration of about 0.1 mM to about 1 mM (e.g., about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, or about 1 mM).

[0102] In some embodiments, the BMP pathway inhibitor is LDN-193189 (CAS No. 1062368-24-4). In some embodiments, the LDN-193189 is present in the cell culture medium at a concentration between about 5 nM to about 10 nM (e.g., about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, or about 10 nM).

[0103] In some embodiments, the MAPK pathway inhibitor is PD0325901 (CAS No. 391210-10-9). In some embodiments, the PD0325901 is present in the cell culture medium at a concentration between about 1 nM to about 10 pM (e.g., about 1 nM to about 10 nM, about 10 nM to about 50 nM, about 50 nM to about 100 nM, about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 pM, about 1 pM PATENT

[0104] Attorney Docket No.: 51763-009WO2 to about 2 |_iM , about 2 |_iM to about 3 |_iM , about 3 |_iM to about 4 |_iM , about 4 |_iM to about 5 |_iM, about 5 pM to about 6 |_iM, about 6 |_iM to about 7 |_iM , about 7 |_iM to about 8 |_iM , about 8 |_iM to about 9 |_iM, about 9 pM to about 10 |_iM) .

[0105] In some embodiments, the nonessential amino acid is one or more nonessential amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, and tyrosine. In some embodiments, the one or more nonessential amino acids are present in the cell culture medium at a concentration between about 0.1 mM to about 1 mM (e.g., about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, or about 1 mM).

[0106] In some embodiments, the sonic hedgehog pathway activator is a smoothened agonist (CAS No. 364590-63-6). In some embodiments, the smoothened agonist is present in the cell culture medium at a concentration of about 0.1 pM to about 1 pM (e.g., about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, or about 1 pM).

[0107] In some embodiments, the keto acid is sodium pyruvate. In some embodiments, the sodium pyruvate is present in the cell culture medium at a concentration between about 1 mM to about 2 mM (e.g., about 1 mM, about 1 .5 mM, or about 2 mM).

[0108] In some embodiments, the Wnt pathway activator is CHIR99021 (CAS No. 252917-06-9). In some embodiments, CHIR99021 is present in the cell culture medium at a concentration between about 1 pM to about 7 pM (e.g..about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, or about 7 pM).

[0109] In some embodiments, the FGF is selected from the group consisting of FGF1 , FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21 , FGF22, FGF23, or a combination thereof. In some embodiments, the FGF is present in the cell culture medium at a concentration between about 1 ng / mL to about 100 ng / ml_(e.g., about 1 ng / mL to about 10 ng / mL, about 10 ng / mL to about 20 ng / mL, about 20 ng / mL to about 30 ng / mL, about 30 ng / mL to about 40 ng / mL, about 40 ng / mL about 50 ng / mL, about 50 ng / mL to about 60 ng / mL, about 60 ng / mL to about 70 ng / mL, about 70 ng / mL to about 80 ng / mL, about 80 ng / mL to about 90 ng / mL, or about 90 ng / mL to about 100 ng / mL). In some embodiments, the FGF is FGF9.

[0110] In some embodiments, the BMP is selected from the group consisting of BMP1 , BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMP9, BMP10, BMP15, BMP16, or a combination thereof. In some embodiments, the BMP is present in the cell culture medium at a concentration between about 1 ng / mL to about 10 ng / mL (e.g., about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, or about 10 ng / mL). In some embodiments, the BMP is BMP4.

[0111] In some embodiments, the cell culture medium includes one or more morphogens and / or growth factors including an IGF, an IGFBP, an EGF, and / or a TGF. In some embodiments, the IGF is IGF1 and / or IGF2. In some embodiments, the IGFBP is selected from the group consisting of IGFBP1 , IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6, IGFBP7, or a combination thereof. In some embodiments, the EGF is EGF and / or amphiregulin. In some embodiments, the TGF is TGF-a and / or an isoform of TGFp, including TGF-p1 , TGF-p2, TGF-p3, or a combination thereof. PATENT

[0112] Attorney Docket No.: 51763-009WO2

[0113] In some embodiments of the above aspects, the extracellular matrix component is a collagen, an elastin, a fibronectin, a vitronectin, a laminin, a cell adhesion protein, a hyaluronic acid, a plant-derived protein or protein polymer, or a combination thereof.

[0114] In some embodiments, the OSC exhibits a gene expression profile that is indicative of an ovarian theca cell. In some embodiments, the gene expression profile includes genes NR2F2 and GATA4. In some embodiments, the OSC expresses a cytochrome P450 enzyme, HSD17B, LHR, SMA, and PDGFRp. In some embodiments, the cytochrome P450 enzyme is CYP11 A1 (P450scc), CYP17A1 (P450c17), and / or CYP19A1 (aromatase).

[0115] In some embodiments, the OSC produces one or more growth factors and / or steroids. In some embodiments, the one or more growth factors and / or steroids is at detectable levels in the cell culture medium. In some embodiments, the one or more growth factors and / or steroids comprises progesterone and / or an androgen. In some embodiments, the androgen comprises androstenedione, testosterone, dihydrotestosterone, or a combination thereof. In some embodiments, the one or more growth factors and / or steroids are produced following contact of the OSC and LH.

[0116] In some embodiments, the OSC has low or undetectable expression of one or more genes associated with pluripotency relative to an iPSC. In some embodiments, the one or more genes associated with pluripotency includes NANOG and / or POU5F1 .

[0117] In a further aspect, the disclosure features a composition including one or more OSCs, wherein the one or more OSCs were previously produced by a method of differentiating one or more iPSCs in an in vitro cell culture including a cell culture medium including an alkanethiol, a MAPK pathway inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, a BMP, or a combination thereof, optionally wherein the one or more iPSCs were induced to express or overexpress one or more transcription factors including NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, GATA4, or any combination thereof, thereby producing the one or more OSCs.

[0118] In some embodiments, the alkanethiol is beta-mercaptoethanol. In some embodiments, the BMP pathway inhibitor is LDN-193189 (CAS No. 1062368-24-4). In some embodiments, the MAPK pathway inhibitor is PD0325901 (CAS No. 391210-10-9). In some embodiments, the nonessential amino acid is one or more nonessential amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, and tyrosine. In some embodiments, the sonic hedgehog pathway activator is a smoothened agonist (CAS No. 364590- 63-6). In some embodiments, the keto acid is sodium pyruvate. In some embodiments, Wnt pathway activator is CHIR99021 (CAS No. 252917-06-9).

[0119] In some embodiments, the FGF is selected from the group consisting of FGF1 , FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21 , FGF22, FGF23, or a combination thereof. In some embodiments, the FGF is FGF9. In some embodiments, the BMP is selected from the group consisting of BMP1 , BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMP9, BMP10, BMP15, BMP16, or a combination thereof. In some embodiments, the BMP is BMP4.

[0120] In some embodiments, the composition includes an extracellular matrix component. In some embodiments, the extracellular matrix component is a collagen, an elastin, a fibronectin, a vitronectin, a PATENT

[0121] Attorney Docket No.: 51763-009WO2 laminin, a cell adhesion protein, a hyaluronic acid, a plant-derived protein or protein polymer, or a combination thereof.

[0122] In some embodiments, the one or more OSCs includes one or more ovarian theca cells.

[0123] In some embodiments, the one or more OSCs express a cytochrome P450 enzyme, 17p- HSD17B, LHR, SMA, PDGFRp, or a combination thereof. In some embodiments, the cytochrome P450 enzyme is CYP11A1 (P450scc), CYP17A1 (P450c17), and / or CYP19A1 (aromatase).

[0124] In some embodiments, the one or more OSCs produce one or more growth factors and / or steroids. In some embodiments, the one or more growth factors and / or steroids comprises progesterone and / or an androgen. In some embodiments, the androgen comprises androstenedione, testosterone, dihydrotestosterone, or a combination thereof. In some embodiments, the one or more growth factors and / or steroids are produced following contact of the one or more OSCs and LH.

[0125] In some embodiments, the one or more OSCs have low or undetectable expression of one or more genes associated with pluripotency relative to an iPSC. In some embodiments, the one or more genes associated with pluripotency includes NANOG and / or POU5F1 .

[0126] In some embodiments, the composition further includes or is suitable for combination with a granulosa cell, an ovarian stroma cell, an ovarian lutein cell, one or more germ cells, one or more uterine cells, and / or an iPSC. In some embodiments, the one or more germ cells include a primordial germ celllike cell (PGCLC), an oogonium, an oocyte, or a combination thereof. In some embodiments, the one or more uterine cells include a uterine endometrial cell, a uterine myometrial cell, a uterine perimetrial cell, or a combination thereof. In some embodiments, the iPSC is a human iPSC (hiPSC). In some embodiments, the iPSC was previously retrieved from a subject. In some embodiments, the subject has or is at risk of having a decline in ovarian function, optionally wherein the decline in ovarian function is a decline in one or more of follicular development, oocyte release, and oocyte maturation. In some embodiments, the subject has or is at risk of developing primary ovarian insufficiency (POI), polycystic ovarian syndrome (PCOS), ovarian cancer, ovarian hyperstimulation syndrome, endometriosis, uterine fibroids, adenomyosis, a gynecological cancer, pelvic inflammatory disease (PID), cervical dysplasia, or pelvic floor prolapse.

[0127] In some embodiments, the composition further includes a pharmaceutically acceptable excipient and is suitable for administration to a subject. In some embodiments, the subject to receive the composition has or is at risk of developing a decline in ovarian function, optionally wherein the decline in ovarian function is a decline in one or more of follicular development, oocyte release, and oocyte maturation. In some embodiments, the subject has or is at risk of developing POI, PCOS, ovarian cancer, ovarian hyperstimulation syndrome, endometriosis, uterine fibroids, adenomyosis, a gynecological cancer, PID, cervical dysplasia, or pelvic floor prolapse.

[0128] In a further aspect, the disclosure features an ovarian or uterine implant that includes a population of OSCs and an extracellular matrix substrate, wherein the OSCs were produced by differentiating one or more iPSCs in an in vitro cell culture including a cell culture medium including an alkanethiol, a MAPK pathway inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, a BMP, or a combination thereof.

[0129] In another aspect, the disclosure features implant for ovarian decline, the implant including at least an engineered OSC, an extracellular matrix substrate, and a delivery apparatus, wherein the PATENT

[0130] Attorney Docket No.: 51763-009WO2 engineered OSC was produced by differentiating one or more iPSCs in an in vitro cell culture including a cell culture medium including an alkanethiol, a MAPK pathway inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, a BMP, or a combination thereof.

[0131] In some embodiments, the alkanethiol is beta-mercaptoethanol. In some embodiments, the BMP pathway inhibitor is LDN-193189 (CAS No. 1062368-24-4). In some embodiments, the MAPK pathway inhibitor is PD0325901 (CAS No. 391210-10-9). In some embodiments, the nonessential amino acid is one or more nonessential amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, and tyrosine. In some embodiments, the sonic hedgehog pathway activator is a smoothened agonist (CAS No. 364590- 63-6). In some embodiments, the keto acid is sodium pyruvate. In some embodiments, Wnt pathway activator is CHIR99021 (CAS No. 252917-06-9).

[0132] In some embodiments, the FGF is selected from the group consisting of FGF1 , FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21 , FGF22, FGF23, or a combination thereof. In some embodiments, the FGF is FGF9. In some embodiments, the BMP is selected from the group consisting of BMP1 , BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMP9, BMP10, BMP15, BMP16, or a combination thereof. In some embodiments, the BMP is BMP4.

[0133] In some embodiments, the extracellular matrix substrate is a collagen, an elastin, a fibronectin, a vitronectin, a laminin, a cell adhesion protein, a hyaluronic acid, a plant-derived protein or protein polymer, or a combination thereof.

[0134] In some embodiments, the engineered OSC or a population thereof includes ovarian theca cells.

[0135] In some embodiments, the engineered OSC or a population thereof has been determined to express one or more of genes NR2F2 and GATA4. In some embodiments, the engineered OSC or a population thereof has been determined to express a cytochrome P450 enzyme, HSD17B), LHR, SMA, PDGFRp, or a combination thereof. In some embodiments, the cytochrome P450 enzyme is CYP11 A1 (P450scc), CYP17A1 (P450c17), and / or CYP19A1 (aromatase).

[0136] In other embodiments, the engineered OSC or a population thereof has been determined to express one or more genes including NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, GATA4, or a combination thereof.

[0137] In some embodiments, the implant expresses a growth factor and / or a hormone. In some embodiments, the one or more growth factors and / or steroids comprises progesterone and / or an androgen. In some embodiments, the androgen comprises androstenedione, testosterone, dihydrotestosterone, or a combination thereof. In some embodiments, the one or more growth factors and / or steroids are produced following contact of the implant and LH.

[0138] In some embodiment, the implant is further assembled as a plurality of microparticles (e.g., semi- permeable microparticles). In some embodiments, the plurality of microparticles comprise a biocompatible material.

[0139] In a further aspect, the disclosure features use of implant of any one of the above aspects and embodiments for treating a subject in need thereof, in which the treatment includes administering the implant to the subject. In some embodiments, the subject has or is at risk of having ovarian decline. In some embodiments, the ovarian decline is a result of menopause or premature menopause. In some PATENT

[0140] Attorney Docket No.: 51763-009WO2 embodiments, the subject has or is at risk of developing POI, POOS, ovarian cancer, ovarian hyperstimulation syndrome, endometriosis, uterine fibroids, adenomyosis, a gynecological cancer, PID, cervical dysplasia, or pelvic floor prolapse.

[0141] In some embodiments, the implant is administered to the subject as a semi-permeable rod, a semi-permeable pouch, an omental pouch, a patch, or an intrauterine device. In some embodiments, the semi-permeable rod is administered subdermally to the arm of the subject. In some embodiments, the semi-permeable pouch, omental pouch, or patch is administered to the abdomen of the subject. In some embodiments, the patch is delivered onto or adjacent to the ovary or fallopian tube of the subject.

[0142] In another aspect, the disclosure features an implant including (i) a population of OSCs and (ii) an extracellular matrix substrate, wherein the OSCs and extracellular matrix substrate are assembled as a plurality of microparticles.

[0143] In some embodiments, the population of OSCs includes one or more ovarian granulosa cells, ovarian lutein cells, and / or ovarian theca cells. In some embodiments, the population of OSCs includes one or more ovarian granulosa cells. In some embodiments, the one or more ovarian granulosa cells express one or more, or all, of proteins FOXL2, CD82, follicle-stimulating hormone receptor (FSHR), FOXL2, NR5A1 , GATA4, RUNX1 , and RUNX2.

[0144] In some embodiments, the implant includes from about 1 x 106and to about 1 x 107ovarian granulosa cells. In some embodiments, the implant includes from about 1 x 106to about 2 x 106ovarian granulosa cells, from about 2 x 106to about 3 x 106ovarian granulosa cells, from about 3 x 106to about 4 x 106ovarian granulosa cells, from about 4 x 106to about 5 x 106ovarian granulosa cells, from about 5 x 106to about 6 x 106ovarian granulosa cells, from about 6 x 106to about 7 x 106ovarian granulosa cells, from about 7 x 106to about 8 x 106ovarian granulosa cells, from about 8 x 106to about 9 x 106ovarian granulosa cells, or from about 9 x 106to about 1 x 107ovarian granulosa cells. In some embodiments, the implant includes about 1 x 106ovarian granulosa cells, about 2 x 106ovarian granulosa cells, about 3 x 106ovarian granulosa cells, about 4 x 106ovarian granulosa cells, about 5 x 106ovarian granulosa cells, about 6 x 106ovarian granulosa cells, about 7 x 106ovarian granulosa cells, about 8 x 106ovarian granulosa cells, about 9 x 106ovarian granulosa cells, or about 1 x 107ovarian granulosa cells.

[0145] In some embodiments, the one or more ovarian granulosa cells secrete an estrogen. In some embodiments, the estrogen is estradiol.

[0146] In some embodiments, the population of OSCs includes one or more ovarian lutein cells. In some embodiments, the one or more ovarian lutein cells express one or more, or all, of proteins KRT19, CYP19A1 , STAR, CYP17A1 , and PGR.

[0147] In some embodiments, the implant includes from about 1 x 106and to about 1 x 107ovarian lutein cells. In some embodiments, the implant includes from about 1 x 106to about 2 x 106ovarian lutein cells, from about 2 x 106to about 3 x 106ovarian lutein cells, from about 3 x 106to about 4 x 106ovarian lutein cells, from about 4 x 106to about 5 x 106ovarian lutein cells, from about 5 x 106to about 6 x 106ovarian lutein cells, from about 6 x 106to about 7 x 106ovarian lutein cells, from about 7 x 106to about 8 x 106ovarian lutein cells, from about 8 x 106to about 9 x 106ovarian lutein cells, or from about 9 x 106to about 1 x 107ovarian lutein cells. In some embodiments, the implant includes about 1 x 106ovarian lutein cells, about 2 x 106ovarian lutein cells, about 3 x 106ovarian lutein cells, about 4 x 106ovarian lutein cells, about 5 x 106ovarian lutein cells, about 6 x 106ovarian lutein cells, about 7 x 106ovarian lutein cells, about 8 x 106ovarian lutein cells, about 9 x 106ovarian lutein cells, or about 1 x 107ovarian lutein cells. PATENT

[0148] Attorney Docket No.: 51763-009WO2

[0149] In some embodiments, the one or more ovarian lutein cells secrete a progestogen. In some embodiments, the progestogen is progesterone.

[0150] In some embodiments, the population of OSCs includes one or more ovarian theca cells. In some embodiments, the one or more ovarian theca cells express one or both of proteins NR2F2 and GATA4.

[0151] In some embodiments, the implant includes from about 1 x 106and to about 1 x 107ovarian theca cells. In some embodiments, the implant includes from about 1 x 106to about 2 x 106ovarian theca cells, from about 2 x 106to about 3 x 106ovarian theca cells, from about 3 x 106to about 4 x 106ovarian theca cells, from about 4 x 106to about 5 x 106ovarian theca cells, from about 5 x 106to about 6 x 106ovarian theca cells, from about 6 x 106to about 7 x 106ovarian theca cells, from about 7 x 106to about 8 x 106ovarian theca cells, from about 8 x 106to about 9 x 106ovarian theca cells, or from about 9 x 106to about 1 x 107ovarian theca cells. In some embodiments, the implant includes about 1 x 106ovarian theca cells, about 2 x 106ovarian theca cells, about 3 x 106ovarian theca cells, about 4 x 106ovarian theca cells, about 5 x 106ovarian theca cells, about 6 x 106ovarian theca cells, about 7 x 106ovarian theca cells, about 8 x 106ovarian theca cells, about 9 x 106ovarian theca cells, or about 1 x 107ovarian theca cells.

[0152] In some embodiments, the one or more ovarian theca cells secrete an androgen. In some embodiments, the androgen is androstenedione.

[0153] In some embodiments, the OSCs include one or more ovarian granulosa cells, ovarian lutein cells, and ovarian theca cells, and wherein the ratio of ovarian granulosa cells to ovarian lutein cells to ovarian theca cells in the implant is from about 10:1 :1 to about 1 ,000:1 :1 . In some embodiments, the ratio of ovarian granulosa cells to ovarian lutein cells to ovarian theca cells in the implant is about 100:1 :1 .

[0154] In some embodiments, the one or more OSCs are obtained by differentiating a population of induced pluripotent stem cells (iPSCs) into one or more ovarian granulosa cells, ovarian lutein cells, and / or ovarian theca cells.

[0155] In some embodiments, the extracellular matrix substrate includes one or more of collagen, an epidermal growth factor (EGF), an elastin, a fibronectin, a vitronectin, a laminin, a cell adhesion protein, or a plant-derived protein or protein polymer. In some embodiments, the collagen is fibrillar collagen. In some embodiments, the collagen is collagen type I, type II, type III, type V, type XI. In some embodiments, the elastin is tropoelastin or mature elastin. In some embodiments, the plant-derived protein polymer is alginate.

[0156] In a further aspect, the disclosure features a method of increasing secretion of an estrogen, progestogen, and / or androgen in a human subject, the method including administering to the subject the implant of any one of the foregoing aspects and embodiments.

[0157] In some embodiments, the subject is a pre-menopausal human subject. In some embodiments, administration of the implant restores secretion of an estrogen, progestogen, and / or androgen in the subject. In some embodiments, the subject is diagnosed as having ovarian dysfunction.

[0158] In some embodiments, prior to administration of the implant to the subject, the subject exhibits consistent, circulating estradiol levels of about 20 pg / ml or less, optionally wherein the subject exhibits consistent, circulating estradiol levels of from about 10 to about 20 pg / ml.

[0159] In some embodiments, prior to administration of the implant to the subject, the subject exhibits consistent, circulating progesterone levels of about 0.5 ng / ml or less, optionally wherein the subject exhibits consistent, circulating progesterone levels of from about 0.01 ng / ml to about 0.5 ng / ml. In some embodiments, the subject has previously undergone an ovariectomy. PATENT

[0160] Attorney Docket No.: 51763-009WO2

[0161] In some embodiments, prior to administration of the implant to the subject, the subject exhibits ovarian decline. In some embodiments, the ovarian decline is a result of menopause. In some embodiments, the ovarian decline is not a result of menopause. In some embodiments, the ovarian decline is a result of premature menopause. In some embodiments, the ovarian decline is a result of a disease or condition selected from primary ovarian insufficiency (POI) , polycystic ovarian syndrome (POOS), endometriosis, uterine fibroids, gynecological cancer, interstitial cystitis, pelvic inflammatory disease (PI D) , vaginitis, cervical dysplasia, and pelvic floor prolapse.

[0162] In some embodiments, the population of OSCs secretes estradiol during an early- to mid-follicular phase of the subject’s menstrual cycle at a serum concentration of from about 20 pg / ml to about 80 pg / ml. In some embodiments, the population of OSCs secretes estradiol during an early- to mid-follicular phase of the subject’s menstrual cycle at a serum concentration of from about 20 pg / ml to about to 30 pg / ml, from about 30 pg / ml to about 40 pg / ml, from about 40 pg / ml to about 50 pg / ml, from about 50 pg / ml to about 60 pg / ml, from about 60 pg / ml to about 70 pg / ml, or from about 70 pg / ml to about 80 pg / ml.

[0163] In some embodiments, the population of OSCs secretes estradiol during a mid- to late-follicular phase of the subject’s menstrual cycle at a serum concentration of from about 20 pg / ml to about 200 pg / ml. In some embodiments, the population of OSCs secretes estradiol during a mid- to late-follicular phase of the subject’s menstrual cycle at a serum concentration of from about 20 pg / ml to about to 50 pg / ml, from about 50 pg / ml to about 70 pg / ml, from about 70 pg / ml to about 90 pg / ml, from about 90 pg / ml to about 110 pg / ml, from about 110 pg / ml to about 130 pg / ml, from about 130 pg / ml to about 150 pg / ml, from about 150 pg / ml to about t170 pg / ml, or from about 170 pg / ml to about 200 pg / ml.

[0164] In some embodiments, the population of OSCs secretes estradiol during a pre-ovulation phase of the subject’s menstrual cycle at a serum concentration of from about 100 pg / ml to about 400 pg / ml. In some embodiments, the population of OSCs secretes estradiol during a pre-ovulation phase of the subject’s menstrual cycle at a serum concentration of from about 100 pg / ml to about to 150 pg / ml, from about 150 pg / ml to about 200 pg / ml, from about 200 pg / ml to about 250 pg / ml, from about 250 pg / ml to about 300 pg / ml, from about 300 pg / ml to about 350 pg / ml, or from about 350 pg / ml to about 400 pg / ml.

[0165] In some embodiments, the population of OSCs secretes estradiol during an ovulation phase of the subject’s menstrual cycle at a serum concentration of from about 250 pg / ml to about 500 pg / ml. In some embodiments, the population of OSCs secretes estradiol during an ovulation phase of the subject’s menstrual cycle at a serum concentration of from about 250 pg / ml to about to 300 pg / ml, from about 300 pg / ml to about 350 pg / ml, from about 350 pg / ml to about 400 pg / ml, from about 400 pg / ml to about 450 pg / ml, or from about 450 pg / ml to about 500 pg / ml.

[0166] In some embodiments, the implant is administered to the subject in the form of a semi-permeable rod, a semi-permeable pouch, an omental pouch, a patch, or an intrauterine device, optionally wherein (a) the semi-permeable rod is administered subdermally to the arm of the subject, (b) the semi-permeable pouch, omental pouch, or patch is administered to the abdomen of the subject, or (c) the patch is delivered onto or adjacent to the ovary or fallopian tube of the subject.

[0167] In some embodiments, the implant is administered to the subject subdermally. In some embodiments, the implant is inserted beneath a segment of skin on the subject’s arm.

[0168] In some embodiments, the implant is administered onto one, or both, of the subject’s ovaries.

[0169] In some embodiments, the implant is administered into the subject’s uterus. In some embodiments, the implant is embedded into the subject’s uterine lining. PATENT

[0170] Attorney Docket No.: 51763-009WO2

[0171] In some embodiments, the implant is administered to the subject at a frequency of no greater than once every 6, 7, 8, 9, 10, 1 1 , or 12 months. In some embodiments, the implant is administered to the subject at a frequency of no greater than once every 24, 36, 48, or 52 weeks. In some embodiments, the implant is administered to the subject at a frequency of no greater than once every one, two, or three years.

[0172] In yet another aspect, the disclosure features a kit including the implant of any one of the foregoing aspects and embodiments, a delivery apparatus, and a package insert. In some embodiments, the insert instructs a user of the kit to perform any one of the foregoing methods.

[0173] BRIEF DESCRIPTION OF THE DRAWINGS

[0174] The accompanying drawings are included to illustrate embodiments of the disclosure and further an understanding of its implementations.

[0175] FIG. 1 A is a series of micrographs showing representative images of the research-use only (RUO)-hiPSC expansion and transcription factor-induced ovarian support cell (OSC) differentiation process. Micrographs show the cells during the hiPSC expansion and days 1 and 5 (d1 and d5, respectively) of OSC differentiation. Scale bar is 250 pm. Below the images is a schematic of a timeline depicting the expansion and differentiation protocol.

[0176] FIG. 1 B is a graph depicting flow cytometry analysis of CD82 expression in a control sample of undifferentiated hiPSCs and RUO-OSC-M differentiated cells.

[0177] FIG. 1C is a series of Uniform Manifold Approximation and Projection (UMAP) projections and bar graphs depicting single cell RNA-sequencing (scRNA-seq) data obtained from six independent batches of hiPSCs after five days of differentiating with three inducible transcription factors: NR5A1 , RUNX2, and GATA4. The resulting gene expression profiles of the batches were partitioned into clusters based on granulosa cell markers (GO), the clusters including Early GO, GO, Atresia / luteolysis, Mitochondrial gene enriched, and Ribosomal gene enriched, and further partitioned into subclusters Early GO I, Early GO II, and Early GO III as well as GO I, GO II, and GO III.

[0178] FIG. 1D is a series of dot plots, depicting the expression of granulosa cell markers, pre-GC I and pre-GC II genes, and estradiol (E2)-related genes in accordance to each cluster. The scale represents mean expression in groups, ranging from 0 to 3, 0 to 1 .5, and 0 to 4, respectively. The circles represent fractions of cells in the indicated groups, ranging from 0 to 100%, 0 to 90%, and 0 to 100%, respectively.

[0179] FIG. 1E is a series of UMAP projections depicting the signature scores for genes corresponding to antral GO genes and pre-ovulatory GO genes. The color scale ranges from -0.1 to 0.2.

[0180] FIG. 1F is a stacked bar graph depicting the amount of each cluster type found in each lot. The overall percentages per group are shown to the right of the bar graph.

[0181] FIG. 2A is a schematic representation of the OSC-enhanced in vitro maturation (IVM) vs. media- only control IVM culture process.

[0182] FIG. 2B is a bar graph depicting the quantification of Mil maturation rate in the control cultures and OSC co-cultured groups. RUO-OSC-M represents the combined maturation rates of three separate batches (lot 6, lot 8, and lot 56). Data is shown as mean ± SEM (p= 0.021 ; lot 6 v. control: 1 .37, lot 8 v. control: 1 .31 , lot 56 v. control: 1 .28). PATENT

[0183] Attorney Docket No.: 51763-009WO2

[0184] FIG. 2C is a dot plot that depicts the expression of ligand-receptor related genes based on each cluster. The scale represents mean expression in groups, ranging from 0 to 2, and the circles represent fractions of cells in the indicated groups, ranging from 0 to 70%.

[0185] FIG. 2D is a dot plot that depicts the expression of growth factor-related genes based on each cluster. The scale represents mean expression in groups, ranging from 0 to 1 , and the circles represent fractions of cells in the indicated groups, ranging from 0 to 100%.

[0186] FIG. 3A is a schematic of the design of experiments (DOE) strategy for optimizing the hiPSC- derived OSC manufacturing process.

[0187] FIG. 3B is a bar graph depicting logworth values of DOE main effect results. The dashed line indicates p<0.01 .

[0188] FIG. 3C is a series of micrographs showing images of OSCs in cultures on day 5 of differentiation performed on a substrate of vitronectin or laminin-521 . Scale bar is 250 pm.

[0189] FIG. 3D is a graph showing flow cytometry analysis of CD82 expression in a control sample of undifferentiated hiPSCs, and OSCs differentiated on a vitronectin matrix (RUO-OSC-V) or a laminin-521 matrix (RUO-OSC-L).

[0190] FIG. 3E is a series of UMAP projections depicting gene clustering data of OSCs cultured on a vitronectin matrix (Vitronectin-OSC) (left) and OSCs cultured on a laminin-521 matrix (Laminin-OSC) (right).

[0191] FIG. 3F is a dot plot depicting the expression of granulosa cell markers in the vitronectin-OSC and laminin-OSC subsets. The scale represents mean expression in groups ranging from 0 to 3, and the circles represent the fraction of cells in the indicated group, ranging from 0 to 100%.

[0192] FIG. 3G is a series of UMAP projections and a stacked bar graph depicting gene clustering data of individual batches of OSCs differentiated on a vitronectin matrix. The overall percentages per group are shown to the right of the bar graph.

[0193] FIG. 3H is a series of UMAP projections and a stacked bar graph depicting gene clustering data of individual batches of OSCs differentiated on a laminin-521 matrix. The overall percentages per group are shown to the right of the bar graph.

[0194] FIG. 4A is a bar graph comparing Mil maturation rates between media-only control groups and OSC co-culture groups. The maturation rates of three separate batches, (lots 41 and 49 manufactured on a vitronectin matrix and lot 86 manufactured on a laminin-521 matrix), are combined in the RUO-OSC-L / V bar, with additional bars depicting the individual maturation rates of each batch. Data is shown as the mean ± SEM (p=0.018; lot 41 / control: 1.08, lot 49 / control: 1.36, lot 86 / control: 1.27).

[0195] FIG. 4B is a graph depicting the relative Mil maturation rates across OSC batches differentiated on different matrices selected from matrigel (M), vitronectin (V), and laminin-521 (L).

[0196] FIG. 4C is a series of dot plots depicting the expression of ligand-receptor genes in the RUO- OSC and laminin-OSC subsets. The scale represents mean expression in groups, ranging from 0 to 1 , and the circles represent the fraction of cells in the indicated group, ranging from 0 to 100%.

[0197] FIG. 4D is a series of dot plots depicting the expression of growth factor genes in the vitronectin- OSC and laminin-OSC subsets. The scale represents mean expression in groups, ranging from 0 to 1 , and the circles represent the fraction of cells in the indicated group, ranging from 0 to 100%. PATENT

[0198] Attorney Docket No.: 51763-009WO2

[0199] FIG. 5A is an image of a gel depicting genotype data from a PCR reaction to assess the relative expression levels of transcription factors NR5A1 , GATA4, and RUNX2 from individual clones following hiPSC reprogramming.

[0200] FIG. 5B is a graph showing relative expression levels of ovarian support cell biomarkers and an hiPSC biomarker expressed in each indicated clone based on a cut-off (dotted line) as measured by flow cytometry.

[0201] FIG. 5C is a bar graph depicting the levels of estradiol (E2) secreted by the indicated clones in response to application of follicle stimulating hormone (FSH) (2), application of androstenedione (3), or application of a combination of FSH and androstenedione (4) to the cell culture media. E2 secretion in cells cultured in unsupplemented cell culture medium is also shown (1). Cells were cultured for 48 hours. Data is shown as the mean ± SEM.

[0202] FIG. 5D is a dot plot depicting the expression of granulosa cell markers in the indicated clones. The scale represents mean expression in groups, ranging from 0 to 1 , and the circles represent the fraction of cells in the indicated group, ranging from 0 to 100%.

[0203] FIG. 5E is a chart relative expression of OSC markers FOXL2 and CD82 as compared to the relative expression of hiPSC marker OCT4, cell viability, relative biomarker expression as shown as a percentage, and measured E2 secretion (pg / mL) of the nine indicated clones of manufactured OSCs.

[0204] FIG. 6A is a series of micrographs showing representative images of clinical grade (CG)-hiPSCs expansion and transcription factor-induced ovarian support cell (OSC) differentiation process on a matrix of laminin-521 . Micrographs show the cells during the hiPSC expansion and day 5 (d5) of OSC differentiation. Scale bar is 250 pm. Below the images is a schematic of a timeline depicting the expansion and differentiation protocol.

[0205] FIG. 6B is a series of graphs showing flow cytometry analysis of OSC biomarkers FOXL2 and CD82 after differentiation depicted in FIG. 6A.

[0206] FIG. 6C is a series of graphs showing flow cytometry analysis of hiPSC biomarkers OCT4 and NANOG after differentiation depicted in FIG. 6A.

[0207] FIG. 6D is a UMAP projection of the clinical-grade (CG)-OSC subset.

[0208] FIG. 6E is a UMAP projection of the individual lots from the CG-OSC subset.

[0209] FIG. 6F is a stacked bar graph depicting the amount of each cluster type found in the indicated individual lots of the CG-OSC subset. Overall percentages per groups are shown to the right of the bar graph.

[0210] FIG. 6G is a dot plot depicting the expression of granulosa cell markers in the CG-OSC subset. The scale represents mean expression in groups, ranging from 0 to 2, and the circles represent the fraction of cells in the indicated group, ranging from 0 to 100%.

[0211] FIG. 6H is a gene ontology (GO) chord plot showing differentially regulated proteins in both RUO- OSCs and CG-OSCs as compared to hiPSCs.

[0212] FIG. 61 is a graph depicting the correlation curve for proteins detected in the secretome of RUO- OSCs as compared to CG-OSCs.

[0213] FIG. 7A is a bar graph comparing Mil maturation rates between media-only control groups and CG-OSC co-culture groups. The maturation rates of three separate batches, (lots 88, 90, and 116), are combined in the CG-OSC-L bar, with additional bars depicting the individual maturation rates of each PATENT

[0214] Attorney Docket No.: 51763-009WO2 batch. Data is shown as the mean ± SEM (p=0.019; lot 88 / control: 1 .24, lot 90 / control: 1 .22, lot 116 / control: 1.29).

[0215] FIG. 7B is a graph depicting the relative Mil maturation rates of different individual batches of RUO-OSCs and CG-OSCs.

[0216] FIG. 7C is a dot plot depicting the expression of ligand-receptor genes in the CG-OSC subset. The scale represents mean expression in groups, ranging from 0 to 1 .5, and the circles represent fraction of cells in the indicated group, ranging from 0 to 100%.

[0217] FIG. 7D is a dot plot depicting the expression of growth factor-related genes in the CG-OSC subset. The scale represents mean expression in groups, ranging from 0 to 1 , and the circles represent fraction of cells in the indicated group, ranging from 0 to 100%.

[0218] FIG. 7E is a GO chord plot showing differentially regulated proteins in both RUO-OSCs and CG- OSCs after 24 hours of culture with a human oocyte as compared to the OSCs prior to culture.

[0219] FIG. 8 is a series of immunofluorescence micrographs depicting protein expression of the indicated biomarkers NANOG, NR2F2, AMHR2, and CYP17A following differentiation of hiPSCs.

[0220] FIG. 9 is a schematic diagram and table depicting the detection of various proteins expressed (e.g., intracellularly expressed or surface-expressed) or secreted by the produced OSCs.

[0221] FIG. 10A is a UMAP projection depicting the gene clusters identified by single-cell RNA- sequencing of the reprogrammed hiPSCs.

[0222] FIG. 10B is a dot plot depicting the expression of indicated theca cell biomarkers CYP11 A1 , CYP19A1 , HSD17B1 , and CGA in each gene cluster identified in (FIG. 10A).

[0223] FIG. 11 A is a series of immunofluorescence micrographs depicting human ovaroid (F66 / N.R1 .G.F #4 granulosa-like cells + hPGCLCs) sections at days 2, 4, 14, and 32 of culture, stained for FOXL2 (granulosa), OCT4 (germ cell / pluripotent), and DAZL (mature germ cell). Scale bars are 40 pm.

[0224] FIG. 11 B is a series of immunofluorescence micrographs depicting mouse ovaroid (fetal mouse ovarian somatic cells + hPGCLCs) sections stained as in FIG. 18A. Scale bars are 40 pm.

[0225] FIG. 11C is a series of graphs showing the fraction of OCT4+ cells (left) and DAZL+ cells (right) relative to the total (DAPI+) over time in human ovaroids and mouse xenovaroids. Counts were performed at 11 time points on images from 2 replicates of human ovaroids (F66 / N.R1 .G.F #4 and F66 / N.R2 #1 granulosa-like cells + hPGCLCs) and 1 replicate of mouse xeno-ovaroids.

[0226] FIG. 11D is a series of immunofluorescence micrographs depicting human ovaroid (F66 / N.R2 #1 granulosa-like cells + hPGCLCs) sections at days 4 and 8 of culture, stained for SOX17 (germ cell), TFAP2C (early germ cell), and AMHR2 (granulosa). Scale bars are 40 pm.

[0227] FIG. 11E is a series of immunofluorescence micrographs depicting day 16 human ovaroid ((F66 / N.R2 #1 granulosa-like cells + hPGCLCs) sections, stained for DAZL and OCT4. Some DAZL+OCT4- cells (arrows) are visible, as well as DAZL+OCT4+ cells (arrows). Ovaroids are also beginning to form follicle-like morphology (arrows). Scale bars are 40 pm.

[0228] FIG. 12A is a series of immunofluorescence micrographs depicting day 35 human ovaroid (F66 / N.R1 .G #7 + hPGCLC) sections stained for FOXL2, OCT4, and AMHR2. Scale bars are 40 pm. Follicle-like structures are marked with triangles.

[0229] FIG. 12B is a series of micrographs that show a whole-ovaroid view of follicle-like structures in human ovaroids (F66 / N.R1 .G #7) at day 6 and day 26. Scale bars are 1 mm. PATENT

[0230] Attorney Docket No.: 51763-009WO2

[0231] FIG. 12C is a series of immunofluorescence micrographs depicting a section of human ovaroid (F66 / N.R1 .G.F #4 + hPGCLC) at day 70 of culture, stained for FOXL2, NR2F2, and AMHR2, showing multiple small follicles (triangles) consisting single layers of FOXL2+AMHR2+ cells. NR2F2+ cells are interspersed between these. Scale bars are 100 pm.

[0232] FIG. 12D is a series of immunofluorescence micrographs depicting a section of human ovaroid (F66 / N.R2 #1 + hPGCLC) at day 70 of culture, stained for FOXL2, NR2F2, and AMHR2, showing an antral follicle consisting of FOXL2+AMHR2+ granulosa-like cells arranged in several layers around a central cavity. NR2F2 staining is visible outside of the follicle (marked ‘Stroma’). Scale bars are 100 pm.

[0233] FIG. 13A is a series of UMAP projections, showing the expression (Iog2 CPM) of selected granulosa (FOXL2), stroma / theca (NR2F2), and germ cell (PRDM1 ) markers. Expression is from scRNA- seq analysis of ovaroids (F66 / N.R1 .G.F #4 granulosa-like cells + hPGCLCs). Data from all samples (days 2, 4, 8, and 14) were combined for joint dimensionality reduction and clustering.

[0234] FIG. 13B is a UMAP projection and a violin plot that shows Leiden clustering of four main clusters and the expression (Iog2 CPM) of marker genes is plotted for each cluster from the scRNA-seq analysis of ovaroids (as in FIG. 13A).

[0235] FIG. 13C is a series of UMAP projections showing the mapping of cells onto a human fetal ovary reference atlas (Garcia-Alonso et al., 2022) and assignment of cell types based on the scRNA-seq analysis described in (FIG. 13A).

[0236] FIG. 13D is a series of graphs showing the proportion of somatic cell types, germ cells, DAZL+ cells, and DDX4+ cells in ovaroids from each day based on the scRNA-seq analysis described in (FIG. 13A).

[0237] FIG. 14A is a bar graph that shows the rate of oocyte maturation of denuded oocytes from standard of care IVM medium or oocytes grown in a culture with OSCs-lVM.

[0238] FIG. 14B is a bar graph that shows the rate of oocyte maturation of COCs from minimal stimulation in standard of care IVM medium or COCs grown in a culture with OSCs-lVM.

[0239] FIG. 14C is a bar graph comparing in vitro maturation rates of oocytes in a culture with standard of care IVM medium or in a culture with OSCs-lVM.

[0240] FIG. 15A is a violin plot that shows the morphological quality of oocytes based on total oocyte score (TOS) metrics grown in in a culture with standard of care IVM medium or in a culture with OSCs-lVM.

[0241] FIG. 15B is a violin plot that show the angle between the polar body 1 (PB1 ) and the spindle of oocytes grown in in a culture with standard of care IVM medium or in a culture with OSCs-lVM.

[0242] FIG. 16A is a series of bar graphs showing the rate of oocyte degradation rate in a toxicity assessment of OSCs-lVM product in denuded oocytes (left) and COCs (right).

[0243] FIG. 16B is a series of micrographs showing the fertilization and blastocysts generation of OSCs- lVM product.

[0244] DEFINTIONS

[0245] Unless otherwise defined herein, scientific, and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The PATENT

[0246] Attorney Docket No.: 51763-009WO2 use of "or" means "and / or" unless stated otherwise. The use of the term "including," as well as other forms, such as "includes" and "included," is not limiting.

[0247] As used herein, the terms “about” or “approximately” refer to a value that is within 10% (10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less) above or below the value being described. For instance, the phrase “about 50 mg” refers to a value between and including 45 mg and 55 mg.

[0248] As used herein, the term “assisted reproductive technology” or “ART” refers to a fertility treatment in which one or more female gametocytes (oocytes) or gametes (ova) are manipulated ex vivo so as to promote the formation of an embryo that can, in turn, be implanted into a subject in an effort to achieve pregnancy. For example, in some embodiments, an oocyte retrieved from a subject undergoing an ART procedure may be matured in vitro using, e.g., co-culturing methodologies described herein. In some embodiments, upon the formation of a mature oocyte (ovum), the ovum may be treated with one or more sperm cells so as to promote the formation of a zygote and, ultimately, an embryo. The embryo may then be transferred to the uterus of a female subject, for instance, using the compositions and methods in the art. Exemplary ART procedures include in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) techniques described herein and known in the art.

[0249] As used herein, the terms “subject” refers to an organism that receives treatment for a particular disease or condition as described herein. Examples of subjects and subjects include mammals, such as humans (e.g., a female human), receiving treatment for diseases or conditions that correspond to a reduced ovarian reserve, release of immature oocytes, or other forms of reproductive dysfunction. Exemplary diseases are primary ovarian insufficiency (POI) , polycystic ovarian syndrome (POOS), ovarian cancer, ovarian hyperstimulation syndrome, endometriosis, uterine fibroids, adenomyosis, a gynecological cancer, pelvic inflammatory disease (PI D) , cervical dysplasia, or pelvic floor prolapse.

[0250] As used herein, the terms “controlled ovarian hyperstimulation” or more simply “ovarian stimulation” refers to a procedure in which ovulation is induced in a subject, such as a human subject, prior to oocyte or ovum retrieval for use in embryo formation, for instance, by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Controlled ovarian hyperstimulation procedures may involve administration of follicle-stimulating hormone (FSH), human chorionic gonadotropin (hCG), and / or a gonadotropin-releasing hormone (GnRH) antagonist to the subject so as to promote follicular maturation. Controlled ovarian hyperstimulation methods are known in the art and are described herein as they pertain to methods for inducing follicular maturation and ovulation in conjunction with assisted reproductive technology.

[0251] As used herein, the term “derived from” in the context of a cell derived from a subject refers to a cell, such as a mammalian ovum, that is either isolated from the subject or obtained from expansion, division, maturation, or manipulation (e.g., ex vivo expansion, division, maturation, or manipulation) of one or more cells isolated from the subject. For instance, an ovum is “derived from” a subject or an oocyte as described herein if the ovum is directly isolated from the subject or obtained from the maturation of an oocyte isolated from the subject, such as an oocyte isolated from the subject from about 1 day to about 5 days following the subject receiving ovarian hyperstimulation procedures (e.g., an oocyte isolated from the subject from about 2 days to about 4 days following ovarian hyperstimulation procedures).

[0252] As used herein, the term “therapeutic agent” refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect. PATENT

[0253] Attorney Docket No.: 51763-009WO2

[0254] As used herein, the term “administering” to a subject includes dispensing, delivering, or applying a composition of the disclosure to a subject by any suitable route for delivery of the composition to the desired location in the subject. Alternatively or in combination, delivery is by the topical, parenteral, or oral route, intramuscular injection, subcutaneous / intradermal injection, intravenous injection, buccal administration, transdermal delivery, and administration by the rectal, colonic, vaginal, intranasal, or respiratory tract route.

[0255] As used herein, the term “dose” refers to the quantity of a therapeutic agent, such as a follicle stimulating agent described herein, that is administered to a subject for the treatment of a disorder or condition, such as to enhance oocyte maturation and / or release and promote retrieval and ex vivo maturation of viable oocytes. A therapeutic agent as described herein may be administered in a single dose or in multiple doses. In each case, the therapeutic agent may be administered using one or more unit dosage forms of the therapeutic agent. For instance, a single dose of 100 mg of a therapeutic agent may be administered using, e.g., two 50 mg unit dosage forms of the therapeutic agent. Similarly, a single dose of 300 mg of a therapeutic agent may be administered using, e.g., six 50 mg unit dosage forms of the therapeutic agent or two 50 mg unit dosage forms of the therapeutic agent and one 200 mg unit dosage form of the therapeutic agent, among other combinations. Similarly, a single dose of 900 mg of a therapeutic agent may be administered using, e.g., six 50 mg unit dosage forms of the therapeutic agent and three 200 mg unit dosage forms of the therapeutic agent or ten 50 mg unit dosage form of the therapeutic agent and two 200 mg unit dosage forms of the therapeutic agent, among other combinations.

[0256] As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0257] As used herein, the term “follicular triggering period” refers to the timepoint for administering a follicular triggering agent. The timepoint for administering a follicular triggering agent (i.e. , the follicular triggering period) to a female subject is on day 1 , day 2, or day 3 of her menstrual cycle, with preference for day 2 of her menstrual cycle. However, if the female subject is taking a hormonal contraceptive, then the timepoint for administering a follicular triggering agent is 4-6 days (e.g., 4 days, 5 days, or 6 days) after consuming the last oral contraception pill, with preference for 5 days following the dosing of her last oral contraception pill.

[0258] As used herein, the term “follicle-stimulating hormone” (FSH) refers to a biologically active heterodimeric human fertility hormone capable of inducing ovulation in a subject. FSH may be purified from post-menopausal human urine or produced as a recombinant protein product. Exemplary recombinant FSH products include follitropin alfa (GONAL-F, Merck Serono / EMD Serono) and follitropin beta (PUREGON / FOLLISTIM, MSD / Scherig-Plough).

[0259] As used herein, the term “human chorionic gonadotropin” (hCG) refers to the polypeptide hormone that interacts with the luteinizing hormone chorionic gonadotropin receptor (LHCGR) to induce follicle maturation and ovulation. hCG may be purified from the urine of pregnant women or produced as a recombinant protein product. Exemplary recombinant hCG products include choriogonadotropin alfa (OVIDREL®, Merck Serono / EMD Serono).

[0260] As used herein, the term “in vitro fertilization” (IVF) refers to a process in which an ovum, such as a human ovum, is contacted ex vivo with one or more sperm cells so as to promote fertilization of the PATENT

[0261] Attorney Docket No.: 51763-009WO2 ovum and zygote formation. The ovum can be derived from a subject, such as a human subject, undergoing various ARTs known in the art. For instance, one or more oocytes may be obtained from the subject following injection of follicular maturation stimulating agents for controlled ovarian hyperstimulation procedures, e.g., from about 1 day to about 5 days prior after injection of said agents (such as from about one day to about 4 days after injection of follicular maturation stimulating agents to the subject). The ovum may also be retrieved directly from the subject, for instance, by transvaginal ovum retrieval procedures known in the art.

[0262] As used herein, the term “intracytoplasmic sperm injection” (ICSI) refers to a process in which a sperm cell is injected directly into an ovum, such as a human ovum, so as to promote fertilization of the ovum and zygote formation. The sperm cell may be injected into the ovum, for instance, by piercing the oolemma with a microinjector so as to deliver the sperm cell directly to the cytoplasm of the ovum. ICSI procedures useful in conjunction with the compositions and methods described herein are known in the art and are described, for instance, in WO 2013 / 158658, WO 2008 / 051620, and WO 2000 / 009674, among others, the disclosures of which are incorporated herein by reference as they pertain to compositions and methods for performing intracytoplasmic sperm injection.

[0263] As used herein, the terms “ovum” and “oocyte” refer to a haploid female reproductive cell or gamete. In the context of assisted reproductive technology as described herein, ova may be produced ex vivo by maturation of one or more oocytes isolated from a subject undergoing ART. Ova may also be isolated directly from the subject, for example, by transvaginal ovum retrieval methods described herein or known in the art. Ovum or oocyte as used in this disclosure may refer to a plurality of oocytes. An oocyte may be in complex with surrounding cells such as a cumulus-oocyte complex (COC).

[0264] As used herein, the terms “mature ova” and “mature oocyte” refer to one or more ovum or oocyte in metaphase II (Mll)-stage of meiosis and typically has morphological or structural features consistent with metaphase II, such as a polar body and other features described herein.

[0265] As used herein, the terms “immature ovum” and “immature oocyte” refer to one or more ovum or oocyte that has not reached MH stage of meiosis. In some embodiments, an immature oocyte may be an oocyte including germinal vesicle (GV)-stage and / or metaphase I (Ml)-stage oocytes as determined by morphological features and / or other indications known in the art.

[0266] As used herein, the term “oocyte maturation” refers to the process by which an immature oocyte developmentally transitions to a mature oocyte. Oocyte maturation occurs as immature oocytes undergo cell signaling events incurred by external and internal stimuli. External stimuli may be produced by neighboring cells or supporting cells described herein. Oocyte maturation may occur prior to the release of an oocyte and retrieval from a subject. Oocyte maturation may occur in vitro as a result of culturing methods and culture compositions described herein.

[0267] As used herein, an “induced pluripotent stem cell (IPSO)” such as a human iPSC (hiPSC) refers to one or more cells that are self-renewing in an undifferentiated state and can differentiate into any one type of differentiated cell types found in an organism. iPSCs can be derived from non-embryonic sources such as a somatic cell and can proliferate without limit. iPSCs can differentiate into each one of the three embryonic germ layers (i.e., the endoderm, mesoderm, and ectoderm) and further cell types therein, depending on the induction of transcription factors and / or use of methods of gene editing known in the art. In one example, iPSCs can differentiate into a population of ovarian support cells, as described herein. The pluripotency or “stem-ness” of an iPSC may be verified by the expression of one or more PATENT

[0268] Attorney Docket No.: 51763-009WO2 pluripotent cell-specific markers including OCT4, SSEA3, SSEA4, TRA1 -60, TRA1 -81 , NANOG, SOX2, and / or POU5F1 , among other pluripotent cell-specific markers known in the art and described herein.

[0269] As used herein, an “ovarian support cell” (OSC) or “support cell” refers to one or more cells that promotes maturation of one or more oocytes. An OSC may be an ovarian theca cell (e.g., a type of theca cell described herein). An OSC may be an ovarian granulosa cell (e.g., a type of granulosa cell described herein). An OSC may be an ovarian stroma cell (e.g., a type of stroma cell described herein). An OSC may be a theca cell (e.g., a type of theca cell described herein). An OSC may be a lutein cell (e.g., a type of lutein cell described herein). An OSC may form a cumulus-oocyte complex (COC) with an oocyte. An OSC may be generated from an exogenous source, such as from progenitor cells including pluripotent stem cells such as induced pluripotent stem cells (iPSCs), e.g., human induced pluripotent stem cells (hiPSCs), as described herein. An OSC may be applied to a retrieved oocyte using in vitro cell culture methods and compositions described herein. An OSC may be a mixture of two or more cell types. An OSC may be a mixture of theca cells and one or more other cell types (e.g., granulosa cells, stroma cells, and / or lutein cells) such that the mixture is approximately a 1 :1 population of theca cells and another cell type (e.g., granulosa cells, stroma cells, or lutein cells). An OSC may be a mixture of theca cells and one or more other cell types such that one cell type is in higher relative abundance compared to one or more cell types such that the mixture is approximately a 2:1 population, a 3:1 population, a 4:1 population, a 5:1 population, among other possible population distributions. An OSC may be a mixture of theca cells and one or more cell types such that one cell type is more abundant in the mixture (e.g., 90% theca cells and 10% granulosa cells, stroma cells, or lutein cells; 80% theca cells and 20% granulosa cells, stroma cells, or lutein cells; 70% theca cells and 30% granulosa cells, stroma cells, or lutein cells; 60% theca cells and 40% granulosa cells, stroma cells, or lutein cells; 40% theca cells and 60% granulosa cells, stroma cells, or lutein cells; 30% theca cells and 70% granulosa cells, stroma cells, or lutein cells; 20% theca cells and 80% granulosa cells, stroma cells, or lutein cells; or 10% theca cells and 90% granulosa cells, stroma cells, or lutein cells; among other possible distributions). In some embodiments, an OSC may be a mixture of theca cells, granulosa cells, ovarian stroma cells, and / or lutein cells in combination with one or more additional cell types.

[0270] As used herein, the term “FOXL2” is the gene name of forkhead box L2 that encodes a transcription factor (NCBI Gene ID: 668) that regulates gene activity in tissues including ovarian tissues and the pituitary gland. FOXL2 may be expressed or overexpressed in a pluripotent progenitor cell (e.g., an iPSC) to produce one or more OSCs described herein or may be measured or detected to confirm the identity of one or more OSCs described herein.

[0271] As used herein, the term “GATA4” is the gene name of GATA binding protein 4 (NCBI Gene ID: 2626) that encodes a zinc-finger transcription factor to regulate genes involved in various physiological processes including embryogenesis. GATA4 may be expressed or overexpressed in a pluripotent progenitor cell (e.g., an iPSC) to produce one or more OSCs described herein or may be measured or detected to confirm the identity of one or more OSCs described herein.

[0272] As used herein, the term “NR2F2” is the gene name of nuclear receptor subfamily 2 group F member 2 (NCBI Gene ID: 7026) that encodes a steroid thyroid hormone nuclear receptor, which is a transcription factor. NR2F2 may be expressed or overexpressed in a pluripotent progenitor cell (e.g., an iPSC) to produce one or more OSCs described herein or may be measured or detected to confirm the identity of one or more OSCs described herein. PATENT

[0273] Attorney Docket No.: 51763-009WO2

[0274] As used herein, the term “NR5A1 ” is the gene name of nuclear receptor subfamily 5 group A member 1 (NCBI Gene ID: 2516) that encodes a transcription factor that regulates the activation of genes related to reproductive glands and adrenal glands. NR5A1 may be expressed or overexpressed in a pluripotent progenitor cell (e.g., an iPSC) to produce one or more OSCs described herein or may be measured or detected to confirm the identity of one or more OSCs described herein.

[0275] As used herein, the term “RUNX1 ” is the gene name of RUNX family transcription factor 1 (NCBI Gene ID: 861) that encodes a transcription factor that regulates the activity of genes related to hematopoiesis, or the development and maturation of blood cells. RUNX1 may be expressed or overexpressed in a pluripotent progenitor cell (e.g., an iPSC) to produce one or more OSCs described herein or may be measured or detected to confirm the identity of one or more OSCs described herein.

[0276] As used herein, the term “RUNX2” is the gene name of RUNX family transcription factor 2 (NCBI Gene ID: 860) that encodes a transcription factor that regulates the activity of genes related to skeletal development. RUNX2 may be expressed or overexpressed in a pluripotent progenitor cell (e.g., an iPSC) to produce one or more OSCs described herein or may be measured or detected to confirm the identity of one or more OSCs described herein.

[0277] As used herein, the term “HSD17B1 ” is the gene name of hydroxysteroid 17-beta dehydrogenase 1 (NCBI Gene ID: 3292) that encodes an enzyme that regulates the oxidation state of steroid hormones (e.g., androgens and / or estrogens). Expression of HSD17B1 may be measured or detected to confirm the identity of one or more OSCs described herein.

[0278] As used herein, the term “LHR” is the gene name of luteinizing hormone receptor (NCBI Gene ID: 3973) that encodes the G-protein coupled receptor that binds leutenizing hormone. Expression of LHR may be measured or detected to confirm the identity of one or more OSCs described herein.

[0279] As used herein, the term “SMA” is an acronym for smooth muscle actin, an intracellularly expressed structural protein. Expression of SMA may be measured or detected to confirm the identity of one or more OSCs described herein. SMA is one protein encoded by the gene ACTA2 (NCBI Gene ID: 59).

[0280] As used herein, the term “PDGFRB” is the gene name of platelet-derived growth factor receptorbeta (NCBI Gene ID: 5159) that encodes a cell surface tyrosine kinase receptor that binds mitogens for cells of mesenchymal origin during development. Expression of PDGFRB may be measured or detected to confirm the identity of one or more OSCs described herein.

[0281] As used herein, the term “CYP11 A1 ” is the gene name of cytochrome P450 family 11 subfamily A member 1 (NCBI Gene ID: 1583), an enzyme that regulates steroidogenesis by catalyzing the conversion of cholesterol into pregnenolone, which is the precursor to all steroid hormones. CYP11 A1 is also known as P450scc in the art. Expression of CYP1 1 A1 may be measured or detected to confirm the identity of one or more OSCs described herein.

[0282] As used herein, the term “CYP17A1 ” is the gene name of cytochrome P450 family 17 subfamily A member 1 (NCBI Gene ID: 1586), an enzyme that is essential for producing steroid hormones. For example, CYP17A1 catalyzes the conversion of progesterone to testosterone to regulate testosterone levels in a subject. CYP17A1 is also known in the art as P450c17. Expression of CYP17A1 may be measured or detected to confirm the identity of one or more OSCs described herein.

[0283] As used herein, the term “CYP19A1 ” is the gene name of cytochrome P450 family 19 subfamily A member 1 (NCBI Gene ID: 1588), an enzyme that catalyzes estrogen biosynthesis. CYP19A1 is also PATENT

[0284] Attorney Docket No.: 51763-009WO2 known in the rat as aromatase. Expression of CYP19A1 may be measured or detected to confirm the identity of one or more OSCs described herein.

[0285] As used herein, an “ovarian theca cell” or a “theca cell” is a somatic cell that differentiates from an ovarian stroma cell and surrounds a granulosa cell. A theca cell may produce one or more steroids in an LH-dependent manner. For example, a theca cell can produce C19 steroids that diffuse into the adjacent granulosa cell and serve as the substrates for estrogen production. A theca cell may express one or more markers consistent with a theca cell type such as nuclear receptor subfamily 2 group F member 2 (NR2F2), GATA binding protein 4 (GATA4), 17p-hydroxysteroid dehydrogenase 1 (HSD17B1), luteinizing hormone receptor (LHR), smooth muscle actin (SMA), platelet-derived growth factor receptorbeta (PDGFRp), and / or a cytochrome P450 enzyme, such as cytochrome P450 family 11 subfamily A member 1 (CYP11 A1 or P450scc), cytochrome P450 family 17 subfamily A member 1 (CYP17A1 or P450c17), cytochrome P450 family 19 subfamily A member 1 (CYP19A1 or aromatase), or a combination thereof, which can be detected or measured by methods known in the art.

[0286] As used herein, an “ovarian granulosa cell” or a “granulosa cell” is a cumulus cell surrounding the oocyte to ensure healthy oocyte and subsequent embryo development. An ovarian granulosa cell may form a COC with an oocyte. An ovarian granulosa cell may express markers consistent with a granulosa subtype such as forkhead box protein L2 (FOXL2), nuclear receptor subfamily 5 group A member 1 (NR5A1), GATA binding protein 4 (GATA4), runt-related transcription factor 1 (RUNX1), CD82, follicle- stimulating hormone receptor (FSHR), among other biomarkers described herein, which can be detected or measured by methods known in the art. An ovarian granulosa cell may be a steroidogenic granulosa cell. An ovarian granulosa cell may be produced from differentiated iPSCs (e.g., hiPSCs) or differentiated progenitor cells as described herein.

[0287] As used herein, a “steroidogenic granulosa cell” is a granulosa cell that may produce one or more steroids such as estradiol, progesterone, or a combination thereof. One or more steroids may be produced in response to hormonal stimulation, such as by FSH, androstenedione, or a combination thereof. One or more steroids may be secreted.

[0288] As used herein, an “ovarian stroma cell” or a “stroma cell” is a cumulus cell surrounding the oocyte to ensure healthy oocyte and subsequent embryo development. An ovarian stroma cell may form a COC with an oocyte. An ovarian stroma cell may express markers consistent with a stroma subtype such as NR2F2, which can be detected or measured by methods known in the art. An ovarian stroma cell may be a steroidogenic stroma cell. An ovarian stroma cell may be produced from differentiated iPSCs (e.g., hiPSCs) or differentiated progenitor cells as described herein.

[0289] As used herein, an “ovarian lutein cell” or a “lutein cell” is a cell of the corpus luteum and secretes progesterone. A lutein cell may express markers consistent with a lutein cell type such as cytokeratin-19 (KRT19), CYP19A1 or aromatase, steroidogenic acute regulatory protein (STAR), CYP17A1 or P450c17, or progesterone receptor (PGR). A lutein cell may be produced from differentiated hiPSCs or otherwise engineered as described herein.

[0290] As used herein, the term “biological sample” or “sample” may refer to a component of an in vitro cell culture system such as one or more isolated cells, a whole population of cells or a portion thereof, and / or cell culture media. Additionally, a biological sample or a sample may refer to a specimen (e.g., blood, blood component (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue PATENT

[0291] Attorney Docket No.: 51763-009WO2

[0292] (e.g., placental or dermal), pancreatic fluid, chorionic villus sample, hair, oocyte, ovum, and / or cells isolated from a subject.

[0293] As used herein, the term "express" refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein. Expression of a gene or a biomarker of interest in a sample (e.g., a biological sample; e.g., a biological sample that comprises one or more iPSCs, OSCs, oocytes, or a combination thereof) can manifest, for example, by detecting: the quantity or concentration of mRNA encoding a corresponding protein (as assessed, e.g., using RNA detection procedures such as quantitative polymerase chain reaction (qPCR), reverse transcription PCR (RT-PCR), and RNA sequencing (RNA-seq) techniques, among other RNA detection methods known in the art), the quantity or concentration of a corresponding protein (as assessed, e.g., using protein detection methods described herein or known in the art, such as enzyme-linked immunosorbent assays (ELISA), immunofluorescence methods, Western blot, or mass spectrometry, among others), and / or the activity of a corresponding protein (e.g., in the case of an enzyme, as assessed using an enzymatic activity assay known in the art) in a sample. In any one of the preceding processes, the expression can increase or decrease relative to a control sample. In some embodiments, a control sample is a cell or population of cells that has not undergone one or more of the procedures that a sample of interest has undergone. Exemplary procedures include a reprogramming or differentiation method such as any one or more types of reprogramming or differentiation methods directed to an iPSC as described herein, an in vitro maturation method such as an in vitro maturation method directed to one or more oocytes as described herein, or an in vitro fertilization method such as an in vitro fertilization method described herein. In some embodiments, a control sample is a cell or a population of cells that is representative of a particular cell type, such as, an established cell line from a manufacturer or an otherwise characterized cell type (e.g., an iPSC or an OSC). In some embodiments, one or more cells are determined to express a gene or biomarker if the expression level is within an acceptable range as compared to the expression level of a control sample (e.g., within 20%, within 15%, within 10%, or within 5% of the expression level of a control sample). In some embodiments, significant expression refers to an expression level relative to a cut-off value or threshold, such as, e.g., a cut-off value or threshold for an RNA-sequencing or flow cytometry method. In some embodiments, a population of cells may be determined to express a gene or biomarker of interest if a significant portion of the population (e.g., about 50% of the population, about 60% of the population, about 70% of the population, about 80% of the population, about 90% of the population, about 95% of the population, or about 99% of the population) has significant expression of a gene or biomarker of interest by meeting or exceeding a particular cut-off or threshold.

[0294] In some instances, no significant expression of one or more target genes or biomarkers is observed. In some embodiments “no significant expression” or “no detectable expression” refers to an expression level that is below the limits of detection for a particular detection method (e.g., an RT-PCR or an ELISA) and / or an expression level that is less than about 95% (e.g., 95%, 96%, 97%, 98%, 99%, or less than 99%) relative to a suitable control, such as a particular cell type (e.g., an undifferentiated iPSC or a typical OSC such as an in vivo OSC). In some embodiments, “no significant expression” or “no detectable expression” refers to an expression level relative to a cut-off value or threshold, such as, e.g., PATENT

[0295] Attorney Docket No.: 51763-009WO2 a cut-off value or threshold for an RNA-sequencing or a flow cytometry method. No significant expression or no detectable expression may refer to the relative expression levels of a population of cells, in which a significant portion of the population (e.g., about 50% of the population, about 60% of the population, about 70% of the population, about 80% of the population, about 90% of the population, about 95% of the population, or about 99% of the population) does not have significant expression of a gene or biomarker of interest by having an expression level that is below a particular cut-off or threshold.

[0296] As used herein, the term “overexpress” refers to expression of a gene or a biomarker that is increased relative to a basal level of expression for a particular cell type (e.g., an iPSC). The expression of a gene or a biomarker may be increased by 5%, by 10%, by 15%, by 20%, by 25%, by 30%, by 35%, by 40%, by 45%, by 50%, by 55%, by 60%, by 65%, by 70%, by 75%, by 80%, by 85%, by 90%, by 95%, by 100%, or in some instances, greater than 100%, such as 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, or greater than 400%. Overexpression of a gene or biomarker may be detected or measured by any suitable methods for detecting or measuring expression of a gene or a biomarker, such as genotyping methods, mRNA detection methods, or protein detection methods, such as methods described herein and known in the art.

[0297] As used herein, the term “doxycycline-responsive transcription regulatory element” refers to a nucleotide sequence such as a promoter that initiates transcription of a gene in the presence of doxycycline (e.g., an effective amount of doxycycline).

[0298] As used herein, the terms “oral contraceptive treatment,” “oral contraception,” “contraception,” or “birth control pill” refer to a hormonal method of treatment typically used to prevent pregnancy. Oral contraceptive treatment may block the release of oocytes from the ovaries and may contain hormones including estrogen and progestin.

[0299] As used herein, the term “ovarian reserve” refers to the number of oocytes in a subject’s ovaries and the quality of said oocytes. The ovarian reserve naturally declines with age and / or medical conditions described herein. Subjects with a diminished ovarian reserve may seek IVF or other ARTs to achieve a successful pregnancy. Levels of anti-Mullerian hormone (AMH), as described herein, may be indicative of a subject’s ovarian reserve.

[0300] As used herein, the term “stimulation protocol” refers to the process of administering to the subject one or more follicular triggering agents during a follicular triggering period.

[0301] As used herein, the terms “follicular triggering agent” or “triggering agent” refer to a chemical or biological composition that stimulates release of oocytes from the ovaries during ovulation. Follicular triggering agents may include hormones such as human chorionic gonadotropin and follicle-stimulating hormone. As used herein, the term “induced pluripotent stem cells” (iPSCs) refer to artificial stem cells that derive from reprogrammed and otherwise manipulated harvested somatic cells. iPSCs may differentiate into other cell types including ovarian support cells or granulosa cells via methods known in the art and methods described herein. iPSCs may be human iPSCs (hiPSCs) or iPSCs from, e.g., other mammalian sources.

[0302] As used herein, the terms “Clomid” or “clomiphene citrate” are interchangeable terms that refer to a nonsteroidal, ovulatory stimulant that is designated chemically as 2-[p-(2-chloro-1 ,2- diphenylvinyl)phenoxy]triethylamine citrate (1 :1 ) with a molecular formula of C26H28CINO • CeHsO? and a molecular weight of 598.01 g / mol. Clomiphene citrate is a mixture of two geometric isomers in the cis (zuclomiphene) and trans (enclomiphene) forms, in which the mixture contains between 30% and 50% PATENT

[0303] Attorney Docket No.: 51763-009WO2

[0304] (e.g., about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, or about 55%) of the cisisomer (zuclomiphene).

[0305] As used herein, the term “cell culture” refers to laboratory methods that enable in vitro cell proliferation and / or cultivation of prokaryotic or eukaryotic cell types.

[0306] As used herein, the term “matrix” in the context of cell culture or methods of in vitro cell cultivation refers to a coating such as a substrate or a membrane on a surface of a cell culture receptacle (e.g., a well plate or a Petri dish) that facilitates immobilization or adhesion of a cell to the surface. A suitable matrix may be a protein such as a glycoprotein, a proteoglycan, or a combination thereof. A suitable matrix may derive from plants or animal products or may be synthetic (e.g., a recombinant protein or a polymer). A suitable matrix may be an extracellular matrix protein, a particular isoform thereof, or a purified or partially purified fraction that is enriched for species based on their molecular weight. A suitable matrix may comprise a mixture of components. Exemplary matrices include alginate, laminin (e.g., laminin-111 , laminin-211 , laminin-121 , laminin-221 , laminin-332, laminin-31 1 , laminin-321 , laminin- 411 , laminin-421 , laminin-511 , laminin-521 , and / or laminin-213), vitronectin (e.g., type I, type II, and / or type III), fibronectin (e.g., type I, type II, and / or type III), collagen (e.g., type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX, type X, type XI, type XII, type XIII, type XIV, type XV, type XVI, type XVII, type XVIII, type XIX, type XX, type XXI, type XXII, type XIII, and / or type XXIV), chitosan, hyaluronic acid, Poly-D-Lactone, and hydrogels. Selection of a matrix may depend on cell type or relative cellular composition (e.g., lipid content, surface protein expression; e.g., relative abundance of one or more adhesion receptors).

[0307] As used herein, the term “encapsulate” or variations thereof refer to a coating of a cell or a plurality of cells in an in vitro culture system, optionally wherein the cell or plurality of cells is delivered (e.g., implanted, dispensed, or deposited) to a cellular niche (e.g., a tissue or an organ of an organism). Encapsulating a cell or a plurality of cells may increase the efficacy of delivery. A suitable reagent for encapsulation may be a protein such as a glycoprotein, a proteoglycan, or a combination thereof. A reagent for encapsulation may derive from plants or animal products or may be synthetic (e.g., a recombinant protein or a polymer). A suitable reagent for encapsulation may be an extracellular matrix protein, a particular isoform thereof, or a purified or partially purified fraction that is enriched for species based on their molecular weight. A suitable reagent for encapsulation comprise a mixture of components. Exemplary encapsulation agents include alginate, laminin (e.g., laminin-111 , laminin-211 , laminin-121 , laminin-221 , laminin-332, laminin-311 , laminin-321 , laminin-411 , laminin-421 , laminin-511 , laminin-521 , and / or laminin-213), vitronectin (e.g., type I, type II, and / or type III), fibronectin (e.g., type I, type II, and / or type III), collagen (e.g., type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX, type X, type XI, type XII, type XIII, type XIV, type XV, type XVI, type XVII, type XVIII, type XIX, type XX, type XXI, type XXII, type XIII, and / or type XXIV), chitosan, hyaluronic acid, Poly-D-Lactone, and hydrogels. Selection of a reagent for encapsulation may depend on cell type or relative cellular composition (e.g., lipid content, surface protein expression; e.g., relative abundance of one or more adhesion receptors) of the cells to be encapsulated or of the cells in a niche that will receive the one or more encapsulated cells.

[0308] As used herein, the term “alkanethiol” refers to an organic compound comprising a sulfur group and an alkyl chain. Exemplary alkanethiols are beta-mercaptoethanol, 1 -dodecanethiol, 1 -hexanethiol, and 1 -octadecanethiol. PATENT

[0309] Attorney Docket No.: 51763-009WO2

[0310] As used herein, the term “keto acid” refers to an organic compound comprising a carboxylic acid and a ketone group. Exemplary keto acids are pyruvate, oxaloacetate, alpha-ketoglutarate, alphaketobutyrate, acetoacetic acid, and 4-oxopentanoic acid.

[0311] As used herein, the term “nonessential amino acid” refers to an amino acid that an organism (e.g., a mammal, e.g., a human) or a cell thereof can synthesize. In contrast, an essential amino acid is an amino acid that must be provided to an organism or a cell thereof (e.g., by diet or supplementation).

[0312] As used herein, the term “mitogen-activated protein kinase inhibitor” refers to any agent (e.g., a nucleic acid, a protein, a lipid, a small molecule, or a combination thereof) that ultimately downregulates, inhibits, abrogates, reduces induction or, or reduces the magnitude of signal of the mitogen-activated protein kinase (MAPK) signaling pathway. A MAPK inhibitor may directly contact a positive regulator of the pathway to downregulate the pathway. A MAPK inhibitor may directly contact a negative regulator of the pathway to downregulate the pathway by way of, e.g., activating a negative regulator or enhancing the function of a negative regulator. Regulators of the MAPK pathway and exemplary inhibitors are known in the art and can be found elsewhere, such as, e.g., Cargnello and Roux, Microbiol Mol Biol Rev. 75(1):50-83, 201 1 ; Johnson and Lapadat, Science. 298(5600) :191 1 -1912, 2002; and English and Cobb, Trends Pharmacol Sci. 23(1):40-45, 2002, each of which is hereby incorporated by reference.

[0313] As used herein, the term “bone morphogenetic protein (BMP) pathway inhibitor” refers to any agent (e.g., a nucleic acid, a protein, a lipid, a small molecule, or a combination thereof) that ultimately downregulates, inhibits, abrogates, reduces induction or, or reduces the magnitude of signal of the BMP signaling pathway. A BMP pathway inhibitor may directly contact a positive regulator of the pathway to downregulate the pathway. A BMP pathway inhibitor may directly contact a negative regulator of the pathway to downregulate the pathway by way of, e.g., activating a negative regulator or enhancing the function of a negative regulator. Regulators of the BMP pathway and exemplary BMPs are known in the art and can be found elsewhere, such as, e.g., Katagiri and Watabe, Cold Spring Harb Perspect Biol. 8(6):a021899, 2016; Guo and Wang, Cell Res. 19(1 ):71 -88, 2009; and Akiyama et al., Genetics. 226(2) :iyad200, 2024, each of which is hereby incorporated by reference.

[0314] As used herein, the term “sonic hedgehog pathway activator” refers to any agent (e.g., a nucleic acid, a protein, a lipid, a small molecule, or a combination thereof) that ultimately upregulates, stimulates, initiates, drives, or induces activation of the sonic hedgehog signaling pathway. A sonic hedgehog pathway activator may directly contact a positive regulator of the pathway to upregulate said pathway. A sonic hedgehog pathway activator may directly contact a negative regulator of the pathway to upregulate said pathway (i.e. , act as an inhibitor of an inhibitor of the sonic hedgehog pathway). Regulators of the sonic hedgehog signaling pathway are known in the art and can be found elsewhere such as, e.g., Carballo et al., Cell Commun Signal. 16(1 ) :11 , 2018, which is hereby incorporated by reference.

[0315] As used herein, the term “Wnt pathway activator” refers to any agent (e.g., a nucleic acid, a protein, a lipid, a small molecule, or a combination thereof) that ultimately upregulates, stimulates, initiates, drives, or induces activation of the Wnt / p-catenin signaling pathway. A Wnt pathway activator may directly contact a positive regulator of the pathway to upregulate said pathway. A Wnt pathway activator may directly contact a negative regulator of the pathway to upregulate said pathway (i.e., act as an inhibitor of an inhibitor of the Wnt / p-catenin pathway). Regulators of the Wnt / p-catenin signaling pathway are known in the art and can be found elsewhere such as, e.g., Nusse and Clevers, Cell. 169(6):985-999, 2017, which is hereby incorporated by reference. PATENT

[0316] Attorney Docket No.: 51763-009WO2

[0317] In some embodiments, a Wnt pathway activator is a Rho-associated protein kinase (ROCK) inhibitor, such as Y-27642 or an equivalent salt or derivative thereof, the structure of which is shown below:

[0318] In some embodiments, a Wnt pathway activator is a glycogen synthase kinase-3 inhibitor, such as CHIR099021 , or an equivalent salt or derivative thereof, the structure of which is shown below:

[0319] As used herein, an “administration site” or implantation site” refers to the site for the application of the implant to the subject in need thereof.

[0320] As used herein, the term “co-culture” refers to a type of cell culture method in which more than one cell type or cell populations are cultivated with some degree of contact between them. In a typical coculture system, two or more cell types may share artificial growth medium.

[0321] As used herein, the terms “adherent co-culture systems” or “adherent cell culture” refer to a cell culture arrangement by which cells are attached to a surface for proper growth and proliferation.

[0322] As used herein, the terms “suspension co-culture systems” or “suspension cell culture” refer to a cell culture arrangement by which cells are cultivated via dispersion in a liquid medium for proper growth and proliferation.

[0323] DETAILED DESCRIPTION

[0324] Described herein are compositions and methods for use in assisted reproductive technology (ART), as well as for the treatment and modeling of various ovarian or uterine conditions. For example, the compositions and methods described herein are directed to producing, engineering, and culturing one or more ovarian support cells (OSCs) (e.g., theca cells, ovarian granulosa, ovarian stroma cells, ovarian lutein cells, or a combination thereof) and producing an organoid or implant of one or more components of the human female reproductive system (e.g., an ovarian organoid (“an ovaroid”) or a uterine organoid (“a uteroid”)).

[0325] The one or more OSCs, organoids, and / or implants of the disclosure may replicate the structural and / or functional features of endogenous human ovarian and / or uterine tissue by, e.g., recapitulating antigen expression and / or hormone secretion profiles of their naturally occurring cell counterparts. The PATENT

[0326] Attorney Docket No.: 51763-009WO2 compositions and methods described herein may be used to treat infertility, ovarian decline, and other forms of reproductive or gynecological dysfunction in a subject. Similarly, the OSCs, organoids, and / or implants of the present disclosure may be used to evaluate candidate therapeutic agents or interventions for safety and / or efficacy in treating one or more conditions associated with reproductive dysfunction. The one or more OSCs, organoids, and / or implants of the disclosure may further be used in methods of in vitro maturation of an oocyte.

[0327] The sections that follow provide a detailed description of exemplary processes that may be used to generate an OSC of the disclosure, as well as methods of using the same for treating and studying a variety of reproductive and gynecological pathologies.

[0328] I. Compositions and cell culture media

[0329] In some embodiments, the disclosure provides an engineered cell culture system. In some embodiments, the engineered cell culture system comprises a population of engineered ovarian supporting cells (OSCs). In some embodiments, the subject matter described herein relates to a method of differentiating a population of progenitor cells to a population of OSCs. In some embodiments, a population of progenitor cells is a population of induced pluripotent stem cells (iPSCs), such as human iPSCs (hiPSCs). In some embodiments, the OSCs are produced in a suspension culture. In some embodiments, the OSCs are produced in an adherent culture. In some embodiments, the engineered OSCs are further combined with one or more other cell types or matrix components, such as any one of the cell types or matrix components described herein, to produce an organoid or an implant.

[0330] A. Ovarian support cells differentiated from progenitor cells

[0331] Any one or more OSCs may be created from progenitor cells (e.g., iPSCs) using transcription factor (TF)-directed protocols. In some embodiments, the iPSCs are mammalian iPSCs. In preferred embodiments, the iPSCs are human iPSCs (hiPSCs). In some embodiments, hiPSCs may be transformed with any one or more plasmids encoding one or more transcription factors. In some embodiments, the differentiation of hiPSCs to OSCs is driven by overexpression of one or more transcription factors. In some embodiments, the one or more TFs comprise NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, GATA4, or any combination thereof. In some embodiments, undifferentiated hiPSCs are reprogrammed using a transposase method (e.g., a piggyBac transposase method) to carry specific inducible transcription factors (e.g., NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, and / or GATA4). In some embodiments, hiPSCs may be transformed via electroporation, liposome-mediated transformation, viral- med iated gene transfer, or another cell transformation methodologies known in the art. In some embodiments, gene expression of desired transcription factors may be induced in a doxycyclinedependent manner. In some embodiments, a plasmid or expression vector used for reprogramming hiPSCs may have a reporter gene such as a fluorescent protein.

[0332] In some embodiments, hiPSCs may differentiate into theca cells with induced expression of transcription factors NR2F2 and / or GATA4. In some embodiments, hiPSCs may differentiate into stroma cells with induced expression of transcription factors including NR2F2. In some embodiments, hiPSCs may differentiate into granulosa with induced expression of transcription factors including FOXL2, NR5A1 , GATA4, RUNX1 , RUNX2, KLF2, TCF21 , NR2F2, or any combination thereof. In some embodiments, PATENT

[0333] Attorney Docket No.: 51763-009WO2 hiPSCs may differentiate into lutein cells with induced expression of transcription factors FOXL2, NR5A1 , GATA4, RUNX1 , RUNX2, KRT19, or a combination thereof.

[0334] The OSCs may be produced using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. “CRISPR” is programmable technology that targets specific stretches of genetic code to edit DNA at precise locations. CRISPR technology may include CRISPR-CAS 9. Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence, allowing for the insertion of exogenous nucleic acids into a cell’s genome. For example, CRISPR-based gene editing techniques can be used to introduce into an iPSC genome, one or more genes encoding for factors that induce differentiation into OSCs (e.g., granulosa cells or stroma cells). These factors include, e.g., NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, and / or GATA4.

[0335] Exemplary CRISPR systems include those that utilize a Cas9 enzyme. Cas9 enzymes, together with CRISPR sequences, form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within organisms. CRISPR technology may include Class 1 CRISPR systems including type I (cas3), type III (cas10), and type IV and 12 subtypes. CRISPR technology may include Class 2 CRISPR systems including type II (cas9), type V (cas12), type VI (cas13), and 9 subtypes. In some embodiments, CRISPR technology may involve CRISPR-Cas design tools which are computer software platforms and bioinformatics tools used to facilitate the design of guide RNAs (gRNAs) for use with the CRISPR / Cas gene editing system. For example, CRISPR-Cas design tools may include: CRISPRon, CRISPRoff, Invitrogen TrueDesign Genome Editor, Breaking-Cas, Cas-OFFinder, CASTING, CRISPy, CCTop, CHOPCHOP, CRISPOR, sgRNA Designer, Synthego Design Tool, and the like. CRISPR technology may also be used as a diagnostic tool. For example, CRISPR-based diagnostics may be coupled to enzymatic processes, such as SHERLOCK-based Profiling of in vitro Transcription (SPRINT). SPRINT can be used to detect a variety of substances, such as metabolites in subject samples or contaminants in environmental samples, with high throughput or with portable point-of-care devices.

[0336] In some embodiments, overexpression of the one or more TFs is driven by any suitable induction agent known in the art. In some embodiments, the induction lasts for 1 day, 2 days, 3, days, 4 days, 5 days or longer than 5 days. In some embodiments, transcription factors are constitutively expressed. In some embodiments, a plasmid or expression vector used for reprogramming hiPSCs may have a reporter gene such as a fluorescent protein. In some embodiments, the cells (e.g., hiPSCs) are treated with an agent that primes mesodermal induction. In some embodiments, during the initial induction of one or more transcription factors, cells (e.g., hiPSCs) are treated with an agent that activates Wnt / p-catenin signaling to prime cells for a mesodermal cell fate. In some embodiments, during the initial induction of one or more transcription factors, cells (e.g., hiPSCs) are treated with an agent that inhibits a serinethreonine protein kinase such as Rho-associated protein kinase (ROCK) (e.g., a small molecule ROCK inhibitor; e.g., Y-27642) or glycogen synthase kinase-3 (GSK3) (e.g., a GSK3 inhibitor; e.g., CHIR099021).

[0337] In addition to the induction of one or more TFs, one or more components or supplements may be added to the cell culture medium to modulate or stabilize the cellular identity of the produced OSCs. In some embodiments, the cell culture medium comprises a MAPK pathway inhibitor (e.g., a small molecule MAPK inhibitor, e.g., PD0325901). In some embodiments, the cell culture medium comprises a BMP pathway inhibitor. In some embodiments, the BMP pathway inhibitor targets activin receptor-like kinase-2 PATENT

[0338] Attorney Docket No.: 51763-009WO2

[0339] (ALK2) and / or activin receptor-like kinase-3 (ALK3) (e.g., a small molecule ALK2 and / or ALK3 inhibitor; e.g., LDN-193189). In some embodiments, the cell culture medium comprises one or more growth factors and / or morphogens such as a fibroblast growth factor (FGF) (e.g., FGF1 , FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21 , FGF22, and / or FGF23), an insulin-like growth factor (IGF) (e.g., IGF1 and / or IGF2), an epidermal growth factor (EGF) (e.g., EGF and / or amphiregulin), a transforming growth factor (TGF) (e.g., TGF-a, TGF-p1 , TGF-p2, and / or TGF-p3), and / or a BMP (e.g., BMP1 , BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMP9, BMP10, BMP15, and / or BMP16). In some embodiments, a growth factor or morphogen is naturally occurring and may be isolated or partially purified from a biological sample. In some embodiments, a growth factor or morphogen is a recombinant growth factor or morphogen or a fragment thereof (e.g., a fragment that retains biological activity). In some embodiments, the cell culture medium comprises a sonic hedgehog pathway activator. In some embodiments, the sonic hedgehog pathway activator stimulates the G protein-coupled receptor smoothened (e.g., a smoothened agonist). In some embodiments, the cell culture medium comprises an alkanethiol (e.g., beta-mercaptoethanol). In some embodiments, the cell culture medium comprises an antioxidant or metabolite that promotes cell proliferation and / or survival, such as sodium pyruvate, pyruvic acid, and / or a nonessential amino acid (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, and / or tyrosine).

[0340] In some embodiments, application of doxycycline to the cells or cell culture maintains activation of one or more TFs in the OSCs (e.g., for doxycycline-dependent induction).

[0341] In some embodiments, the resulting OSCs comprise a population of cells with functional and biological similarity to human theca cells (cells that express NR2F2, GATA4, one or more cytochrome P450 enzymes, 17p-hydroxysteroid dehydrogenase (HDS17B), luteinizing hormone receptor (LHR), smooth muscle actin (SMA), platelet-derived growth factor receptor-beta (PDGFRp), among other theca cell biomarkers known in the art and described herein). In some embodiments, the resulting OSCs comprise a population of cells with functional and biological similarity to human granulosa cells (cells that express FOXL2 and AMHR2, among other granulosa biomarkers known in the art and described herein). In some embodiments, the resulting OSCs comprise a population of cells with functional and biological similarity to human ovarian stroma cells (cells that express NR2F2, among other stroma cell biomarkers known in the art and described herein). In some embodiments, the resulting OSCs comprise a population of cells with functional and biological similarity to human lutein cells (cells that express KRT19, CYP19A1 , STAR, CYP17A1 , PGR, among other lutein cells known in the art and described herein). In some embodiments, the resulting OSCs comprise a population of cells comprising a mixture of theca cells, granulosa cells, stroma cells, and lutein cells in any combination. In some embodiments, a population of OSCs primarily comprises theca cells such that the population of OSCs comprise more than 50% theca cells, more than 60% theca cells, more than 70% theca cells, more than 80% theca cells, more than 90% theca cells, or more than 95% theca cells.

[0342] Reprogramming of hiPSCs to OSCs may be determined by genotyping methods and biomarker expression analyses. Validating the cell identity of an OSC produced by reprogramming hiPSCs may include evaluating the relative expression of mRNA transcripts and / or proteins (including intracellular proteins and proteins expressed on the cells surface), as compared to an undifferentiated hiPSC or an established OSC (e.g., a naturally occurring OSC or an OSC with a known identity). Reprogramming of PATENT

[0343] Attorney Docket No.: 51763-009WO2 hiPSCs to an OSC may be determined by one or more functional assays, such as evaluating hormone secretion into the cell culture medium in the presence of a supplemented protein or hormone or oocyte maturation. Additionally, validating cell identity of an OSC produced by reprogramming hiPSCs may include evaluating the cell shape, size, and / or determining the presence of one or more structural features.

[0344] Differentiation of hiPSCs to OSCs (such as theca cells) may be determined by relative expression of biomarkers or genes, such as genes associated with cell adherence, chemotaxis, growth factors and / or growth factor receptors, steroids and / or steroid receptors, or a combination thereof, among other genes or biomarkers associated with one or more types of OSCs. Differentiation of hiPSCs to OSCs (such as theca cells) may be determined by relative expression of biomarkers typical of a theca cell type including NR2F2, GATA4, one or more steroidogenic enzymes such as cytochrome P450 enzymes (e.g., CYP11 A1 (also referred to as P450scc), CYP17A1 (also referred to as P450c17), and CYP19A1 (also referred to as aromatase)), HDS17B, LHR, SMA, and / or PDGFRp. In some embodiments, an engineered theca cell expresses a higher level of NR2F2, GATA4, CYP11 A1 , CYP17A1 , CYP19A1 , HDS17B, LHR, SMA, PDGFRp, or a combination thereof, as compared to an accepted reference ovarian cell or other cell type known in the art. In some embodiments, the functional screening of individual clones identifies a stable line harboring the optimal balance of expression of each transcription factor or biomarker. In some embodiments, differentiation of hiPSCs to OSCs (such as theca cells) may be determined by hormone production and / or secretion (i.e., secretion into the cell culture medium). In some embodiments, a hormone is produced and / or secreted by a particular OSC in the presence of luteinizing hormone (LH) (e.g., LH that is added to the cell culture medium). For example, a theca cell may secrete a detectable amount of one or more androgens (e.g., androstenedione, testosterone, and / or dihydrotestosterone) and / or progesterone in the presence of LH.

[0345] Differentiation of hiPSCs to OSCs (such as granulosa cells) may be determined by relative expression of biomarkers typical of a granulosa cell type including FOXL2, AMHR2, CD82, FSHR, IGFBP7, KRT19, STAR, WNT4, or a combination thereof among other granulosa cell biomarkers known in the art. In some embodiments, an engineered granulosa cell expresses a higher level of FOXL2, AMHR2, CD82, FSHR, IGFBP7, KRT19, STAR, WNT4, or a combination thereof, as compared to an accepted reference ovarian cell or other cell type known in the art. hiPSCs that are differentiated to OSCs (such as granulosa cells) may be categorized into one or more clusters based on transcriptome profiling. Differentiation of hiPSCs to OSCs may be determined by relative expression of biomarkers typical of an OSC (e.g., a granulosa cell), including GJA1 , MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1 , COL4A2, LAMC1 , ITGAV, and / or ITGB. In some embodiments, the functional screening of individual clones identifies a stable line harboring the optimal balance of expression of each transcription factor or biomarker. In some embodiments, differentiation of hiPSCs to OSCs (such as a granulosa cell) may be determined by hormone production and / or secretion, such as the production of estradiol and / or progesterone. For example, a granulosa cell may secrete a detectable amount of estradiol and / or progesterone in the presence of one or more hormones such as an androgen (e.g., androstenedione), follicle-stimulating hormone (FSH), and / or forskolin.

[0346] Differentiation of hiPSCs to OSCs (such as ovarian stroma cells) may be determined by relative expression of biomarkers typical of a stroma cell type including NR2F2. In some embodiments, an ovarian stroma cell is distinguished from a theca cell based on detectable production of one or more androgens. PATENT

[0347] Attorney Docket No.: 51763-009WO2

[0348] Differentiation of hiPSCs to OSCs (such as lutein cells) may be determined by relative expression of biomarkers typical of a lutein cell type including KRT19, CYP19A1 , STAR, CYP17A1 , PGR, or a combination thereof among other lutein cell biomarkers known in the art. In some embodiments, the functional screening of individual clones identifies a stable line harboring the optimal balance of expression of each transcription factor or biomarker. In some embodiments, a lutein cell may secrete a detectable amount of estradiol and / or progesterone in the presence of one or more hormones such as an androgen (e.g., androstenedione), FSH, and / or forskolin.

[0349] Expression of one or more genes, transcription factors, and / or biomarkers may be detected or measured by methods of protein and / or mRNA expression that are routine in the art. Exemplary methods of detecting or measuring the relative expression of one or more transcription factors or biomarkers include flow cytometry, RNA-sequencing (RNA-seq) (e.g., single-cell RNA-seq), real-time reverse transcription polymerase chain reaction (RT-PCR), quantitative PCR (qPCR), RT-qPCR, Northern blot analysis, mass spectrometry and proteomic modalities, Western blot analysis, enzyme-linked immunosorbent assay (ELISA), immunofluorescence or other microscopy-based immunodetection methods, among other detection methods known in the art. In some embodiments, the expression of one or more biomarkers of a produced OSC may be measured based on the expression of one or more biomarker of a naturally occurring cell counterpart. In some embodiments, the production of one or more hormones by an engineered OSC may be compared to the relative production of one or more hormones produced by a naturally occurring cell counterpart.

[0350] In some embodiments, reprogramming of hiPSCs to OSCs yield one or more OSCs that share strong gene expression similarity to in vivo theca cells (e.g., cells that express NR2F2, GATA4, CYP1 1 A1 , CYP17A1 , CYP19A1 , HDS17B, LHR, SMA, and / or PDGFRp). In some embodiments, reprogramming of hiPSCs to OSCs yield one or more OSCs that share strong gene expression similarity to in vivo granulosa cells (e.g., cells that express FOXL2 and AMHR2, among other biomarkers described herein). In some embodiments, the OSCs share strong gene expression similarity to in vivo stroma cells (e.g., cells that express NR2F2). In some embodiments, the OSCs share strong gene expression similarity to in vivo lutein cells (e.g., cells that express KRT19, CYP19A1 , STAR, CYP17A1 , and / or PGR).

[0351] In some embodiments, the OSCs recapitulate folliculogenesis progression in vitro through follicle formation. In some embodiments, reprogramming of hiPSCs to one or more OSCs may be determined by production of growth factors and / or hormones that may adequately support in vitro maturation of retrieved oocyte via paracrine and juxtacrine cell signaling. In some embodiments, the OSCs produce one or more growth factors including insulin-like growth factor (IGF), stem cell factor (SCF), epidermal growth factor (EGF), leukemia inhibitory factor (LIF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), bone morphogenetic proteins (BMPs), C-type natriuretic peptide (CNP), or any combination thereof. In some embodiments, the one or more growth factors are secreted from the OSCs. In some embodiments, the OSCs are steroidogenic and produce hormones including estradiol and / or progesterone. In some embodiments, the OSCs are steroidogenic in the presence of an exogenously supplied reagent. In some embodiments, the OSCs such as granulosa cells produce estradiol and / or progesterone upon stimulation of androstenedione and FSH and / or forskolin. In some embodiments, secretion of estradiol and / or progesterone may be detected or measured by one or more protein detection methods known in the art. In some embodiments, application of doxycycline to the OSCs maintains cell identity and drives steroidogenic activity. In some embodiments, doxycycline is applied to the OSCs or PATENT

[0352] Attorney Docket No.: 51763-009WO2

[0353] OSC media upon thawing and seeding OSCs derived from reprogrammed iPSCs after cryopreservation and frozen storage (e.g., for doxycycline-dependent induction).

[0354] B. Cell culture media

[0355] OSCs derived from iPSCs (e.g., hiPSCs) or transgenic OSCs may be provided as a composition further containing a cell culture medium. Said OSCs or precursors (e.g., hiPSCs prior to reprogramming) may be cultivated in a cell culture medium. In some embodiments, the engineered OSCs can be added to a commercially available reproductive media (e.g., IVF, IVM, (e.g., MEDICULT IVM® medium), or LAG medium). In some embodiments, cell culture medium comprises DMEM / F12 supplemented with Knockout Serum Replacement (KSR). In some embodiments, the cell culture medium comprises L-glutamine analogs such as, e.g., GLUTAMAX™ (GIBCO™, Thermo Fisher Scientific, Waltham, MA), optionally wherein the GLUTAMAX™ has been adapted to use animal origin-free reagents. In some embodiments, an embryology lab procures a suitable IVF cell culture medium. In some embodiments, the cell culture medium comprises Medicult IVM medium. In some embodiments, an embryology lab procures a suitable cell culture plate. In some embodiments, the cell culture plate is a GPS Universal dish. In some embodiments, an embryology lab procures an ART-grade mineral oil. In some embodiments, the coculture is achieved by preparation of an IVM media. In some embodiments, the IVM medium comprises a base medium formulation. In some embodiments, the base medium formulation comprises MEDICULT IVM® medium.

[0356] The cell culture medium may comprise a supplement or additive to promote differentiation of hiSPCs into a population of OSCs. The supplement or additive may influence the cell fate of the population of OSCs and / or maintain cell identity of the population of OSCs.

[0357] In some embodiments, the cell culture medium comprises an alkanethiol at a concentration of about 0.1 mM to about 1 mM (e.g., about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, or about 1 mM). In some embodiments, the alkanethiol is beta-mercaptoethanol.

[0358] In some embodiments, the cell culture medium comprises an antioxidant or a metabolite that promotes cell proliferation and / or survival. In some embodiments the antioxidant is present in the cell culture medium at a concentration of about 1 mM to about 4 mM (e.g., about 1 mM, about 2 mM, about 3 mM, or about 4 mM). In some embodiments the antioxidant is sodium pyruvate or pyruvic acid.

[0359] In some embodiments, the cell culture medium comprises a metabolite that promotes cell proliferation and / or survival, such as a nonessential amino acid, at a concentration of about 0.1 mM to about 2 mM (e.g., about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 mM, about 1.1 mM, about 1 .2 mM, about 1 .3 mM, about 1 .4 mM, about 1 .5 mM, about 1 .6 mM, about 1 .7 mM, about 1 .8 mM, about 1 .9 mM, or about 2 mM). In some embodiments, the nonessential amino acid is added to the media as a supplement comprising a mixture of one or more nonessential amino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, and / or tyrosine).

[0360] In some embodiments, the cell culture medium comprises a BMP at a concentration of about 1 ng / mL to about 10 ng / mL (e.g., about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, or about 10 ng / mL). In some embodiments, the BMP is BMP1 , BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMP9, PATENT

[0361] Attorney Docket No.: 51763-009WO2

[0362] BMP10, BMP 12, BMP13, BMP14, BMP15, BMP16, or a combination thereof. In some embodiments, the BMP is BMP2 and / or BMP4. In some embodiments, the BMP is BMP5, BMP6, BMP7 and / or BMP8. In some embodiments, the BMP is BMP9 and / or BMP10. In some embodiments, the BMP is BMP12, BMP13, and / or BMP14. In some embodiments, the BMP is a combination of BMP4 and one or more additional BMPs. In some embodiments, the BMP is BMP4.

[0363] In some embodiments, the cell culture medium comprises a fibroblast growth factor (FGF) at a concentration of about 1 to about 100 ng / mL (e.g., about 1 ng / mL to about 10 ng / mL, about 10 ng / mL to about 20 ng / mL, about 20 ng / mL to about 30 ng / mL, about 30 ng / mL to about 40 ng / mL, about 40 ng / mL about 50 ng / mL, about 50 ng / mL to about 60 ng / mL, about 60 ng / mL to about 70 ng / mL, about 70 ng / mL to about 80 ng / mL, about 80 ng / mL to about 90 ng / mL, or about 90 ng / mL to about 100 ng / mL). In some embodiments the FGF is FGF1 , FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21 , FGF22, FGF23, or a combination thereof. In some embodiments, the FGF is FGF1 and / or FGF2. In some embodiments, the FGF is FGF3, FGF7, FGF10, and / or FGF22. In some embodiments, the FGF is FGF4, FGF5, and / or FGF6. In some embodiments, the FGF is FGF8, FGF17, and / or FGF18. In some embodiments, the FGF is FGF9, FGF16, and / or FGF20. In some embodiments, the FGF is FGF21 and / or FGF23. In some embodiments, the FGF is a combination of FGF9 and one or more additional FGFs. In some embodiments, the FGF is FGF9.

[0364] In some embodiments, the cell culture medium comprises an insulin-like growth factor binding protein (IGFBP) at a concentration of about 1 ng / mL to about 100 ng / mL (e.g., about 1 ng / mL to about 10 ng / mL, about 10 ng / mL to about 20 ng / mL, about 20 ng / mL to about 30 ng / mL, about 30 ng / mL to about 40 ng / mL, about 40 ng / mL about 50 ng / mL, about 50 ng / mL to about 60 ng / mL, about 60 ng / mL to about 70 ng / mL, about 70 ng / mL to about 80 ng / mL, about 80 ng / mL to about 90 ng / mL, or about 90 ng / mL to about 100 ng / mL). In some embodiments, the IGFBP is IGFBP1 , IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6, or a combination thereof.

[0365] In some embodiments, the cell culture medium comprises an epidermal growth factor (EGF) and / or a transforming growth factor (TGF) at a concentration of about 1 ng / mL to about 100 ng / mL (e.g., about 1 ng / mL to about 10 ng / mL, about 10 ng / mL to about 20 ng / mL, about 20 ng / mL to about 30 ng / mL, about 30 ng / mL to about 40 ng / mL, about 40 ng / mL about 50 ng / mL, about 50 ng / mL to about 60 ng / mL, about 60 ng / mL to about 70 ng / mL, about 70 ng / mL to about 80 ng / mL, about 80 ng / mL to about 90 ng / mL, or about 90 ng / mL to about 100 ng / mL). In some embodiments, the EGF is EGF and / or amphiregulin. In some embodiments, the TGF is TGF-a, TGFpl , TGFp2, TGFp3, or a combination thereof.

[0366] In some embodiments, one or more supplemented proteins are recombinant proteins.

[0367] In some embodiments, the cell culture medium comprises an agent that inhibits the MAPK pathway at a concentration of about 1 nM to about 10 pM (e.g., about 1 nM to about 10 nM, about 10 nM to about 50 nM, about 50 nM to about 100 nM, about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 pM, about 1 pM to about 2 pM, about 2 pM to about 3 pM, about 3 pM to about 4 pM, about 4 pM to about 5 pM, about 5 pM to about 6 pM, about 6 pM to about 7 pM, about 7 pM to about 8 pM, about 8 pM to about 9 pM, about 9 pM to about 10 pM). In some embodiments, the MAPK pathway inhibitor is a small molecule such as PD0325901 (CAS No. 391210-10-9). PATENT

[0368] Attorney Docket No.: 51763-009WO2

[0369] In some embodiments, the cell culture medium comprises an agent that activates the sonic hedgehog pathway at a concentration of about 0.1 pM to about 1 pM (e.g., about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, or about 1 pM). In some embodiments, the sonic hedgehog pathway activator is a smoothened agonist (CAS No. 364590-63-6).

[0370] In some embodiments, the cell culture medium comprises an agent that inhibits the BMP pathway at a concentration of about 5 nM to about 10 nM (e.g., about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, or about 10 nM). In some embodiments, the BMP pathway inhibitor of ALK2 and ALK3. In some embodiments, the BMP pathway inhibitor is LDN-193189 (CAS No. 1062368-24-4).

[0371] In some embodiments, the cell culture medium is supplemented with an agent that that activates Wnt / p-catenin signaling at a concentration of about 1 pM to about 20 pM (e.g., about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, about 10 pM, about 11 pM, about 12 pM, about 13 pM, about 14 pM, about 15 pM, about 16 pM, about 17 pM, about 18 pM, about 19 pM, or about 20 pM. A Wnt pathway activator may be a ROCK inhibitor or a GSK3 inhibitor. In some embodiments, the Wnt pathway activator is CHIR99021 (CAS No. 252917-06-9).

[0372] In some embodiments, the hiPSCs and / or the resulting OSCs described herein are cultured on a matrix or encapsulated during induction, culturing, or co-culturing with another cell (e.g., a germ cell, e.g., an oocyte). In some embodiments, the hiPSCs and / or the OSCs are cultured on a matrix or encapsulated in a matrix comprising alginate. In some embodiments, the hiPSCs and / or the OSCs are cultured on a matrix or encapsulated in a matrix comprising laminin. In some embodiments, the hiPSCs and / or the OSCs are cultured on a matrix or encapsulated in a matrix comprising laminin-521 . In some embodiments, the hiPSCs and / or the OSCs are cultured on a matrix or encapsulated in a matrix comprising vitronectin. In some embodiments, the hiPSCs and / or the OSCs are cultured on a matrix or encapsulated in a matrix comprising collagen. In some embodiments, the hiPSCs and / or the OSCs are cultured on a matrix or encapsulated in a matrix comprising chitosan. In some embodiments, the hiPSCs and / or the OSCs are cultured on a matrix or encapsulated in a matrix comprising hyaluronic acid. In some embodiments, the hiPSCs and / or the OSCs are cultured on a matrix or encapsulated in a matrix comprising dextran hydrogel. In some embodiments, the hiPSCs and / or the OSCs are cultured on a matrix or encapsulated in matrix comprising a MATRIGEL® matrix.

[0373] In some embodiments, the supplemented cell culture media forms an in vitro maturation (IVM) media. In some embodiments, the IVM media is utilized by placement of about 100 pL of the media into the suitable cell culture dish, with a mineral oil overlay the day before oocyte retrieval from a subject. In some embodiments, about 2 to about 4 hours prior to IVM culture, the engineered OSCs are thawed. In some embodiments, the thawed engineered OSCs are centrifuged. In some embodiments, the engineered OSCs are washed with IVM media. In some embodiments, the engineered OSCs are seeded to a cell culture droplet. In some embodiments, the engineered OSCs are seeded at a final concentration of about 1 ,000 cells per 1 pl (e.g., about 500-1 ,000 cells / pL, about 700-1 ,000 cells / pL, about 1 ,000-1 ,200 cells / pL, about 1 ,000-1 ,500 cells / pL, or about 1 ,000-2,000 cells / pL).

[0374] C. Production and storage of ovarian support cells

[0375] In some embodiments, one or more OSCs described herein may be produced in multiple batches. In some embodiments, the OSCs may be frozen and thawed prior to co-culture methods. In PATENT

[0376] Attorney Docket No.: 51763-009WO2 some embodiments, the OSCs are freshly reprogrammed from a population of iPSCs prior to an in vitro maturation method. In some embodiments, the OSCs may be seeded and equilibrated for 2-8 hours (e.g., 2-3 hours, 2-4 hours, 3-4 hours, 4-6 hours, 5-7 hours, 6-8 hours; e.g., 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours) before the addition of oocytes for in vitro maturation. In some embodiments, the OSCs may be seeded and equilibrated for about 25-90 minutes (e.g., about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, or about 90 minutes).

[0377] In some embodiments, a subject may donate hiPSCs. A subject that donates hiPSCs may have or be at risk of ovarian decline or a disorder that causes or leads to reproductive dysfunction, such as one of the disorders described herein. hiPSCs donation may precede application or administration of an implant comprising a population of engineered OSCs. A subject participating in hiPSCs donation may be different, or the same, from the subject receiving the implant. hiPSCs donation may follow an oocyte retrieval process such as an oocyte retrieved for in vitro maturation. A subject participating in hiPSCs donation may be different, or the same, from the subject from whom the one or more oocytes were retrieved. In some embodiments, a hiPSC donor may undergo a stimulation protocol to stimulate oocyte release for oocyte retrieval. Select hiPSC clones may be expanded to generate an intermediate cell bank. Select hiPSC clones may be expanded and fully differentiated for immediate use.

[0378] In some embodiments, the hiPSCs (e.g., undifferentiated hiPSCs; e.g., intermediate cell banks) or the engineered OSCs described herein are provided in a cryovial. In some embodiments, the cryovial is composed of -125,000 cells (e.g., hiPSCs or OSCs). In some embodiments, the hiPSCs or the engineered OSCs are suspended in a cryoprotectant solution. In some embodiments, the cryoprotectant solution comprises CryoStor CS10. In some embodiments, the cryovial is a plastic vial. In some embodiments, the cryovial is an internal thread liquid nitrogen-suited plastic vial. In some embodiments, the hiPSCs or the engineered OSCs are provided in one or more aggregates. In some embodiments, the hiPSCs or the engineered OSCs are provided in one or more single cell suspensions.

[0379] In some embodiments, the hiPSCs or the engineered OSCs (e.g., OSCs produced from reprogrammed hiPSCs) are prepared and then frozen or cryopreserved for later use. Cryopreservation or freezing methods may include using a cryoprotective agent such as dimethyl sulfoxide and / or any other freezing method known in the art. In some embodiments, the hiPSCs or the engineered OSCs are stored in liquid nitrogen. In some embodiments, the hiPSCs or the engineered OSCs are stored in liquid nitrogen until use. In some embodiments, cryopreserved undifferentiated hiPSCs (e.g., intermediate cell banks) are stored in liquid nitrogen. Undifferentiated hiPSCs (e.g., intermediate cell banks) can be thawed for further differentiation steps. In some embodiments, the engineered OSCs are stored in liquid nitrogen until use for IVM methods and applications.

[0380] In some embodiments, frozen cells are submitted to different sets of batch release assays, in which cells undergo one or more tests to determine cell quality. In some embodiments, one or more aliquots of frozen cells undergo a panel of tests to determine cell count and / or viability upon thawing, to verify genetic stability, to verify genotype, to confirm sterility, or a combination thereof. One or more aliquots of undifferentiated cells (e.g., undifferentiated hiPSCs; e.g., an intermediate cell bank) may further undergo a panel of tests to measure the presence and / or relative abundance of one or more pluripotency markers (e.g., NANOG, POU5F1 , SOX2, OCT4). One or more aliquots of one or more OSC PATENT

[0381] Attorney Docket No.: 51763-009WO2 populations (e.g., final target cell banks) may undergo a panel of tests to measure the presence and / or relative abundance of a marker associated with one or more types of OSCs (e.g., a granulosa or a stroma cell) or the potency of secreted proteins or steroids (e.g., estradiol and / or progesterone production). In further embodiments, one or more aliquots of frozen cells undergo tests to determine risks of embryo toxicity (e.g., the presence of one or more chemicals or agents that may disrupt normal growth, development, or genetic differentiation of an embryo).

[0382] D. Production of in vivo implants from ovarian support cells

[0383] A population of OSCs produced by a progenitor cell (e.g., a hiPSC), such as any one of the OSCs described herein (e.g., theca cells, lutein cells, granulosa cells, and / or stroma cells), may be further assembled as an implant that comprises a plurality of OSCs and proteinaceous components that replicate the structural and functional properties of female reproductive tissues and organs (e.g., ovarian and / or uterine tissues). An OSC implant may be delivered to a subject and regulate the subject’s cyclic gonadal hormone production (e.g., the production of estrogen (including estrone (E1), estradiol (E2), estriol (E3), estetrol (E4), or a combination thereof), progesterone, and / or androstenedione). Upon delivery to the subject, an OSC implant may respond to endogenous hormones and signals that are periodically released by the subject’s hypothalamus and pituitary gland on a cyclic basis (e.g., over the course of a typical 28-32 day menstrual cycle). Thus, such implants generated from one or more OSCs described herein leverage the natural, periodic secretion of hormones on the hypothalamic-pituitary-gonadal (HPG) axis in order to supplement - or altogether recapitulate - ovarian activity. Without being limited by mechanism, in this way, the implants of the disclosure may ameliorate ovarian decline or reproductive dysfunction in a subject. By responding to the body’s natural HPG axis fluctuations, OSC implants herein offer a new and improved form of personalized treatment to a wide variety of subjects seeking to supplement ovarian function or ameliorate ovarian decline. The compositions and method of the disclosure may reduce the need for continuous medical interventions, such as hormonal injection therapies, thereby reducing certain health risks that are associated with traditional hormone replacement therapy. Chief among these risks is the risk of cancer that is associated with continuous exposure to high levels of exogenously injected hormones.

[0384] / '. Components of an ovarian support cell implant

[0385] An OSC implant may include theca cells, granulosa cells, stroma cells and / or ovarian lutein cells, optionally in combination with one or more additional cell types. The distribution of cells in an OSC implant may depend on a subject’s particular hormonal, biological, or physiological needs, which, if necessary, may be determined by a skilled practitioner (e.g., a physician, an OB / GYN, a nurse practitioner, or another skilled professional). In some embodiments, an OSC implant comprises an equal or relatively equal distribution of theca, granulosa, and lutein cells. In some embodiments, an OSC implant comprises a distribution of cells in which one cell type is more abundant than the other cell types in the population. In other embodiments, an OSC implant comprises a distribution of cells in which each cell type is represented at a different relative abundance such that no two cell types have an equivalent relative distribution.

[0386] An OSC implant may include a predetermined number of ovarian theca cells for implantation. In some embodiments, theca cells in an OSC are implanted at a range between 1 x 106and 1 x 107cells PATENT

[0387] Attorney Docket No.: 51763-009WO2

[0388] (e.g., between 1 x 106and 2 x 106, between 2 x 106and 3 x 106, between 3 x 106and 4 x 106, between 4 x 106and 5 x 106, between 5 x 106and 6 x 106, between 6 x 106and 7 x 106, between 7 x 106and 8 x 106, between 8 x 106and 9 x 106, or between 9 x 106and 1 x 107; e.g., about 1 x 106cells, about 2 x 106cells, about 3 x 106cells, about 4 x 106cells, about 5 x 106cells, about 6 x 106cells, about 7 x 106cells, about 8 x 106cells, about 9 x 106cells, about 1 x 107cells). In the ovary, theca cells convert cholesterol to androstenedione, which is the substrate for estradiol production in granulosa cells. An ovarian theca cell in an OSC implant may secrete androstenedione during the luteal phase and / or ovulation described herein.

[0389] An OSC implant may include a predetermined number of granulosa cells for implantation. In some embodiments, granulosa cells in an OSC are implanted at a range between 1 x 106and 1 x 107cells (e.g., between 1 x 106and 2 x 106, between 2 x 106and 3 x 106, between 3 x 106and 4 x 106, between 4 x 106and 5 x 106, between 5 x 106and 6 x 106, between 6 x 106and 7 x 106, between 7 x 106and 8 x 106, between 8 x 106and 9 x 106, or between 9 x 106and 1 x 107; e.g., about 1 x 106cells, about 2 x 106cells, about 3 x 106cells, about 4 x 106cells, about 5 x 106cells, about 6 x 106cells, about 7 x 106cells, about 8 x 106cells, about 9 x 106cells, about 1 x 107cells). A granulosa cell is a cumulus cell surrounding the oocyte to ensure healthy oocyte and subsequent embryo development. An ovarian granulosa cell may be a steroidogenic granulosa cell. An ovarian granulosa cell in an OSC implant may secrete levels of E2 consistent with various phases of the menstrual cycle in healthy, pre-menopausal ovaries as described herein.

[0390] An OSC implant may include a predetermined number of lutein cells for implantation. In some embodiments, lutein cells in an OSC are implanted at a range between 1 x 106and 1 x 107cells (e.g., between 1 x 106and 2 x 106, between 2 x 106and 3 x 106, between 3 x 106and 4 x 106, between 4 x 106and 5 x 106, between 5 x 106and 6 x 106, between 6 x 106and 7 x 106, between 7 x 106and 8 x 106, between 8 x 106and 9 x 106, or between 9 x 106and 1 x 107; e.g., about 1 x 106cells, about 2 x 106cells, about 3 x 106cells, about 4 x 106cells, about 5 x 106cells, about 6 x 106cells, about 7 x 106cells, about 8 x 106cells, about 9 x 106cells, about 1 x 107cells). A lutein cell secretes progesterone during the luteal phase of the menstrual cycle at levels described in detail in the following section.

[0391] An OSC implant may include a predetermined number of ovarian stroma cells for implantation. In some embodiments, stroma cells in an OSC are implanted at a range between 1 x 106and 1 x 107cells (e.g., between 1 x 106and 2 x 106, between 2 x 106and 3 x 106, between 3 x 106and 4 x 106, between 4 x 106and 5 x 106, between 5 x 106and 6 x 106, between 6 x 106and 7 x 106, between 7 x 106and 8 x 106, between 8 x 106and 9 x 106, or between 9 x 106and 1 x 107; e.g., about 1 x 106cells, about 2 x 106cells, about 3 x 106cells, about 4 x 106cells, about 5 x 106cells, about 6 x 106cells, about 7 x 106cells, about 8 x 106cells, about 9 x 106cells, about 1 x 107cells).

[0392] An OSC implant may comprise inactive ingredients, including extracellular matrix (ECM) components that provide biochemical and biomechanical structure and support for the active cellular components. In preferred embodiments, the inactive ingredients of the OSC implant (such as the one or more ECM components) encapsulate the OSCs, such that the OSCs are embedded into the inactive ingredients.

[0393] In some embodiments, an OSC implant comprises a population of OSCs that are embedded into an ECM comprising one or more ECM proteins and associated molecules such as binding partners or ions (e.g., divalent ions; e.g., calcium ions and / or magnesium ions). In some embodiments, an OSC PATENT

[0394] Attorney Docket No.: 51763-009WO2 implant comprises ECM proteins, protein polymers, and molecules that can be found in connective tissue. In some embodiments, the ECM comprises one or more types of collagen (e.g., fibrillar collagen; e.g., collagen I, II, III, V, XI), one or more epidermal growth factors (EGFs), elastin (e.g., tropoelastin or mature elastin), fibronectin, vitronectin, laminin, among other glycoproteins, cell adhesion proteins, or plant- derived proteins or protein polymers (e.g., alginate). In some embodiments, the ECM comprises particles or polymers that range from 100 nm to 100 pm in size (e.g., 100 to 200 nm, 200 to 300 nm, 300 to 400 nm, 400 to 500 nm, 500 to 600 nm, 600 to 700 nm, 700 to 800 nm, 800 to 900 nm, 900 nm to 1 pm, 1 to 10 pm, 10 to 20 pm, 20 to 30 pm, 30 to 40 pm, 40 to 50 pm, 50 to 60 pm, 60 to 70 pm, 70 to 80 pm, 80 to 90 pm, or 90 to 100 pm). In some embodiments, the ECM comprises particles or polymers that range from 100 to 500 pm in size (e.g., 100 to 150 pm, 150 to 200 pm, 200 to 250 pm, 250 to 300 pm, 300 to 350 pm, 350 to 400 pm, 400 to 450 pm, or 450 to 500 pm). In some embodiments, the ECM comprises particles or polymers ranging from 500 to 800 pm in size (e.g., 500 to 550 pm, 550 to 600 pm, 600 to 650 pm, 650 to 700 pm, 700 to 750 pm, or 750 to 800 pm; e.g., 500 pm, 510 pm, 520 pm, 530 pm, 540 pm, 550 pm, 560 pm, 570 pm, 580 pm, 590 pm, 600 pm, 610 pm, 620 pm, 630 pm, 640 pm, 650 pm, 660 pm, 670 pm, 680 pm, 690 pm, 700 pm, 710 pm, 720 pm, 730 pm, 74 pm, 750 pm, 760 pm, 770 pm, 780 pm, 790 pm, or 800 pm). Other embodiments of an ECM of an OSC implant include proteoglycans, heparan sulfate, chondroitin sulfate, keratan sulfate, hyaluronic acid, elastin, dermatan sulfate, extracellular vesicles, nanoparticles, microparticles, proteins, cell adhesion proteins, proteoglycans, carbohydrate polymers, non-proteoglycan polysaccharides, and other forms of substrates one skilled in the art may understand as applicable to embedding and supporting an OSC implant. In some embodiments, one or more ECM components are derived from purified or partially purified animal or plant tissue. In other embodiments, one or more ECM components are derived from in vitro cell culturing conditions.

[0395] In some embodiments, an OSC implant is further encapsulated as a microparticle or a plurality of microparticles following embedding into one or more ECM components. In some embodiments, the microparticle is a spherical microparticle (e.g., a microsphere) and has a diameter that ranges from 100 nm to 100 pm in size (e.g., 100 to 200 nm, 200 to 300 nm, 300 to 400 nm, 400 to 500 nm, 500 to 600 nm, 600 to 700 nm, 700 to 800 nm, 800 to 900 nm, 900 nm to 1 pm, 1 to 10 pm, 10 to 20 pm, 20 to 30 pm, 30 to 40 pm, 40 to 50 pm, 50 to 60 pm, 60 to 70 pm, 70 to 80 pm, 80 to 90 pm, or 90 to 100 pm). A microparticle is comprised of a core substance that is enclosed by a membrane or shell. In some embodiments, a microparticle is enclosed by more than one membrane or shell (e.g., 2 or 3 membranes). In some embodiments, the core substance comprises one or more ECM components (e.g., one or more ECM components described herein) as a homogenous matrix or as a heterogenous matrix. In some embodiments, the membrane or shell comprises one or more ECM components and / or a synthetic polymer (e.g., a water repelling material including a-hydroxy acid) such as polyvinylchloride (PVC), polyethylene (PE), polypropylene (PP), polymethyl methacrylate (PMMA), polystyrene (PS), polytetrafluoroethylene (PTFE), polyurethane (PU), polyamide (nylon), polyethylene terephthalate (PET), polyether sulfone (PES), or a combination thereof. In some embodiments, the membrane or shell comprises one or more synthetic amino acid polymers or amino acid polymers (e.g., poly-L-ornithine (PLO) or poly-L-lysine (PLL)). In some embodiments, a microparticle is formed by a core substance and a membrane or a shell, in which the active ingredients (e.g., the OSCs) are dispersed throughout the matrix. PATENT

[0396] Attorney Docket No.: 51763-009WO2

[0397] As an example, an OSC implant may be assembled as a plurality of microparticles in which each microparticle comprises a population of OSCs encapsulated in an alginate matrix core, a first shell comprising poly-L-ornithine, and an outer shell comprising alginate. The OSC implant in this configuration may then be further assembled into a device for delivery to a subject (see, e.g., Section 11(A)) .

[0398] In some embodiments, following embedding the OSCs into one or more ECM components and / or a microparticle, the OSC implant is further assembled to comprise one or more additional materials for delivery (e.g., a delivery apparatus). In preferred embodiments, a delivery apparatus consists of biocompatible materials such that the delivery apparatus is non-toxic to cells or tissues (e.g., the OSC implant and / or cells or tissues of a subject following delivery) or has a low risk of inducing an immune response following delivery of the OSC implant to a subject. Exemplary biocompatible materials are ECM components (such as any one of the ECM components described above), a synthetic polymer (e.g., a water-repelling material including an a-hydroxy acid), PVC, PE, PP, PMMA, PS, PTFE, PU, nylon, PET, PES, or a combination thereof. Other exemplary biocompatible materials are composite material (e.g., plastic fiberglass or bone), ceramic material (e.g., alumina (AI2O3), biocompatible zirconia (ZrOz), or bioglass (NazOCaOPzOs-SiO)), and other biocompatible materials known in the art. Further considerations for inactive ingredients for encapsulation of cells for therapeutic purposes are described elsewhere, such as, e.g., Liu et al. Engineering. 13:53-70, 2022 and Fanse et al. Adv Drug Deliv Rev. 191 :114581 , 2022, each of which is hereby incorporated by reference.

[0399] In some embodiments, the materials that encapsulate an OSC implant are porous (e.g., semi- permeable) to permit the bi-directional flow of molecules following delivery. Particularly, the porosity of the OSC implant enables the OSCs to have contact and respond to hormones and signals that are released by the subject’s hypothalamus and pituitary gland (e.g., endogenous signals over the course of a typical 28-32 day menstrual cycle) by releasing one or more hormones or steroids (e.g., progesterone, one or more androgens (including androstenedione, testosterone, dihydrotestosterone, or a combination thereof), and / or estrogen (including estrone (E1), estradiol (E2), estriol (E3), estetrol (E4), or a combination thereof). In some embodiments, the pore size of the materials that encapsulate the OSCs (e.g., the one or more ECM components, a microparticle (including the core and the shell), and / or a delivery apparatus) is about 10 nm to 500 nm (e.g., 10 to 50 nm, 50 to 100 nm, 100 to 150 nm, 150 to 200 nm, 200 to 250 nm, 250 to 300 nm, 300 to 350 nm, 350 to 400 nm, 400 to 450 nm, or 450 to 500 nm; e.g., about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, about 200 nm, about 210 nm, about 220 nm, about 230 nm, about 240 nm, about 250 nm, about 260 nm, about 270 nm, about 280 nm, about 290 nm, about 300 nm, about 310 nm, about 320 nm, about 330 nm, about 340 nm, about 350 nm, about 360 nm, about 370 nm, about 380 nm, about 390 nm, about 400 nm, about 410, about 420, about

[0400] 430 nm, about 440 nm, about 450 nm, about 460 nm, about 470 nm, about 480 nm, about 490 nm, or about 500 nm). In some embodiments, the pore size of the materials that encapsulate the OSCs (e.g., the one or more ECM components, a microparticle (including the core and the shell), and / or a delivery apparatus) is 50 nm to 150 nm (e.g., 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm). In some embodiments, the porosity of each material that encapsulates the OSCs is uniform such that the one or more ECM components and / or the core and the shell of the microparticle have pore sizes PATENT

[0401] Attorney Docket No.: 51763-009WO2 that are the same or nearly the same (e.g., have a size within an acceptable range). In some embodiments, the porosity of each material that encapsulates the OSCs are non-uniform and have pores that are different sizes.

[0402] Encapsulation of an OSC implant may serve several purposes. Encapsulation of an OSC implant may protect the OSCs during delivery to a subject. For instance, encapsulation may reduce the effect of shear forces applied to the membrane of an OSC during delivery via injection and thus reduce the likelihood of the OSC lysing or bursting during delivery. Encapsulation of an OSC implant sequesters the population of OSCs and may prevent diffusion of the OSCs into a surrounding area (e.g., an adjacent area or surrounding tissue following implantation). Reducing the likelihood of OSC diffusion via encapsulation of an OSC implant maintains the local concentration of OSCs in the implant and may maintain expression of a growth factor or a hormone more effectively compared to a population of OSCs that are not encapsulated prior to delivery to a subject. In addition to maintaining the concentration of OSCs in the implant, encapsulation may further reduce the likelihood of migration of the implant following delivery to a subject, such that a skilled practitioner may easily retrieve the OSC implant for removal or replacement. Encapsulation of the OSC implant may shield the OSCs from degradation following implantation into a subject by reducing contact of the OSCs with factors that promote cell death or degradation including proteolytic enzymes, cytokines, and / or immune cells. Considerations for delivery of an OSC implant to a subject, including delivery modalities, are described in further detail in Section I l(A) , below.

[0403] / ' / . Cyclic hormone release

[0404] Cyclic hormone release refers to the physiological phenomenon of natural fluctuations of hormone secretions in the body over a period of time. Upon ovarian decline, the ovarian cells have a reduced capacity of responding to the body’s continued cyclic release of hormones. Cyclic hormone release is controlled by the hypothalamus and pituitary gland and results in temporally increased secretion of FSH, LH, and gonadotropin-releasing hormone (GnRH). Upon administration of an OSC implant, the implant may help to restore the subject’s ability to respond to cyclic fluctuations in HPG axis hormones by providing a synchronized, time-dependent release of estrogen, progesterone, and androstenedione in a manner that approximates the cyclic secretion of these hormones by a healthy, premenopausal subject.

[0405] The OSC may work in tandem with the ovaries to produce sufficient hormone levels that can be measured using methods known in the art. For example, a subject in ovarian decline may still produce estrogen but not at levels sufficient for fertility, and the OSC implant may secrete a dose of estrogen that matches the levels of healthy, pre-menopausal ovaries. In some embodiments, the implant may be the sole source of the hormone. In some embodiments, the implant recapitulates the variable, fluctuating estrogen levels during the different phases of the menstrual and follicular cycles. In some embodiments, the OSC implant secretes estradiol levels that are consistent with the early to mid-follicular phase (i.e. , the first week of the menstrual cycle or menses) and range from 20 to 80 pg / mL (e.g., 20 to 30 pg / mL, 30 to 40 pg / mL, 40 to 50 pg / mL, 50 to 60 pg / mL, 60 to 70 pg / mL, or 70 to 80 pg / mL; e.g., 20 pg / mL, 21 pg / mL, 22 pg / mL, 23 pg / mL, 24 pg / mL, 25 pg / mL, 26 pg / mL, 27 pg / mL, 28 pg / mL, 29 pg / mL, 30 pg / mL, 35 pg / mL, 40 pg / mL, 45 pg / mL, 50 pg / mL, 55 pg / mL, 60 pg / mL, 65 pg / mL, 70 pg / mL, 75 pg / mL, or 80 pg / mL). PATENT

[0406] Attorney Docket No.: 51763-009WO2

[0407] In some embodiments, the OSC implant secretes gradually increasing levels of estradiol consistent with the mid to late follicular phase (i.e., the second week of the menstrual cycle) until the preovulatory phase. In some embodiments, the OSC implant secretes levels of estradiol consistent with the mid to late follicular phase at level between 20 and 200 pg / mL (e.g., between 20 and 50 pg / mL, between 50 and 70 pg / mL, between 70 and 90 pg / mL, between 90 and 1 10 pg / mL, between 110 and 130 pg / mL, between 130 and 150 pg / mL, between 150 and 170 pg / mL, or between 170 and 200 pg / mL; e.g., 20 pg / mL, 21 pg / mL, 22 pg / mL, 23 pg / mL, 24 pg / mL, 25 pg / mL, 26 pg / mL, 27 pg / mL, 28 pg / mL, 29 pg / mL, 30 pg / mL, 35 pg / mL, 40 pg / mL, 45 pg / mL, 50 pg / mL, 55 pg / mL, 60 pg / mL, 65 pg / mL, 70 pg / mL, 75 pg / mL, 80 pg / mL, 90 pg / mL, 100 pg / mL, 110 pg / mL, 120 pg / mL, 130 pg / mL, 140 pg / mL, 150 pg / mL, 160 pg / mL, 170 pg / mL, 180 pg / mL, 190 pg / mL, or 200 pg / mL).

[0408] At the time of pre-ovulation (a period of about 24 to 48 hours), estradiol levels may rise. In some embodiments, the OSC implant secretes estradiol levels consistent with the pre-ovulation phase at a level ranging from 130 to 200 pg / mL (e.g., 130 to 140 pg / mL, 140 to 150 pg / mL, 150 to 160 pg / mL, 160 to 170 pg / mL, 170 to 180 pg / mL, 180 to 190 pg / mL, 190 to 200 pg / mL, or above 200 pg / mL; e.g., 130 pg / mL, 131 pg / mL, 132 pg / mL, 133 pg / mL, 134 pg / mL, 135 pg / mL, 136 pg / mL, 137 pg / mL, 138 pg / mL, 139 pg / mL, 140 pg / mL, 145 pg / mL, 150 pg / mL, 155 pg / mL, 160 pg / mL, 165 pg / mL, 170 pg / mL, 175 pg / mL, 180 pg / mL, 185 pg / mL, 190 pg / mL, 195 pg / mL, 200 pg / mL, or higher). In some embodiments, the OSC implant secretes estradiol levels consistent with higher circulating levels of estradiol observed in some subjects during the pre-ovulation phase, such that the OSC implant secretes levels of estradiol between 300 and 400 pg / mL (e.g., 300 to 310 pg / mL, 310 to 320 pg / mL, 320 to 330 pg / mL, 330 to 340 pg / mL, 340 to 350 pg / mL, 350 to 360 pg / mL, 360 to 370 pg / mL, 370 to 380 pg / mL, 380 to 390 pg / mL, 390 to 400 pg / mL, or above 400 pg / mL; e.g., 300 pg / mL, 301 pg / mL, 302 pg / mL, 303 pg / mL, 304 pg / mL, 305 pg / mL, 306 pg / mL, 307 pg / mL, 308 pg / mL, 309 pg / mL, 310 pg / mL, 315 pg / mL, 320 pg / mL, 325 pg / mL, 330 pg / mL, 335 pg / mL, 340 pg / mL, 345 pg / mL, 350 pg / mL, 355 pg / mL, 360 pg / mL, 365 pg / mL, 370 pg / mL, 375 pg / mL, 380 pg / mL, 385 pg / mL, 390 pg / mL, 395 pg / mL, 400 pg / mL, or higher).

[0409] At the time of ovulation, concentrations of secreted estradiol may surge. In some embodiments, the OSC implant secretes estradiol levels consistent with an ovulatory surge between 250 and 500 pg / mL (e.g., between 250 and 275 pg / mL, between 275 and 300 pg / mL, between 300 and 325 pg / mL, between 325 and 350 pg / mL, between 350 and 375 pg / mL, between 375 and 400 pg / mL, between 400 and 425 pg / mL, between 425 and 450 pg / mL, between 450 and 475 pg / mL, between 475 and 500 pg / mL, or higher; e.g., 250 pg / mL, 260 pg / mL, 270 pg / mL, 280 pg / mL, 290 pg / mL, 300 pg / mL, 310 pg / mL, 320 pg / mL, 330 pg / mL, 340 pg / mL, 350 pg / mL, 360 pg / mL, 370 pg / mL, 380 pg / mL, 390 pg / mL, 400 pg / mL, 410 pg / mL, 420 pg / mL, 430 pg / mL, 440 pg / mL, 450 pg / mL, 460 pg / mL, 470 pg / mL, 480 pg / mL, 490 pg / mL, 500 pg / mL, or higher).

[0410] Following ovulation (or mid-cycle) and during the latter half of the menstrual cycle or the luteal phase, estradiol levels plateau and fluctuate during the early and mid-luteal phase. In some embodiments, the OSC implant secretes estradiol levels consistent with the early and mid-luteal phase at a range between 100 and 150 pg / mL (e.g., 100 to 110 pg / mL, 110 to 120 pg / mL, 120 to 130 pg / mL, 130 to 140 pg / mL, or 140 to 150 pg / mL; e.g., 100 pg / mL, 101 pg / mL, 102 pg / mL, 103 pg / mL, 104 pg / mL, 105 pg / mL, 106 pg / mL, 107 pg / mL, 108 pg / mL, 109 pg / mL, 110 pg / mL, 115 pg / mL, 120 pg / mL, 125 pg / mL, 130 pg / mL, 135 pg / mL, 140 pg / mL, 145 pg / mL, or 150 pg / mL). In other embodiments, the OSC implant secretes levels of estradiol consistent with that of the late luteal phase (i.e., a few days before PATENT

[0411] Attorney Docket No.: 51763-009WO2 menstruation or menses), such that the secreted level of estradiol is between 60 and 200 pg / mL (e.g., 60 and 80 pg / mL, between 80 and 100 pg / mL, between 100 and 120 pg / mL, between 120 and 140 pg / mL, between 140 and 160 pg / mL, between 160 and 180 pg / mL, between 180 and 200 pg / mL; e.g., 60 pg / mL, 61 pg / mL, 62 pg / mL, 63 pg / mL, 64 pg / mL, 65 pg / mL, 66 pg / mL, 67 pg / mL, 68 pg / mL, 69 pg / mL, 70 pg / mL, 75 pg / mL, 80 pg / mL, 85 pg / mL, 90 pg / mL, 95 pg / mL, 100 pg / mL, 105 pg / mL, 110 pg / mL, 115 pg / mL, 120 pg / mL, 125 pg / mL, 130 pg / mL, 135 pg / mL, 140 pg / mL, 145 pg / mL, 150 pg / mL, 155 pg / mL, 160 pg / mL, 165 pg / mL, 170 pg / mL, 175 pg / mL, 180 pg / mL, 185 pg / mL, 190 pg / mL, 195 pg / mL, or 200 pg / mL).

[0412] Progesterone levels are relatively low during the pre-ovulatory phase of the menstrual cycle, rise after ovulation, and are elevated during the luteal phase. Progesterone levels tend to be less than 2 ng / mL prior to ovulation and greater than 5 ng / mL after ovulation. In some embodiments, an OSC implant secretes progesterone at levels consistent with a pre-ovulatory phase such that an OSC secretes progesterone at levels less than 2 ng / mL (e.g., less than 0.1 ng / mL, between 0.1 and 0.5 ng / mL, between 0.5 and 1 ng / mL, between 1 .0 and 1 .5 ng / mL, or between 1 .5 and 1 .9 ng / mL; e.g., 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.6 ng / mL, 0.7 ng / mL, 0.8 ng / mL, 0.9 ng / mL, 1 .0 ng / mL, 1.1 ng / mL, 1 .2 ng / mL, 1 .3 ng / mL, 1 .4 ng / mL, 1 .5 ng / mL, 1 .6 ng / mL, 1 .7 ng / mL, 1 .8 ng / mL, or 1 .9 ng / mL). In other embodiments, an OSC implant secretes progesterone at levels consistent with post-ovulation phase at a range greater than 5 ng / mL (e.g., greater than 5 ng / mL, greater than 6 ng / mL, greater than 7 ng / mL, greater than 8 ng / mL, greater than 9 ng / mL, greater than 10 ng / mL, or higher). If pregnancy occurs, human chorionic gonadotropin is released, maintaining the corpus luteum and allowing it to maintain levels of progesterone. Between 7 and 9 weeks, the placenta begins to produce progesterone in place of the corpus luteum in a process called the luteal-placental shift.

[0413] Androstenedione levels are higher during the mid-follicular and luteal phases compared to the early follicular phase. In some embodiments, an OSC implant secrets androstenedione at levels between 1 and 3 ng / mL (e.g., between 1 and 1 .5 ng / mL, between 1 .5 and 2 ng / mL, between 2 and 2.5 ng / mL, or between 2.5 and 3 ng / mL; e.g., 1 ng / mL, 1 .5 ng / mL, 2 ng / mL, 2.5 ng / mL, or 3 ng / mL).

[0414] LH levels are normally low during childhood and, in women, are high after menopause. In some embodiments, the subject endogenously secretes LH consistent with levels observed during reproductive years, such as levels between 1 and 20 IU / L (e.g., between 1 and 5 IU / L, between 5 and 10 IU / L, between 10 and 15 IU / L, between 15 and 20 IU / L; e.g., about 1 IU / L, about 2 IU / L, about 3 IU / L, about 4 IU / L, about 5 IU / L, about 6 IU / L, about 7 IU / L, about 8 IU / L, about 9 IU / L, about 10 IU / L, about 11 IU / L, about 12 IU / L, about 13 IU / L, about 14 IU / L, about 15 IU / L, about 16 IU / L, about 17 IU / L, about 18 IU / L, about 19 IU / L, about 20 IU / L). Physiologic high LH levels are seen during the LH surge and typically last 48 hours.

[0415] FSH levels are typically measured in the early follicular phase of the menstrual cycle, day three to five, counted from last menstruation. At this time, the levels of estradiol (E2) and progesterone are at the lowest point of the menstrual cycle. FSH levels in this time is often called basal FSH levels, to distinguish from the increased levels when approaching ovulation. In some embodiments, the subject endogenously secretes levels of FSH consistent with secreted levels before ovulation at a range between

[0416] 3.8 to 8.8 mIU / mL (e.g., from 3.8 to 4.8 mIU / mL, from 4.8 to 5.8 mIU / mL, from 5.8 to 6.8 mIU / mL, from

[0417] 7.8 to 8.8. mIU / mL; e.g., 3.8 mIU / mL, 3.9 mIU / mL, 4.0 mIU / mL, 4.1 mIU / mL, 4.2 mIU / mL, 4.3 mIU / mL, 4.4 mIU / mL, 4.5 mIU / mL, 4.6 mIU / mL, 4.7 mIU / mL, 4.8 mIU / mL, 4.9 mIU / mL, 5.0 mIU / mL, 5.1 mIU / mL, 5.2 PATENT

[0418] Attorney Docket No.: 51763-009WO2 mIU / mL, 5.3 mIU / mL, 5.4 mIU / mL, 5.5 mIU / mL, 5.6 mIU / mL, 5.7 mIU / mL, 5.8 mIU / mL, 5.9 mIU / mL, 6.0 mIU / mL, 6.1 mIU / mL, 6.2 mIU / mL, 6.3 mIU / mL, 6.4 mIU / mL, 6.5 mIU / mL, 6.6 mIU / mL, 6.7 mIU / mL, or 6.8 mIU / mL). In some embodiments, the subject endogenously secretes FSH at levels consistent with levels secreted after ovulation between 1 .8 and 5.1 mIU / mL (e.g. between 1 .8 and 3.3 mIU / mL, between 3.3 and 3.8 mIU / mL, between 3.8 and 4.3 mIU / mL, between 4.3 and 4.8 mIU / mL, or between 4.8 and 5.1 mIU / mL; e.g., 1 .8 mIU / mL, 1 .9 mIU / mL, 2.0 mIU / mL, 2.1 mIU / mL, 2.3 mIU / mL, 2.4 mIU / mL, 2.5 mIU / mL, 2.6 mIU / mL, 2.7 mIU / mL, 2.8 mIU / mL, 3.1 mIU / mL, 3.5 mIU / mL, 3.8 mIU / mL, 4.1 mIU / mL, 4.5 mIU / mL, 4.8 mIU / mL, or 5.1 mIU / mL). In other embodiments, the subject endogenously secretes FSH at levels consistent with levels secreted in the middle of the menstrual cycle or menses at levels between 4.5 and 22.5 mIU / mL (e.g., between 4.5 and 9.5 mIU / mL, between 9.5 and 13.5 mIU / mL, between 13.5 and 18.5 mIU / mL, or between 18.5 and 22.5 mIU / mL; e.g., 4.5 mIU / mL, 5.0 mIU / mL, 5.5 mIU / mL, 6.0 mIU / mL, 6.5 mIU / mL, 7.0 mIU / mL, 7.5 mIU / mL, 8.0 mIU / mL, 8.5 mIU / mL, 9.0 mIU / mL, 9.5 mIU / mL, 10 mIU / mL, 10.5 mIU / mL, 1 1 mIU / mL, 1 1.5 mIU / mL, 12.0 mIU / mL, 12.5 mIU / mL, 13.0 mIU / mL, 13.5 mIU / mL, 14.0 mIU / mL, 14.5 mIU / mL, 15.0 mIU / mL, 15.5 mIU / mL, 16.0 mIU / mL, 16.5 mIU / mL, 17.0 mIU / mL, 17.5 mIU / mL, 18.0 mIU / mL, 18.5 mIU / mL, 19.0 mIU / mL, 19.5 mIU / mL, 20.0 mIU / mL, 20.5 mIU / mL, 21.0 mIU / mL, or 21.5 mIU / mL).

[0419] Regarding the cyclic dosing schedule, during the first week of subject’s menstrual cycle, and / or the follicular phase, the subject may secrete FSH in specific, characteristic amounts. The follicular phase starts on the first day of menstruation and ends with ovulation. Prompted by the hypothalamus, the pituitary gland releases FSH. This hormone stimulates the ovary to produce around five to 20 follicles (tiny nodules or cysts), which bead on the surface. In some embodiments, during the first week of subject’s menstrual cycle, and / or the follicular phase, the subject endogenously secretes FSH in an amount of from 1 .4 to 9.9 lU / mL (e.g., 1 .4 to 1 .9 lU / mL, 1 .9 to 2.4 lU / mL, 2.4 to 2.9 lU / mL, 2.9 to 3.4 lU / mL, 3.4 to 3.9 lU / mL, 3.9 to 4.5 lU / mL, 4.5 to 4.9 lU / mL, 4.9 to 5.4 lU / mL, 5.4 to 5.9 lU / mL, 5.9 to 6.4 lU / mL, 6.4 to 6.9 lU / mL, 6.9 to 7.4 lU / mL, 7.4 to 7.9 lU / mL, 7.9 to 8.4 lU / mL, 8.4 to 8.9 lU / mL, 8.9 to 9.4 lU / mL, or 9.4 to 9.9 lU / mL; e.g., 1 .4 lU / mL, 1 .5 lU / mL, 1 .6 lU / mL, 1 .7 lU / mL, 1 .8 lU / mL, 1 .9 lU / mL, 2.0 lU / mL, 2.1 lU / mL, 2.2 lU / mL, 2.3 lU / mL, 2.4 lU / mL, 2.5 lU / mL, 2.9 lU / mL, 3.0 lU / mL, 3.5 lU / mL, 3.9 lU / mL, 4.0 lU / mL, 4.5 lU / mL, 4.9 lU / mL, 5.0 lU / mL, 5.5 lU / mL, 5.9 lU / mL, 6.0 lU / mL, 6.5 lU / mL, 6.9 lU / mL, 7.0 lU / mL, 7.5 lU / mL, 7.9 lU / mL, 8.0 lU / mL, 8.5 lU / mL, 8.9 lU / mL, 9.0 lU / mL, 9.5 lU / mL, or 9.9 lU / mL).

[0420] In some embodiments, the OSC implant secretes a level of estradiol consistent with the follicular phase of the menstrual cycle at a range between 550 and 2800 pmol / L (e.g., between 550 and 650 pmol / L, between 650 and 750 pmol / L, between 750 and 850 pmol / L, between 850 and 950 pmol / L, between 950 and 1050 pmol / L, between 1050 and 1150 pmol / L, between 1150 and 1250 pmol / L, between 1250 and 1350 pmoL / L, between 1350 and 1450 pmol / L, between 1450 and 1550 pmol / L, between 1550 and 1650 pmol / L, between 1650 and 1750 pmol / L, between 1750 and 1850 pmol / L, between 1850 and 1950 pmol / L, between 1950 and 2050 pmol / L, between 2050 and 2150 pmol / L, between 2150 and 2250 pmol / L, between 2250 and 2350 pmol / L, between 2350 and 2450 pmol / L, between 2450 and 2550 pmol / L, between 2550 and 2650 pmol / L, between 2650 and 2750 pmol / L, or between 2750 and 2800 pmol / L; e.g., 550 pmol / L, 555 pmol / L, 560 pmol / L, 565 pmol / L, 570 pmol / L, 575 pmol / L, 580 pmol / L, 585 pmol / L, 590 pmol / L, 595 pmol / L, 600 pmol / L, 650 pmol / L, 700 pmol / L, 750 pmol / L, 800 pmol / L, 850 pmol / L, 900 pmol / L, 950 pmol / L, 1000 pmol / L, 1050 pmol / L, 1100 pmol / L, 1150 PATENT

[0421] Attorney Docket No.: 51763-009WO2 pmol / L, 1200 pmol / L, 1250 pmol / L, 1300 pmol / L, 1350 pmol / L, 1400 pmol / L, 1450 pmol / L, 1500 pmol / L,

[0422] 1550 pmol / L, 1600 pmol / L, 1650 pmol / L, 1700 pmol / L, 1750 pmol / L, 1800 pmol / L, 1850 pmol / L, 1900 pmol / L, 1950 pmol / L, 2000 pmol / L, 2050 pmol / L, 2100 pmol / L, 2150 pmol / L, 2200 pmol / L, 2250 pmol / L,

[0423] 2300 pmol / L, 2350 pmol / L, 2400 pmol / L, 2450 pmol / L, 2500 pmol / L, 2550 pmol / L, 2600 pmol / L, 2650 pmol / L, 2700 pmol / L, 2750 pmol / L, or 2800 pmol / L).

[0424] During the second week and / or ovulation phase of the menstrual cycle, LH may be secreted by the subject’s endogenous pituitary glands. Ovulation is the release of a mature egg from the surface of the ovary. This usually occurs mid-cycle, around two weeks or so before menstruation starts. During the follicular phase, the developing follicle causes a rise in the level of estrogen. The hypothalamus in the brain recognizes these rising levels and releases GnRH. This hormone prompts the pituitary gland to produce raised levels of LH and FSH. In response to this signaling, the subject may endogenously secrete levels of LH between 20 and 60 IU / L (e.g., between 20 and 25 IU / L, between 25 and 30 IU / L, between 30 and 35 IU / L / between 35 and 40 IU / L, between 40 and 45 IU / L, between 45 and 50 IU / L, between 50 and 55 IU / L, or between 55 and 60 IU / L; e.g., 20 IU / L, 21 IU / L, 22 IU / L, 23 IU / L, 24 IU / L, 25 IU / L, 26 IU / L, 27 IU / L, 28 IU / L, 29 IU / L, 30 IU / L, 31 IU / L, 32 IU / L, 33 IU / L, 34 IU / L, 35 IU / L, 36 IU / L, 37

[0425] IU / L, 38 IU / L, 39 IU / L, 40 IU / L, 41 IU / L, 42 IU / L, 43 IU / L, 44 IU / L, 45 IU / L, 46 IU / L, 47 IU / L, 48 IU / L, 49

[0426] IU / L, 50 IU / L, 51 IU / L, 52 IU / L, 53 IU / L, 54 IU / L, 55 IU / L, 56 IU / L, 57 IU / L, 58 IU / L, 59 IU / L, or 60 IU / L).

[0427] During ovulation, the egg is released from its follicle, but the ruptured follicle stays on the surface of the ovary. For the next two weeks or so, the follicle transforms into a structure known as the corpus luteum. This structure starts releasing progesterone, along with small amounts of estrogen. This combination of hormones maintains the thickened lining of the uterus, waiting for a fertilized egg to stick (implant). In some embodiments, the OSC implant may primarily secrete levels of progesterone that is consistent with ovulation at a range from 2 to 25 ng / mL of progesterone (e.g., 2 to 5 ng / mL, 5 to 10 ng / mL, 10 to 15 ng / mL, 15 to 20 ng / mL, or 20 to 25 ng / mL; e.g., 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, or 25 ng / mL).

[0428] In some embodiments, the secretion of one or more hormones may stop based on chemical signaling transmitted from the hypothalamus through the bloodstream.

[0429] In some embodiments, an OSC implant secretes hormones that correlate with a subject’s bodymass index (BMI), such that a subject with a higher BMI has an OSC implant that secretes higher levels of hormones compared to a reference subject with a lower BMI. In some embodiments, an OSC implant comprises a greater number of cells compared to a reference subject with a lower BMI.

[0430] E. Production of ovarian or uterine organoids from ovarian support cells

[0431] Further construction of OSCs (e.g., a population of the one or more OSCs described herein) into an organoid that replicates female reproductive tissues or organs may be used for research or therapeutic purposes. An organoid may replicate the structural and functional properties of ovarian tissue or a cellular niche therein as an ovarian organoid (“an ovaroid”). Additionally or alternatively, an organoid may replicate the structural and functional properties of uterine tissue or a cellular niche therein as a uterine organoid (“a uteroid”). Such organoids may be used to evaluate treatments for conditions such as PATENT

[0432] Attorney Docket No.: 51763-009WO2 infertility, ovarian decline, and other forms of reproductive dysfunction, such as any one of the diseases and conditions described herein.

[0433] In some embodiments, the organoids described herein are constructed, at least in part, from a population of OSCs (e.g., theca cells, granulosa cells, ovarian stroma cells, lutein cells, or a combination thereof). An organoid may be constructed to replicate healthy ovarian or uterine tissue. Alternatively, an organoid may be constructed to replicate the structural and functional features of ovarian or uterine tissue characterized by a particular indication, such as a condition or disorder that causes or leads to reproductive dysfunction (e.g., POI, POOS, ovarian cysts, premature menopause, endometriosis, uterine fibroids, adenomyosis, gynecological cancers, PID, vaginitis, cervical dysplasia, pelvic floor prolapse, and interstitial cystitis, among other forms of reproductive dysfunction). In some embodiments, an organoid is constructed to replicate a subject’s phenotype for the design of a personalized treatment plan or therapeutic regimen. The distribution of cells in an ovaroid may depend on or reflect a subject’s particular hormonal, biological, or physiological needs or compositions, as determined by a skilled practitioner (e.g., a physician, a clinician, an OB / GYN, a nurse practitioner, or another skilled professional). In some embodiments, an ovaroid comprises an equal or relatively equal distribution of theca cells, granulosa, ovarian stroma cells, and / or lutein cells. In some embodiments, an ovaroid comprises a distribution of cells in which one cell type is more abundant than the other cell types in the population. In other embodiments, an ovaroid comprises a distribution of cells in which each cell type is represented at a different relative abundance such that no two cell types have an equivalent relative distribution.

[0434] In some embodiments, the cellular content of an organoid (e.g., an ovaroid or a uteroid) may be composed entirely of a population of OSCs derived from a progenitor cell (e.g., an hiPSC). In some embodiments, an organoid may comprise a population of OSCs that are aggregated with, complexed with, or co-cultured with additional cell types such as a germ cell (e.g., a primordial germ cell, a primordial germ cell-like cell, an oogonium, or an oocyte). An organoid may comprise, in addition to a population of OSCs (e.g., theca cells, granulosa cells, ovarian stroma cells, lutein cells, or a combination thereof, as determined by the one or more biomarkers described herein), one or more cell types differentiated from a progenitor cell (e.g., an hiPSC).

[0435] An ovaroid may include one or more human primordial germ cell-like cells (hPGCLCs). An hPGCLC may express higher levels of biomarkers NANOS3, CD38, ITGA6, EpCAM, BLIMP1 , TFAP2C and / or SOX17 compared to an accepted reference ovarian cell or other cell type known in the art. An hPGCLC may derive from hiPSCs, e.g., as described herein.

[0436] An ovaroid may include one or more oogonia. An oogonium may express higher levels of biomarkers DDX4, DAZL, and / or STRA8 compared to an accepted reference ovarian cell or other cell type known in the art. An oogonia may derive from hiPSCs, e.g., as described herein.

[0437] An ovaroid may include one or more oocytes. An oocyte may express higher levels of biomarkers SYCP1 , ZP1 , ZP2, REC8, LHX8, and / or SOHLH1 compared to an accepted reference ovarian cell or other cell type known in the art. An oocyte may be immature or mature. An oocyte may be harvested from a subject (e.g., a subject with a gynecological or reproductive condition discussed herein, a subject undergoing IVF or another form of ART, a subject by which the ovaroid is modeled, or a healthy female subject). An oocyte may derive from hiPSCs, e.g., as described herein.

[0438] In some embodiments, the organoid recapitulates structural and physiological qualities of one or more types of uterine tissue to form a uterine organoid, or a uteroid. In some embodiments, a uteroid PATENT

[0439] Attorney Docket No.: 51763-009WO2 comprises endometrial-like cells of an endometrial layer to model the endometrium, the inner-most lining of the uterus that prevents adhesions between opposed walls of the myometrium and maintains patency of the uterine cavity. In some embodiments, a uteroid comprises myometrial-like cells of the uterine myometrium, the smooth muscle tissue of the uterus that mediates uterine contractions and expansion. In other embodiments, a uteroid comprises perimetrial cells of the uterine perimetrium or serosa layer, the outer layer of epithelial cells of the uterus that provides structural support and reduces friction between the uterus and neighboring pelvic organs.

[0440] An organoid, such as an ovaroid or uteroid, may comprise inactive ingredients, including extracellular matrix (ECM) components that provide biochemical and biomechanical structure and support for the active cellular components.

[0441] In some embodiments, an organoid is embedded into an ECM containing one or more ECM proteins and / or associated molecules, such as binding partners or ions (e.g., divalent ions, such as calcium ions and / or magnesium ions). In some embodiments, an ovaroid comprises ECM proteins, protein polymers, and / or molecules that can be found in connective tissue. In some embodiments, the ECM comprises one or more types of collagen (e.g., fibrillar collagen; e.g., collagen I, II, III, V, XI), one or more epidermal growth factors (EGFs), elastin (e.g., tropoelastin or mature elastin), fibronectin, vitronectin, laminin, among other glycoproteins, cell adhesion proteins, or plant-derived proteins or protein polymers (e.g., alginate).

[0442] In some embodiments, the ECM comprises polymers ranging from 500 to 800 pm in size (e.g., from 500 to 550 pm, 550 to 600 pm, 600 to 650 pm, 650 to 700 pm, 700 to 750 pm, or 750 to 800 pm; e.g., 500 pm, 510 pm, 520 pm, 530 pm, 540 pm, 550 pm, 560 pm, 570 pm, 580 pm, 590 pm, 600 pm, 610 pm, 620 pm, 630 pm, 640 pm, 650 pm, 660 pm, 670 pm, 680 pm, 690 pm, 700 pm, 710 pm, 720 pm, 730 pm, 74 pm, 750 pm, 760 pm, 770 pm, 780 pm, 790 pm, or 800 pm). In some embodiments, the ECM of an ovaroid may include proteoglycans, heparan sulfate, chondroitin sulfate, keratan sulfate, hyaluronic acid, elastin, dermatan sulfate, extracellular vesicles, nanoparticles, microparticles, proteins, cell adhesion proteins, proteoglycans, carbohydrate polymers, non-proteoglycan polysaccharides, and / or other forms of substrates one skilled in the art may understand as applicable to embedding and supporting an ovaroid. In some embodiments, one or more ECM components are derived from purified or partially purified animal or plant tissue. In other embodiments, one or more ECM components are derived from in vitro cell culturing conditions.

[0443] II. Methods and applications of use of ovarian support cell compositions

[0444] Described below are methods and applications for using the engineered OSCs, CSC implants, and organoids that comprise a population of engineered OSCs. These applications include selecting a subject for receiving an OSC implant and delivering the OSC implant to the subject, methods of evaluating a candidate therapeutic or intervention using an OSC organoid (e.g., an ovaroid and / or a uteroid), and methods of in vitro maturation. PATENT

[0445] Attorney Docket No.: 51763-009WO2

[0446] A. Administering an OSC implant to treat ovarian decline and other gynecologic conditions

[0447] / '. Subject selection

[0448] An OSC implant described herein may be administered to a subject seeking IVF treatment options due to ovarian decline, among other reasons. In general, a subject is a female with a low oocyte retrieval number or a subject with many immature oocytes or reduced ovarian hormone levels due to reduced response to natural hormone fluctuations in the body. A subject may be between 20 and 45 years old, and a subject is typically 35 years of age or older. A subject may have ovarian decline and / or a reduced ovarian reserve due to advancing age and / or a genetic or medical condition (e.g., primary ovarian insufficiency (POI) , polycystic ovarian syndrome (POOS), premature menopause, endometriosis, uterine fibroids, gynecological cancer, interstitial cystitis, pelvic inflammatory disease (PI D) , vaginitis, cervical dysplasia, uterine fibroids, pelvic floor prolapse, and / or interstitial cystitis) that leads to a reduced ovarian reserve. A subject may have an ovarian reserve of 20 or fewer oocytes such that a subject has 1 to 5 oocytes, 4 to 10 oocytes, 8 to 16 oocytes, or 15 to 20 oocytes, e.g., the subject has 1 oocyte, 2 oocytes, 3 oocytes, 4 oocytes, 5 oocytes, 6 oocytes, 7 oocytes, 8 oocytes, 9 oocytes, 10 oocytes, 11 oocytes, 12 oocytes, 13 oocytes, 14 oocytes, 15 oocytes, 16 oocytes, 17 oocytes, 18 oocytes, 19 oocytes, or 20 oocytes. A subject may have anti-Mullerian hormone (AMH) levels that are consistent with reduced ovarian reserve. A subject may have their AMH levels measured by a blood test and other methods known in the art. A subject may have AMH levels between 1 and 6 ng / mL (e.g., 1 -2 ng / mL, 2-4 ng / mL, or 4-6 ng / mL; e.g., 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, or 6 ng / mL). A subject may have measured estradiol levels between 20 and 50 pg / mL (e.g., 20-30 pg / mL, 25-35 pg / mL, 30-40 pg / mL, 35-45 pg / mL, or 40-50 pg / mL; e.g., 20 pg / mL, 21 pg / mL, 22 pg / mL, 23 pg / mL, 24 pg / mL, 25 pg / mL, 30 pg / mL, 35 pg / mL, 40 pg / mL, 45 pg / mL, or 50 pg / mL).

[0449] A physician or skilled practitioner may evaluate a subject for the methods of administering an OSC implant by taking a biological sample from the subject. A biological sample may include a laboratory specimen held by a biorepository for research. In some embodiments, a biological sample may include bodily fluids including blood, saliva, urine, semen (seminal fluid), vaginal secretions, cerebrospinal fluid (CSF), synovial fluid, pleural fluid (pleural lavage), pericardial fluid, peritoneal fluid, amniotic fluid, saliva, nasal fluid, optic fluid, gastric fluid, breast milk, cell culture supernatants, and the like. A biological sample may include a medical diagnosis, user input describing how a user is feeling and / or a symptomatic complaint, information collected from a wearable device pertaining to a user and the like. For example, a biological sample may include information obtained from a visit with a medical professional such as a health history. In yet another non-limiting example, a biological sample may include information such as data collected from a wearable device worn by a user and designed to collect information relating to a user’s sleep patterns, exercise patterns, and the like. In an embodiment, a biological sample collected at a particular date and / or time of a user’s menstrual cycle. For instance, and without limitation, a biological sample may be collected on the second day of a user’s menstrual cycle to evaluate one or more hormone levels. The biological sample may be utilized to determine markers of a subject’s ovarian reserve that may be measured by a subject’s AMH levels and / or other hormone levels or other indications. AMH levels of 1 ng / mL or less may be used to indicate a low ovarian reserve. A subject with a low ovarian reserve may have measured AMH levels of 1 .0 ng / mL, 0.9 ng / mL, 0.8 ng / mL, 0.7 ng / mL, 0.6 ng / mL, 0.5 ng / mL, 0.4 ng / mL, 0.3 ng / mL, 0.2 ng / mL, or 0.1 ng / mL. Other biological samples that may be utilized to PATENT

[0450] Attorney Docket No.: 51763-009WO2 determine one or more markers of a subject’s overall health include without limitation menstrual cycle progression, and / or monitor circulating hormone levels such as estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone (P4), estrone (E1), estriol (E3), testosterone, androgens, dehydroepiandrosterone (DHEA), triiodothyronine (T3), tetraiodothyronine (T4), calcitonin, melatonin, insulin, cortisol, human growth hormone (HGH), adrenaline levels, and other hormones.

[0451] Other biological sample data taken from a subject may include an oocyte. As used in this disclosure, “biological sample data” is data that provides a characterization of the biological, genetic, biochemical and / or physiological properties, compositions, or activities of biological samples. In some embodiments, an oocyte may be an immature oocyte. As is described in the definitions recited above, an “immature oocyte” as used in this disclosure is an immature reproductive cell originating in the ovaries. In some embodiments, an immature oocyte may be an oocyte including GV and / or Ml oocytes. In some embodiments, an immature oocyte may be a plurality of oocytes. An immature oocyte may be immature cumulus-oocyte complexes (COCs) taken from the subject. As used in this disclosure, a “cumulus-oocyte complex” is an oocyte surrounded by specialized granulosa cells, which are a cumulus cells surrounding the oocyte to ensure healthy oocyte and embryo development.

[0452] In some embodiments of the method, the biological sample may be extracted from the user through an extraction device. An “extraction device” is a device and / or tool capable of obtaining, recording and / or ascertaining a measurement associated with a sample. The extraction device may include a needle, syringe, vial, lancet, Evacuated Collection Tubes (ECT), tourniquet, vacuum extraction tube systems, any combination thereof and the like. For example, the extraction device may comprise a butterfly needle set. Data from a biological sample may include measurements, for example, of serum calcium, phosphate, electrolytes, blood urea nitrogen and creatinine, uric acid, and the like.

[0453] In an embodiment of the method, biological sample information of a subject may be obtained from an ultrasound. An “ultrasound,” as used in this disclosure, is any procedure that utilizes sound waves to generate one or more images of a user’s body. For example, an ultrasound may be utilized to obtain an image of a subject’s reproductive organs and / or tissues. In an embodiment, an ultrasound may be performed at a particular time of a subject’s menstrual cycle. For example, a subject may receive an ultrasound on day 2 of her cycle and this may be utilized to determine follicle size and / or follicle count. Selection of a stimulation protocol and / or adjustment to a stimulation protocol may be made utilizing this information. For example, a subject with an ultrasound that shows POOS may have a dose adjustment made to one or more medications received and / or utilized during a stimulation protocol. In addition, the length of her stimulation protocol may be modified based on her POOS diagnosis. In an embodiment, an ultrasound may be repeated one or more times throughout a subject’s stimulation protocol, and information obtained may be utilized to adjust her stimulation protocol in real time.

[0454] Prior to receiving an OSC implant, a subject’s serum may be evaluated for levels of hormones or other relevant compounds. A subject may have low serum levels of estradiol with levels of estradiol lower than 20 pg / mL (e.g., lower than 20 pg / mL, lower than 19 pg / mL, lower than 18 pg / mL, lower than 17 pg / mL, lower than 16 pg / mL, lower than 15 pg / mL, lower than 14 pg / mL, lower than 13 pg / mL, lower than 12 pg / mL, lower than 11 pg / mL, lower than 10 pg / mL, or lower) prior to receiving an OSC implant. A subject who has had an ovariectomy, or the removal or partial removal of their ovaries may have estradiol levels lower than 10 pg / mL (e.g., lower than 10 pg / mL, lower than 9 pg / mL, lower than 8 pg / mL, lower than 7 pg / mL, lower than 6 pg / mL, lower than 5 pg / mL, lower than 4 pg / mL, lower than 3 pg / mL, lower PATENT

[0455] Attorney Docket No.: 51763-009WO2 than 2 pg / mL, lower than 1 pg / mL, or even lower) prior to receiving an OSC implant. A subject may have low serum levels of progesterone prior to receiving an OSC implant. In some embodiments a subject may have serum levels of progesterone lower than 0.5 ng / mL (e.g., lower than 0.5 ng / mL, lower than 0.4 ng / mL, lower than 0.3 ng / mL, lower than 0.2 ng / mL, lower than 0.1 ng / mL, or lower) prior to receiving an OSC implant. Further, a subject may have serum levels of LH from about 1 .0 mIU / mL to about 2.5 mIU / mL (e.g., from about 1 .0 mIU / mL to about 1 .5 mIU / mL, from about 1 .5 mIU / mL to about 2.0 mIU / mL, or from about 2.0 mIU / mL to about 2.5 mIU / mL; e.g., about 1 .0 mIU / mL, about 1 .25 mIU / mL, about 1 .5 mIU / mL, about 1 .75 mIU / mL, about 2 mIU / mL, about 2.25 mIU / mL, or about 2.5 mIU / mL) prior to receiving an OSC implant. A subject may have serum levels of FSH from about 11 mIU / mL to about 14 mIU / mL (e.g., from about 1 1 mIU / mL to about 12 mIU / mL, from about 12 mIU / mL to about 13 mIU / mL, or from about 13 mIU / mL to about 14 mIU / mL; e.g., about 11 mIU / mL, about 12 mIU / mL, about 13 mIU / mL, or about 14 mIU / mL) prior to receiving an OSC implant.

[0456] / ' / . Sites of administration of an OSC implant and OSC implant half-life

[0457] An OSC implant may be applied by a healthcare provider (e.g., a physician, an OB / GYN, a nurse practitioner, or other skilled practitioner) using multiple possible routes of administration. A healthcare provider may use a subject’s preference or biological or medical data to determine the best site for OSC implantation.

[0458] A physician or skilled practitioner may determine the administration of an OSC implant directed to a subject using the described biological parameters. Such biological parameters include hormone levels (e.g., baseline hormone levels and / or hormone levels due to use of contraceptives), subject anatomy (e.g., follicle size, follicle count, ovarian morphology, and / or uterine morphology), subject body-mass index (BMI), among other biological parameters known to a skilled practitioner. A skilled practitioner may administer a stimulation protocol with any one or a combination of triggering agents, or compositions directed to stimulate follicular maturation and oocyte release, including those known in the art and described herein.

[0459] Hormone levels or concentrations of other relevant compounds of the biological sample may include estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone (P4), estrone (E1), estriol (E3), testosterone, androgens, dehydroepiandrosterone (DHEA), triiodothyronine (T3), tetraiodothyronine (T4), calcitonin, melatonin, insulin, cortisol, human growth hormone (HGH), adrenaline levels and the like. In some embodiments, the measurement of hormone levels may be based on blood analysis of the biological sample. For example, blood analysis may include plasma hormone analysis techniques. In some embodiments, measurement of hormone levels may be based on saliva hormone testing techniques. Measurement of hormone levels may be based on other forms of analysis such as hair, urine, and any other form of biological samples described throughout this disclosure. A subject may have a baseline serum level of estradiol from about 30 pg / mL to about 60 pg / mL (e.g., from about 30 pg / mL to about 45 pg / mL, from about 40 pg / mL to about 55 pg / mL, or from about 45 pg / mL to about 60 pg / mL; e.g., about 30 pg / mL, about 35 pg / mL, about 40 pg / mL, about 45 pg / mL, about 50 pg / mL, about 55 pg / mL, or about 60 pg / mL) prior to the follicular triggering period. A subject may have a baseline serum level of progesterone from about 0.5 ng / mL to about 2.5 ng / mL (e.g., from about 0.5 ng / mL to about 1 .0 ng / mL, from about 1 .0 ng / mL to about 1 .5 ng / mL, from about 1 .5 ng / mL to about 2.0 PATENT

[0460] Attorney Docket No.: 51763-009WO2 ng / mL, or from about 2.0 ng / mL to about 2.5 ng / mL; e.g., about 1 .0 ng / mL, about 1 .5 ng / mL, about 2.0 ng / mL, or about 2.5 ng / mL) prior to the follicular triggering period.

[0461] Additionally, a subject’s contraception (e.g., hormonal contraception) usage may affect administration of an OSC implant. Consideration for contraception may aid in determining the administration of an OSC implant.

[0462] Insertion of an OSC implant may follow health and safety protocols known in the art. Considerations related to health and safety may include one or more extractions of a biological sample for a measurement of a hormone described herein. Other considerations may include sterilizing the administration site, numbing the administration site, and / or administering local or systemic analgesics to the subject prior to or upon implantation.

[0463] In some embodiments, an OSC implant may be inserted as a semi-permeable rod. In some embodiments, the semi-permeable rod comprising the OSC implant may be inserted subdermally in the upper arm or the abdomen of a subject. In some embodiments, the OSC implant may be inserted at the inner side of the non-dominant upper arm about 8-10 cm or 3-4 inches (e.g., about 8 cm, about 8.5 cm, about 9 cm, about 9.5 cm, about 10 cm) above the medial epicondyle of the humerus to reduce the risk of neural or vascular injury. In some embodiments, the OSC implant is applied to the subject with a needle (e.g., a hypodermic needle) or other applicator compatible with subdermal insertion.

[0464] In some embodiments, an OSC implant may be inserted or administered onto the ovary. In other embodiments, an OSC implant may be inserted or administered onto or adjacent to the fallopian tubes. In some embodiments, an OSC implant is applied as a patch onto the desired tissue (e.g., a microneedle patch, a reservoir patch, a single layer patch, a multi-layer patch, among other examples). In some embodiments, an OSC implant is applied as a semi-permeable pouch onto or adjacent to the ovary and / or fallopian tubes. In some embodiments, an OSC implant is applied to the desired tissue via direct injection with a tissue-specific applicator (e.g., a vaginal cylinder applicator or an ovoid applicator).

[0465] In some embodiments, an OSC implant may be inserted or administered as an omental pouch in the abdomen of a subject. An omental pouch is a delivery modality in which cells (e.g., OSCs delivered as an implant) are administered to a subject’s omentum, which is a thin layer of tissue that covers and connects the abdominal organs of an animal (e.g., a human). Methods of implanting cells via an omental pouch is known in the art and is described elsewhere such as, e.g., Berman et al. Am J Transplant. 9(1):91 -104, 2009, and Deng et al. Cell Rep Med. 4(3):100959, 2023, each of which is hereby incorporated by reference.

[0466] In some embodiments, an OSC implant may be inserted as an intrauterine device (IUD), which is a small, often T-shaped contraceptive device that is implanted into the uterus using, e.g., methods known in the art. In some embodiments, the IUD can be hormonal or non-hormonal are long-acting, reversible. In some embodiment, the OSC implant may be porous implant (e.g., comprised of porous material such as titanium), which is characterized by the presence of regular or stochastic voids in the metallic or ceramic matrix depending on their manufacture.

[0467] An OSC implant is designed to be surgically inserted into a subject’s body as described in the above section and is, as such, designed to be long lasting as compared to presently available treatment options for those seeking ART or HRT, which involve frequent medical appointments and continuous injections of hormones. An OSC implant, in contrast, offers many practical benefits and is therefore a PATENT

[0468] Attorney Docket No.: 51763-009WO2 potentially more attractive treatment option with fewer doctor appointments, fewer prescription refills, and no daily or weekly injections of hormones, among other potential benefits to the subject.

[0469] In some embodiments, an OSC implant retains its hormone-secreting function following implantation into a subject for a time period that is at least 200 days (e.g., at least 200 days, at least 210 days, at least 220 days, at least 230 days, at least 240 days, at least 250 days, at least 260 days, at least 270 days, at least 280 days, at least 290 days, at least 300 days, or longer). In some embodiments, an OSC implant retains its hormone-secreting function following implantation into a subject for a time period that is at least 6 months (e.g., at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 13 months, at least 14 months, at least 15 months, or longer). In some embodiments an OSC implant retains its hormone-secreting function following implantation into a subject for a time period between 12 and 24 months (e.g., between 12 and 15 months, between 15 and 18 months, between 18 and 21 months, or between 21 and 24 months; e.g., 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months), such as 3 months and / or 3 years. In some embodiments, the OSC implant is removed and / or by a skilled medical practitioner after a certain period of time.

[0470] B. Applications of organoids that include ovarian support cells

[0471] An organoid that recapitulates the structural and / or functional properties of a female reproductive organ can be used, e.g., for modeling the toxicology and efficacy effects of candidate therapeutic interventions on endogenous human tissues. Moreover, the organoids of the disclosure provide the benefit of being more useful, reliable models of drug tolerability and efficacy as compared to existing in vivo animal models, particularly given the ability to generate organoids from iPSCs (e.g., hiPSCs) that strongly approximate, with a high degree of fidelity, the naturally occurring ovarian and uterine niches.

[0472] In addition to serving as close replicas of the human female reproductive organs, the organoid models of the disclosure can provide the additional benefit of facilitating high-throughput screens for the tolerability and efficacy of candidate therapeutic interventions, while minimizing potentially confounding interactions that may otherwise be obtained from an in vivo animal study. Consequently, the organoids of the disclosure provide a unique method of rapidly evaluating the safety and efficacy of candidate interventions.

[0473] In some embodiments, an organoid of the disclosure, such as an ovaroid or uteroid, replicates the ovarian structure and / or function of an individual subject. This may be useful, e.g., for the design of personalized treatment plans and / or for evaluating the safety and efficacy of a candidate therapeutic intervention in the context of a personalized therapeutic regimen. In other embodiments, an organoid of the disclosure (such as an ovaroid or uteroid) replicates the ovarian and / or uterine structure and function of a broader population of patients. This latter context is particularly useful in the design of therapeutic regimens that are more broadly applicable, e.g., to classes of patients having like disorders.

[0474] / '. Disease indications

[0475] An organoid of the disclosure may be developed to study the effects of therapeutic interventions on a variety of disease states or conditions that may alter ovarian, uterine, or other forms of reproductive function. The compositions and methods of the disclosure may also be used to replicate diseases or PATENT

[0476] Attorney Docket No.: 51763-009WO2 conditions that result in impaired ovarian or uterine function (e.g., POI, POOS, ovarian cysts, premature menopause, endometriosis, uterine fibroids, gynecological cancer, interstitial cystitis, PID, vaginitis, and cervical dysplasia, among others). In some embodiments, the compositions and methods of the disclosure may be used to study interventions that facilitate in vitro oocyte maturation, oocyte production and release, oocyte or embryo implantation, and other aspects of assisted reproductive technology.

[0477] Without being limited by mechanism, the structure and hormone secretion activity of the organoids described herein may recapitulate native ovarian functions in vivo and provide a useful model for replicating ovarian decline in subjects due to a natural, timewise reduction in the ovarian reserve (e.g., due to menopause). In some embodiments, an ovaroid may recapitulate ovarian physiology consistent with underlying diseases or conditions with impairments or abnormalities related to ovulation follicular development, oocyte release, oocyte maturation, or a combination thereof. In some embodiments, an ovaroid may recapitulate ovarian structure or function related to diseases such as POI, POOS, premature menopause, ovarian cancer, age-related ovarian decline, ovarian hyperstimulation syndrome, among other related conditions. In some embodiments, an ovaroid may have genetic mutations or abnormalities associated with an individual subject or a broader patient population.

[0478] Similarly, the structure and hormone secretion activity of the organoids may recapitulate native uterine functions in vivo and provide a useful model for replicating uterine complications identified in subjects or broad patient populations. In some embodiments, a uteroid may recapitulate uterine physiology consistent with underlying diseases or conditions with impairments or abnormalities related to menstruation, embryo implantation, carrying a pregnancy, or giving birth. Such diseases or conditions include uterine fibroids, heavy menstruation, retrograde menstruation, endometriosis, adenomyosis, uterine cancer, pre-eclampsia, Asherman syndrome, miscarriage, among other conditions. In some embodiments, a uteroid may have genetic mutations or abnormalities associated with an individual subject or a broader patient population.

[0479] An organoid may be configured to replicate an individual subject’s reproductive organ structure or function based on biological sample data from the subject. A subject may be seeking ART, IVF, or HRT interventions. A subject may be between 20 and 45 years old or older. A subject may have a reduced ovarian reserve due to advancing age and / or a genetic or medical condition (e.g., POOS) that leads to a reduced ovarian reserve. A subject may have an ovarian reserve of 20 or fewer oocytes such that a subject has 1 to 5 oocytes, 4 to 10 oocytes, 8 to 16 oocytes, or 15 to 20 oocytes, e.g., the subject has 1 oocyte, 2 oocytes, 3 oocytes, 4 oocytes, 5 oocytes, 6 oocytes, 7 oocytes, 8 oocytes, 9 oocytes, 10 oocytes, 11 oocytes, 12 oocytes, 13 oocytes, 14 oocytes, 15 oocytes, 16 oocytes, 17 oocytes, 18 oocytes, 19 oocytes, or 20 oocytes. A subject may have anti-Mullerian hormone (AMH) levels that are consistent with reduced ovarian reserve. A subject may have their AMH levels measured by a blood test and other methods known in the art. A subject may have AMH levels between 1 and 6 ng / mL (e.g., 1 -2 ng / mL, 2-4 ng / mL, or 4-6 ng / mL; e.g., 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, or 6 ng / mL). A subject may have measured estradiol levels between 20 and 50 pg / mL (e.g., 20-30 pg / mL, 25-35 pg / mL, 30-40 pg / mL, 35-45 pg / mL, or 40-50 pg / mL; e.g., 20 pg / mL, 21 pg / mL, 22 pg / mL, 23 pg / mL, 24 pg / mL, 25 pg / mL, 30 pg / mL, 35 pg / mL, 40 pg / mL, 45 pg / mL, or 50 pg / mL). A subject may be using oral contraception.

[0480] A physician or skilled practitioner may evaluate a subject for the methods of making organoids, such as ovaroids, by taking a biological sample from the subject. A biological sample may include a PATENT

[0481] Attorney Docket No.: 51763-009WO2 laboratory specimen held by a biorepository for research. In some embodiments, a biological sample may include bodily fluids including blood, saliva, urine, semen (seminal fluid), vaginal secretions, cerebrospinal fluid (CSF), synovial fluid, pleural fluid (pleural lavage), pericardial fluid, peritoneal fluid, amniotic fluid, saliva, nasal fluid, optic fluid, gastric fluid, breast milk, cell culture supernatants, one or more oocytes, and the like. A biological sample may include a medical diagnosis, a medical testimonial by a subject and / or a symptomatic complaint, information collected from a wearable device pertaining to a subject and the like. For example, a biological sample may include information obtained from a visit with a medical professional such as a health history. In yet another non-limiting example, a biological sample may include information such as data collected from a wearable device worn by a user and designed to collect information relating to a user’s sleep patterns, exercise patterns, and the like. In an embodiment, a biological sample collected at a particular date and / or time of a user’s menstrual cycle. For instance, and without limitation, a biological sample may be collected on the second day of a user’s menstrual cycle to evaluate one or more hormone levels. The biological sample may be utilized to determine markers of a subject’s ovarian reserve that may be measured by a subject’s AMH levels and / or other hormone levels or other indications. Other biological samples that may be utilized to determine one or more markers of a subject’s overall health include without limitation menstrual cycle progression, and / or monitor circulating hormone levels such as estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone (P4), estrone (E1), estriol (E3), testosterone, androgens, dehydroepiandrosterone (DHEA), triiodothyronine (T3), tetraiodothyronine (T4), calcitonin, melatonin, insulin, cortisol, human growth hormone (HGH), adrenaline levels, and other hormones.

[0482] In some embodiments of the method, the biological sample may be extracted from the user through an extraction device. An “extraction device” is a device and / or tool capable of obtaining, recording and / or ascertaining a measurement associated with a sample. The extraction device may include a needle, syringe, vial, lancet, Evacuated Collection Tubes (ECT), tourniquet, vacuum extraction tube systems, any combination thereof and the like. For example, the extraction device may comprise a butterfly needle set. Data from a biological sample may include measurements, for example, of serum calcium, phosphate, electrolytes, blood urea nitrogen and creatinine, uric acid, and the like.

[0483] In an embodiment of the method, biological sample information of a subject may be obtained from an ultrasound. An “ultrasound,” as used in this disclosure, is any procedure that utilizes sound waves to generate one or more images of a user’s body. For example, an ultrasound may be utilized to obtain an image of a subject’s reproductive organs and / or tissues. In an embodiment, an ultrasound may be performed at a particular time of a subject’s menstrual cycle. For example, a subject may receive an ultrasound on day 2 of her cycle and this may be utilized to determine follicle size and / or follicle count.

[0484] / ' / . Therapeutic screening and drug safety profiles

[0485] The organoids, such as ovaroids and uteroids, described herein may be utilized to screen effective therapies or treatment options for individual subjects or patient populations that have a disease or condition described in the preceding section. An organoid that recapitulates a disease state through genetic, epigenetic, biochemical, or mechanical manipulation may be administered a drug or compound (e.g., a small molecule, an antibody or antibody derivative, a peptide, or an oligonucleotide) to evaluate its treatment efficacy, dosing regimen, suitable formulations, pharmacokinetics, or pharmacodynamic profile. Additionally, an organoid may be exposed to a cell-based therapy (e.g., one or more stem cells, such as PATENT

[0486] Attorney Docket No.: 51763-009WO2 hematopoietic stem cells, chimeric antigen receptor T cells (CAR-T cells), or other cell therapy known in the art or described herein) to determine treatment efficacy, dosing regimen, targeting or homing strategies, pharmacokinetics, or pharmacodynamic profiles, among other parameters for a candidate pharmaceutical intervention.

[0487] A therapeutic screen using the organoids of the disclosure may be used to identify suitable treatment options to improve ovulation, release of oocytes, oocyte maturation, or other processes related to fertility or healthy reproduction. In some embodiments, the efficacy of a potential therapeutic is evaluated based on changes in released oocytes from an ovaroid. In some embodiments, the efficacy of a potential therapeutic is evaluated based on changes in the number of mature oocytes released from the ovaroid. In some embodiments, the efficacy of a potential therapeutic is evaluated based on oocyte fertilization rates.

[0488] Alternatively or additionally, a therapeutic screen using the organoids of the disclosure may be used to identify suitable treatment options to improve embryo implantation and / or subsequent embryo development (e.g., to support healthy pregnancy). In some embodiments, the efficacy of a potential therapeutic is evaluated based on changes in embryo implantation or embryo development in a uteroid. In some embodiments, an efficacious treatment promotes improved embryo implantation compared to a reference sample. In some embodiments, the efficacy of a potential therapeutic is evaluated based on embryonic development after implantation in a uteroid. In some embodiments, embryo health and development is measured based on hallmarks observed in different developmental stages. In some embodiments, an embryo is a zygote or a single-celled fertilized egg. In some embodiments, an embryo is a blastocyst and contains a zona pellucida (ZP), trophectoderm (TE), blastocoel (BL), and inner cell mass (ICM), among other developmental signatures.

[0489] In some embodiments, the efficacy of a potential therapeutic is evaluated based on changes in ovaroid or uteroid morphology (e.g., size, lipid content, levels of hydration, cell surface structure, glycan content, protein content, among other indications of cell morphology).

[0490] In some embodiments, the efficacy of a potential therapeutic is evaluated based on changes in hormone secretion (e.g., estrogens, progestins, androgens) or metabolites in the ovaroid or uteroid media compared to a reference organoid sample that is not contacted with a candidate therapeutic intervention. Changes in hormone secretion may be evaluated by sequencing methods (e.g., mass spectrometry) or hormone-specific detection methods with an antibody or hormone-detecting compound (e.g., enzyme- linked immunosorbent assay (ELISA), Western blot analysis, lateral flow assay, immunoprecipitation, among other detection methods).

[0491] In some embodiments, the efficacy of a potential therapeutic is evaluated based on changes in cell populations or cellular distributions in the ovaroid or uteroid as detected by changes in cellular biomarkers (e.g., surface proteins, transcription factors, cell morphology, or broader gene expression profile) compared to a relevant control that was not administered the therapeutic. Cell populations or distributions may be evaluated by flow cytometry, mass cytometry, microscopy, among other methods known in the art.

[0492] In some embodiments, the efficacy of a potential therapeutic is evaluated based on changes in gene expression in an ovaroid or uteroid. Changes in gene expression may be evaluated by RNA-seq, qRT-PCR, next-generation sequencing (NGS) modalities, epigenetic profiling, metabolomic profiling, among other non-limiting methodologies known in the art. PATENT

[0493] Attorney Docket No.: 51763-009WO2

[0494] In some embodiments, the efficacy of a potential therapeutic is evaluated based on its cytotoxicity to an organoid. In some embodiments, cytotoxicity is evaluated through cell viability assays, cell proliferation assays, apoptosis assays, autophagy assays, among other non-limiting examples.

[0495] In some embodiments, potential treatments or therapeutic options are directed to treat reproductive diseases or conditions related to ovarian or uterine health. In some embodiments, an ovaroid or uteroid therapeutic screen is used to determine suitable treatments for an individual subject with any one or more of the foregoing diseases or conditions. In other embodiments, an ovaroid or uteroid therapeutic screen is used to determine suitable treatment options for a broader disease indication.

[0496] In addition to their use as platform for evaluating potential treatment options for diseases or conditions related to reproductive health, organoids such as ovaroids or uteroids may be used to screen the safety profile of novel drugs and compounds for unrelated diseases and their potential impact on fertility and reproductive health. In some embodiments, ovaroids or uteroids may be used to screen novel drugs or compounds based on their effect on ovarian or uterine tissue and their functionality to assess potentially adverse effects on fertility prior to a clinical trial.

[0497] C. Methods of in vitro maturation of oocytes with ovarian support cells

[0498] The methods described herein may be indicated for a subject who desires to increase the number of usable oocytes from any standard ART that utilizes controlled ovarian hyperstimulation (COH). The increased number of usable oocytes can result from co-culturing immature oocytes with one or more OSCs (e.g., one or more produced OSCs as described herein) to promote maturation of the immature oocytes that are commonly obtained in typical COH and oocyte retrieval procedures. Current standard of care is to discard retrieved immature oocytes. The OSCs described herein can promote maturation of immature oocytes, thus increasing the number of mature and usable oocytes from a population of retrieved oocytes.

[0499] Additionally, the methods described herein may be indicated for a subject seeking assisted reproductive technology procedures but may have limited access due to prohibitively high costs and / or risks associated with traditional methods of ovarian stimulation for oocyte retrieval (e.g., risks of ovarian hyperstimulation syndrome (OHSS)). Traditional methods typically require administering gonadotropins to a subject for ovarian stimulation and retrieval of mature oocytes. Delivery of administered gonadotropins is typically inefficient and requires high concentrations of gonadotropins to ensure that sufficient levels of gonadotropins are delivered to the ovarian follicle for oocyte maturation and release following systemic injection. By performing IVM methods described herein, reduced quantities of gonadotropin can be administered for oocyte retrieval, thereby providing a method that circumvents the costly and potentially dangerous side effects associated with systemic administration of high levels of gonadotropins. Immature oocytes can be retrieved, exposed to conditions that lead to optimized maturation ex vivo, and the resulting mature oocytes can be used for subsequent fertilization, embryo development, blastocyst formation, implantation, and gestation to ultimately become healthy offspring.

[0500] / '. Subject selection

[0501] The methods of stimulating oocyte release described herein are directed to a subject seeking IVF treatment options. In general, a subject is a female with a low oocyte retrieval number or a subject with many immature oocytes. A subject may be between 20 and 45 years old, and a subject is typically 35 PATENT

[0502] Attorney Docket No.: 51763-009WO2 years of age or older. A subject may have a reduced ovarian reserve due to advancing age and / or a genetic or medical condition (e.g., POOS) that leads to a reduced ovarian reserve. A subject may have an ovarian reserve of 20 or fewer oocytes such that a subject has 1 to 5 oocytes, 4 to 10 oocytes, 8 to 16 oocytes, or 15 to 20 oocytes, e.g., the subject has 1 oocyte, 2 oocytes, 3 oocytes, 4 oocytes, 5 oocytes, 6 oocytes, 7 oocytes, 8 oocytes, 9 oocytes, 10 oocytes, 11 oocytes, 12 oocytes, 13 oocytes, 14 oocytes, 15 oocytes, 16 oocytes, 17 oocytes, 18 oocytes, 19 oocytes, or 20 oocytes. A subject may have anti- Mullerian hormone (AMH) levels that are consistent with reduced ovarian reserve. A subject may have their AMH levels measured by a blood test and other methods known in the art. A subject may have AMH levels between 1 and 6 ng / mL (e.g., 1 -2 ng / mL, 2-4 ng / mL, or 4-6 ng / mL; e.g., 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, or 6 ng / mL). A subject may have measured estradiol levels between 20 and 50 pg / mL (e.g., 20-30 pg / mL, 25-35 pg / mL, 30-40 pg / mL, 35-45 pg / mL, or 40-50 pg / mL; e.g., 20 pg / mL, 21 pg / mL, 22 pg / mL, 23 pg / mL, 24 pg / mL, 25 pg / mL, 30 pg / mL, 35 pg / mL, 40 pg / mL, 45 pg / mL, or 50 pg / mL).

[0503] A physician or skilled practitioner may evaluate a subject for the methods of stimulating oocyte release by taking a biological sample from the subject. A biological sample may include a laboratory specimen held by a biorepository for research. In some embodiments, a biological sample may include bodily fluids including blood, saliva, urine, semen (seminal fluid), vaginal secretions, cerebrospinal fluid (CSF), synovial fluid, pleural fluid (pleural lavage), pericardial fluid, peritoneal fluid, amniotic fluid, saliva, nasal fluid, optic fluid, gastric fluid, breast milk, cell culture supernatants, and the like. A biological sample may include a medical diagnosis, a personal account such as a symptomatic complaint by a subject, information collected from a wearable device pertaining to a subject and the like. For example, a biological sample may include information obtained from a visit with a medical professional such as a health history. In yet another non-limiting example, a biological sample may include information such as data collected from a wearable device worn by a subject that is designed to collect information relating to a subject’s sleep patterns, exercise patterns, and the like. In an embodiment, a biological sample collected at a particular date and / or time of a user’s menstrual cycle. For instance, and without limitation, a biological sample may be collected on the second day of a subject’s menstrual cycle to evaluate one or more hormone levels. The biological sample may be utilized to determine markers of a subject’s ovarian reserve that may be measured by a subject’s AMH levels and / or other hormone levels or other indications. AMH levels of 1 ng / mL or less may be used to indicate a low ovarian reserve. A subject with a low ovarian reserve may have measured AMH levels of 1 .0 ng / mL, 0.9 ng / mL, 0.8 ng / mL, 0.7 ng / mL, 0.6 ng / mL, 0.5 ng / mL, 0.4 ng / mL, 0.3 ng / mL, 0.2 ng / mL, or 0.1 ng / mL. Other biological samples that may be utilized to determine one or more markers of a subject’s overall health include without limitation menstrual cycle progression, and / or monitor circulating hormone levels such as estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone (P4), estrone (E1), estriol (E3), testosterone, androgens, dehydroepiandrosterone (DHEA), triiodothyronine (T3), tetraiodothyronine (T4), calcitonin, melatonin, insulin, cortisol, human growth hormone (HGH), adrenaline levels, and other hormones. Additional data from a biological sample may include measurements, for example, of serum calcium, phosphate, electrolytes, blood urea nitrogen and creatinine, uric acid, and the like.

[0504] Other biological sample data taken from a subject includes at least an oocyte. In some embodiments, an oocyte may be an immature oocyte. In some embodiments, an immature oocyte may be an oocyte including GV and / or Ml oocytes. An immature oocyte may be immature cumulus-oocyte PATENT

[0505] Attorney Docket No.: 51763-009WO2 complexes (COCs) taken from the subject. In some embodiments, the immature oocyte may contain an oocyte wherein the an OSC (e.g., an OSC reprogrammed into one or more cell types by differentiating an iPSC) is added to mature the oocyte in a cell culture (e.g., a co-culture) and thus create a COC.

[0506] In an embodiment of the method, biological sample information of a subject may be obtained from an ultrasound. An “ultrasound,” as used in this disclosure, is any procedure that utilizes sound waves to generate one or more images of a user’s body. For example, an ultrasound may be utilized to obtain an image of a subject’s reproductive organs and / or tissues. In an embodiment, an ultrasound may be performed at a particular time of a subject’s menstrual cycle. For example, a subject may receive an ultrasound on day 2 of her cycle and this may be utilized to determine follicle size and / or follicle count. Selection of a stimulation protocol and / or adjustment to a stimulation protocol may be made utilizing this information. For example, a subject with an ultrasound that shows POOS may have a dose adjustment made to one or more medications received and / or utilized during a stimulation protocol. In addition, the length of her stimulation protocol may be modified based on her POOS diagnosis. In an embodiment, an ultrasound may be repeated one or more times throughout a subject’s stimulation protocol, and information obtained may be utilized to adjust her stimulation protocol in real time.

[0507] / ' / . Oocyte stimulation protocols

[0508] A physician or skilled practitioner may determine the stimulation protocol of oocyte release directed to a subject using the described biological parameters. Such biological parameters include hormone levels (e.g., baseline hormone levels and / or hormone levels due to use of contraceptives), subject anatomy (e.g., follicle size, follicle count, ovarian morphology, and / or uterine morphology), among other biological parameters known to a skilled practitioner. A skilled practitioner may administer a stimulation protocol with any one or a combination of triggering agents, or compositions directed to stimulate follicular maturation and oocyte release, described herein.

[0509] Hormone levels or concentrations of other relevant compounds of the biological sample may include estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone (P4), estrone (E1), estriol (E3), testosterone, androgens, dehydroepiandrosterone (DHEA), triiodothyronine (T3), tetraiodothyronine (T4), calcitonin, melatonin, insulin, cortisol, human growth hormone (HGH), adrenaline levels and the like. In some embodiments, the measurement of hormone levels may be based on blood analysis of the biological sample. For example, blood analysis may include plasma hormone analysis techniques. In some embodiments, measurement of hormone levels may be based on saliva hormone testing techniques. Measurement of hormone levels may be based on other forms of analysis such as hair, urine, and any other form of biological samples described throughout this disclosure. A subject may have a baseline serum level of estradiol from about 30 pg / mL to about 60 pg / mL (e.g., from about 30 pg / mL to about 45 pg / mL, from about 40 pg / mL to about 55 pg / mL, or from about 45 pg / mL to about 60 pg / mL; e.g., about 30 pg / mL, about 35 pg / mL, about 40 pg / mL, about 45 pg / mL, about 50 pg / mL, about 55 pg / mL, or about 60 pg / mL) prior to the follicular triggering period. A subject may have a baseline serum level of progesterone from about 0.5 ng / mL to about 2.5 ng / mL (e.g., from about 0.5 ng / mL to about 1 .0 ng / mL, from about 1 .0 ng / mL to about 1 .5 ng / mL, from about 1 .5 ng / mL to about 2.0 ng / mL, or from about 2.0 ng / mL to about 2.5 ng / mL; e.g., about 1 .0 ng / mL, about 1 .5 ng / mL, about 2.0 ng / mL, or about 2.5 ng / mL) prior to the follicular triggering period. PATENT

[0510] Attorney Docket No.: 51763-009WO2

[0511] Additionally, a subject’s contraception (e.g., hormonal contraception) usage may affect assignment of a stimulation protocol. Consideration for contraception may aid in determining the follicular triggering period in the woman’s menstrual cycle. For instance, and without limitation, a subject who is not using any form of contraception may begin her stimulation protocol with recombinant follicle stimulating hormone (rFSH) between the first and third day of her menstrual cycle, with preference for the second day of her menstrual cycle. In yet another non-limiting example, a subject who is using contraception may begin her stimulation protocol with rFSH 4-6 days (e.g., 4 days, 5 days, or 6 days) after consuming her last oral contraception pill, with preference for 5 days following the dosing of her last oral contraception pill. In an embodiment, rFSH stimulation may be utilized for 2 to 3 days (e.g., 2 days or 3 days), depending on a subject’s tolerance, follicle size, and / or growth dynamics. After this 2- or 3-day window, a coasting period of 1 to 3 days (e.g., 1 day, 2 days, or 3 days) may be utilized to monitor follicle size and allow for further follicle maturation and development. A “coasting period,” as used in this disclosure, is any period of time when a medication used throughout a stimulation protocol is not administered and / or consumed. A coasting period may last for example for 1 day, 2 days, 3 days, or more if medically necessary. During a coasting period, a subject may continue to receive one or more ultrasounds to monitor her progression.

[0512] Once a follicle size has reached anywhere from between about 8-10 mm (e.g., 7.5 mm, 8 mm, 8.5 mm, 9mm, 9.5 mm, 10 mm, 10.5 mm, or more), a subject may be triggered with a dose of a triggering agent, such as human chorionic gonadotropin (hCG). A “follicle measurement” as used in this disclosure, is any measurement of an ovarian follicle. A follicle may include any sac found in an ovary that contains an unfertilized egg. A follicle measurement may be obtained using any methodology as described herein, including for example an ultrasound, a manual measurement, an automated measurement and the like. In an embodiment, a double hCG injection may be utilized, to induce follicle maturation to prepare one or more follicles for retrieval. A double hCG injection may be two or three injections of hCG. A blood test for one or more hormone levels such as E2, P4, and LH may be performed on the trigger day of the double dose of hCG injection to monitor hormone levels. After the day of the double dose of hCG, one or more hormone levels may be measured such as for example with a blood test to determine and examine levels of E2, P4, and LH.

[0513] A “triggering agent” is a chemical that triggers cell generation in the ovaries. A triggering agent (e.g., a follicular triggering agent) may include any substance including any non-prescription and / or prescription product. A triggering agent (e.g., a follicular triggering agent) may include any one or combination of the non-limiting examples such as LUPRON DEPOT® (Abbott Laboratories, North Chicago, IL), Ganirelix (Ferring Pharmaceuticals, Saint-Prex, Switzerland), Cetrotide (Merck Global, Readington Township, NJ), GONAL-F® (Merck Global), FOLLISTIM® (Merck Global), BRAVELLE® (Ferring Pharmaceuticals), CLOMID® (Patheon Pharmaceuticals Inc., Waltham, MA), Serephene (Teva, Tel Aviv-Yafo, Israel), GLUCOPHAGE® (Merck Global), FORTAMET® (Mylan, Canonsburg, PA), PREGNYL® (Schering Plough, Kenilworth, NJ), NOVAREL® (Ferring Laboratories, Parsippany, NJ), Repronex (Ferring Pharmaceuticals), FACTREL® (Zoetis Canada Inc., Kirkland, Canada), MENOPUR® (Ferring Pharmaceuticals), and other drugs that induce cell generation in ovaries that one skilled in the art would understand as applicable. A triggering agent (e.g., a follicular triggering agent) may include human serum albumin, FSH, hCG, androstenedione, and doxycycline among other triggering agents known in the art. PATENT

[0514] Attorney Docket No.: 51763-009WO2

[0515] In one embodiment, a subject may not receive a triggering agent (e.g., a follicular triggering agent) to stimulate oocyte production. In one embodiment, a subject may receive multiple injections of a triggering agent over 1 to 4 days (e.g., 1 day, 2 days, 3 days, or 4 days) but no more than 5 days in the preferred stimulation protocol. A subject may receive multiple injections over multiple days such that a subject receives five dose injections of one or multiple triggering agents. For example, a subject may receive three days of stimulation using 300 IU to 700 IU of rFSH per injection (e.g., 300-500 IU, 400-600 IU, 500-700 IU, 300-350 IU, 350-400 IU, 400-450 IU, 450-500 IU, 500-550 IU, 550-600 IU, 600-650 IU, 650-700 IU; e.g., 300 IU, 325 IU, 350 IU, 375 IU, 400 IU, 425 IU, 450 IU, 475 IU, 500 IU, 525 IU, 550 IU, 575 IU, 600 IU, 625 IU, 650 IU, 675 IU, or 700 IU) with one or more injections per day. A subject may receive injections of hCG as a triggering agent (e.g., a follicular triggering agent) using 200-700 pg or 2,500-10,000 IU hCG (e.g., 200-500 pg, 300-600 pg, 400-700 pg, 200-300 pg, 300-400 pg, 400-500 pg, 500-600 pg, or 600-700 pg), with a preferred stimulation dose of 500 pg. A subject may receive one or more administrations (e.g., by oral administration or by injection) of clomiphene citrate in combination with other triggering agents with a dose of 50-150 mg (e.g., 50-75 mg, 60-80 mg, 75-100 mg, 90-115 mg, 11 Q- 130 mg, 125-150 mg; e.g., 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 1 10 mg, 120 mg, 130 mg, 140 mg, 150 mg) of clomiphene citrate per injection for up to 8 days (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days).

[0516] Prior to receiving a triggering agent, a subject’s serum may be evaluated for levels of hormones or other relevant compounds. A subject may have serum levels of estradiol from about 250 pg / mL to about 400 pg / mL (e.g., from about 250 pg / mL to about 275 pg / mL, from about 275 pg / mL to about 300 pg / mL, from about 300 pg / mL to about 325 pg / mL, from about 325 pg / mL to about 350 pg / mL, from about 350 pg / mL to about 375 pg / mL, or from about 375 pg / mL to about 400 pg / mL; e.g., about 250 pg / mL, about 260 pg / mL, about 270 pg / mL, about 280 pg / mL, about 290 pg / mL, about 300 pg / mL, about 310 pg / mL, about 320 pg / mL, about 330 pg / mL, about 340 pg / mL, about 350 pg / mL, about 360 pg / mL, about 370 pg / mL, about 380 pg / mL, about 390 pg / mL, or about 400 pg / mL) prior to receiving a triggering agent. A subject may have serum levels of progesterone from about 0.25 ng / mL to about 0.75 ng / mL (e.g., from about 0.25 ng / mL to about 0.35 ng / mL, from about 0.35 ng / mL to about 0.45 ng / mL, from about 0.45 ng / mL to about 0.55 ng / mL, from about 0.55 ng / mL to about 0.65 ng / mL, or from about 0.65 ng / mL to about 0.75 ng / mL; e.g., about 0.25 ng / mL, about 0.30 ng / mL, about 0.35 ng / mL, about 0.40 ng / mL, about 0.45 ng / mL, about 0.50 ng / mL, about 0.55 ng / mL, about 0.60 ng / mL, about 0.65 ng / mL, about 0.70 ng / mL, or about 0.75 ng / mL) prior to receiving a triggering agent. A subject may have serum levels of LH from about 1 .0 mIU / mL to about 2.5 mIU / mL (e.g., from about 1 .0 mIU / mL to about 1 .5 mIU / mL, from about 1 .5 mIU / mL to about 2.0 mIU / mL, or from about 2.0 mIU / mL to about 2.5 mIU / mL; e.g., about 1 .0 mIU / mL, about 1 .25 mIU / mL, about 1 .5 mIU / mL, about 1 .75 mIU / mL, about 2 mIU / mL, about 2.25 mIU / mL, or about 2.5 mIU / mL) prior to receiving a triggering agent. A subject may have serum levels of FSH from about 11 mIU / mL to about 14 mIU / mL (e.g., from about 11 mIU / mL to about 12 mIU / mL, from about 12 mIU / mL to about 13 mIU / mL, or from about 13 mIU / mL to about 14 mIU / mL; e.g., about 1 1 mIU / mL, about 12 mIU / mL, about 13 mIU / mL, or about 14 mIU / mL) prior to receiving a triggering agent.

[0517] The triggering agent (e.g., a follicular triggering agent) may be administered over a course of time to produce a follicle stimulation protocol that is a minimal stimulation protocol. The minimal stimulation protocol is configured by a skilled practitioner to trigger the release of a cell in the span of about 3 days. A “minimal stimulation protocol” is a stimulation process spanning over a shortened period of time, PATENT

[0518] Attorney Docket No.: 51763-009WO2 compared to average in vitro fertilization (IVF) stimulation protocols, to aid in inducing an ovary to produce an oocyte. Typically, the average span of time for a stimulation protocol using standard IVF is approximately 8-14 days. The minimal stimulation protocol may induce the release of a cell in a span of 8 days or less (e.g. 8 days or less, 7 days or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, or 1 day; e.g., between 1 -3 days, between 2-4 days, between 3-5 days, between 4-6 days, between 5-7 days, or between 6-8 days; e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days), which is a shorted period of time compared to the 8-14 days of standard IVF stimulation protocols. The average time for performing a minimal stimulation protocol may be 2 days. The average time for performing a minimal stimulation protocol may be 3 days. The average time for performing a minimal stimulation protocol may be 4 days. The average time for performing a minimal stimulation protocol may be 5 days. The average time for performing a minimal stimulation protocol may be 6 days. In an embodiment, the minimal stimulation protocol may not require administration of a follicular triggering agent for successful retrieval and subsequent maturation of an oocyte. In an embodiment, the minimal stimulation protocol may include selecting a first triggering agent (e.g., a follicular triggering agent) and selecting a second triggering agent (e.g., a follicular triggering agent) as a function of a follicle measurement and / or other biological sample data.

[0519] Hi. Oocyte retrieval

[0520] Following follicular stimulation, oocytes (or a group of cells containing an oocyte) are retrieved from the subject. Approximately 24-48 hours (e.g., between 24-32 hours, between 32-40 hours, between 40-48 hours; e.g., about 24 hours, about 28 hours, about 32 hours, about 36 hours, about 40 hours, about 44 hours, about 48 hours) after final dose of triggering agent (e.g., a follicular triggering agent) that is administered, a subject may undergo an oocyte retrieval. On the day of oocyte retrieval, a blood test for one or more hormone levels such as E2, LH, FSH and / or P4 may be performed to ensure quality metrics, hormone levels are within range, and / or that hOG dose was ingested. Hormone levels of E2 may be from about 300 pg / mL to about 450 pg / mL (e.g., from about 300 pg / mL to about 350 pg / mL, from about 350 pg / mL to about 400 pg / mL, or from about 400 pg / mL to about 450 pg / mL; e.g., about 300 pg / mL, about 325 pg / mL, about 350 pg / mL, about 375 pg / mL, about 400 pg / mL, about 425 pg / mL, or about 450 pg / mL) on the day of oocyte retrieval. Hormone levels of LH may be from about 3 mIU / mL to about 6 mIU / mL (e.g., from about 3 mIU / mL to about 4 mIU / mL, from about 4 mIU / mL to about 5 mIU / mL, or from about 5 mIU / mL to about 6 mIU / mL; e.g., about 3 mIU / mL, about 3.5 mIU / mL, about 4 mIU / mL, about 4.5 mIU / mL, about 5 mIU / mL, about 5.5 mIU / mL, or about 6 mIU / mL) on the day of oocyte retrieval. Hormone levels of FSH may be from about 6 mIU / mL to about 9 mIU / mL (e.g., from about 6 mIU / mL to about 7 mIU / mL, from about 7 mIU / mL to about 8 mIU / mL, or from about 8 mIU / mL to about 9 mIU / mL; e.g., about 6 mIU / mL, about 6.5 mIU / mL, about 7 mIU / mL, about 7.5 mIU / mL, about 8 mIU / mL, about 8.5 mIU / mL, or about 9 mIU / mL) on the day of oocyte retrieval. Hormone levels of P4 may be from about 0.5 ng / mL to about 1 .5 ng / mL (e.g., from about 0.5 ng / mL to about 1 .0 ng / mL, from about 0.75 ng / mL to about 1 .0 ng / mL, from about 1 .0 ng / mL to about 1 .5 ng / mL, or from about 1 .25 ng / mL to about 1 .5 ng / mL; e.g., about 0.5 ng / mL, about 0.75 ng / mL, about 1 .0 ng / mL, about 1 .25 ng / mL, or about 1 .5 ng / mL) on the day of oocyte retrieval.

[0521] Oocytes (or a group of cells containing an oocyte) are retrieved from the subject using methods known in the art. For example, oocytes may be retrieved via aspiration using a transvaginal ultrasound PATENT

[0522] Attorney Docket No.: 51763-009WO2 with a needle guide on the probe to suction released follicular contents. Follicular aspirates may then be examined using a dissection microscope and washed with HEPES media (G-MOPS Plus, VITROLIFE®) and filtered with a 70-micron cell strainer (FALCON®, Corning). Oocytes and / or COCs are then transferred to culture dishes and media to begin co-culturing and appropriate controls, as described herein. Other retrieval methods may include an extraction device, such as a needle, syringe, vial, lancet, Evacuated Collection Tubes (ECT), tourniquet, vacuum extraction tube systems, any combination thereof and the like. For example, the extraction device may comprise a butterfly needle set.

[0523] A retrieved oocyte may include but is not limited to an immature oocyte, a mature oocyte, a group of one or more oocytes, a group of one or more cells, such as a cumulus oocyte complex (e.g., a cumulus oocyte complex that contains a mature or an immature oocyte), among other examples.

[0524] At the time of retrieval, any one or more of the retrieved oocytes or cells described herein may be appropriately frozen and stored using methods known in the art for future use, analysis, or experimentation. Additionally, any one or more of the retrieved oocytes or cells described herein may be used fresh (i.e., ready for immediate use such as use for in vitro maturation or any one or more analyses or experimentation described herein).

[0525] Following oocyte retrieval, one or more COCs may require oocyte denudation. As described in this disclosure, “oocyte denudation” refers to the removal of cumulus cells or other cell types from the oocyte by means of mechanical separation, chemical separation, or combinations thereof. Several methods of oocyte denudation are known in the art. In some embodiments, denudation may occur in a IVM well, by gently mechanically disassociating cells by pipetting to remove most cumulus and / or granulosa cells. If enzymatic disassociation is needed, the cells may be transferred to a separate dish for hyaluronidase treatment. COCs may be stripped with stripper tips and washed in IVM media or MOPS plus media to clean the oocyte for imaging and if needed inactivate hyaluronidase. Stripper tips may include 200 micron and / or 400 microns for fine cleaning. In some embodiments, germinal vesical (GV)- stage) and metaphase I (Ml)-stage oocytes may be formulated and utilized in cultivation following denudation of the COCs. Denuded COCs may be transferred to a separate culture dish for imaging. iv. Co-culture contents and timing

[0526] In some embodiments of the method, a cell culture may be formed by combining an immature oocyte with a population of engineered OSCs (e.g., theca cells, granulosa cells, ovarian stroma cells, and / or lutein cells), which is added to mature the oocyte in the cell culture and thus create a COC after extraction of one or more oocytes following the minimal stimulation protocol. In an embodiment, one or more specialized granulosa cells and / or specialized stroma cells may be thawed during a resting period of one or more COCs. In an embodiment, anywhere from between 50,000-150,000 specialized granulosa cells (e.g., 50,000-60,000 cells, 60,000-70,000 cells, 70,000-80,000 cells, 80,000-90,000 cells, 90,000- 100,000 cells, 100,000-110,000 cells, 110,000-120,000 cells, 120,000-130,000 cells, 130,000-140,000 cells, or 140,000-150,000 cells; e.g., 50,000 cells, 55,000 cells, 60,000 cells, 65,000 cells, 70,000 cells, 75,000 cells, 80,000 cells, 85,000 cells, 90,000 cells, 95,000 cells, 100,000 cells, 105,000 cells, 110,000 cells, 1 15,000 cells, 120,000 cells, 125,000 cells, 130,000 cells, 135,000 cells, 140,000 cells, 145,000 cells, or 150,000 cells) may be combined with a COC during culturing. In an embodiment, thawed specialized granulosa cells may be placed into a culture media prior to COC retrieval, including anywhere from about 24-120 hours beforehand (e.g., about 24-48 hours, about 48-72 hours, about 72-96 hours, PATENT

[0527] Attorney Docket No.: 51763-009WO2 about 96-120 hours; e.g., about 24-36 hours, about 30-40 hours, about 36-48 hours, about 48-56 hours, about 56-72 hours, about 72-84 hours, about 80-96 hours, about 90-100 hours about 96-108 hours, about 108-120 hours; e.g., about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 56 hours, about 60 hours, about 65 hours, about 72 hours, about 78 hours, about 86 hours, about 92 hours, about 96 hours, about 102 hours, about 110 hours, about 115 hours, about 120 hours). A COC may be transferred into culture media containing thawed specialized granulosa cells to form a group culture as described below in more detail. In an embodiment, a group culture may be cultured in an incubator ranging in time from anywhere between 12-48 hours (e.g., 12-16 hours, 12-20 hours, 18-24 hours, 18-36 hours, 24-36 hours, 36-48 hours; e.g., 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 hours). The co-culture may be conducted at a biologically suitable temperature, e.g., 37°C.

[0528] In some embodiments of the method, a retrieved oocyte, including immature cumulus-oocyte complexes, may be cultured in a group culture. A “group culture” is an extracted COC combined with one or more additional cells. An additional cell may include any cell grown together with an extracted COC. An additional cell may include a specialized stroma cell. An additional cell may include a specialized granulosa cell. In an embodiment, a group culture may be cultured and / or incubated for a particular length of time, such as from between 12-120 hours (e.g., 12-24 hours, 12-36 hours, 24-48 hours, 36-60 hours, 54-72 hours, 68-96 hours, 96-120 hours; e.g., 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 hours, 50 hours, 52 hours, 54 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, 72 hours, 74 hours, 76 hours, 78 hours, 80 hours, 82 hours, 84 hours, 86 hours, 88 hours, 90 hours, 92 hours, 94 hours, 96 hours, 98 hours, 100 hours, 102 hours, 104 hours, 106 hours, 108 hours, 110 hours, 1 12 hours, 114 hours, 116 hours, 118 hours, or 120 hours). For example, group culturing may include culturing the COCs with a granulosa co-culture as described further below. In some embodiments, group culturing may include culturing a control group of COCs with no co-culture, as described further below. In some embodiments, a user may donate immature oocytes, such as GV-stage and Ml-stage oocytes that may be used in medium as part of the group culture to help grow COCs. Oocyte donation may follow an oocyte retrieval process as discussed above. A subject participating in oocyte donation may be different, or the same, from the subject related to the second biological sample containing immature COCs. In some embodiments, an oocyte donation subject may undergo a stimulation protocol as disclosed above.

[0529] In some embodiments, the maturity of the oocyte retrieved from the subject may dictate the length of time during which the oocyte is co-cultured with one or more OSCs (e.g., theca cells, granulosa cells, ovarian stroma cells, and / or lutein cells). For example, less mature oocytes (e.g., GV oocytes) may require longer co-culturing periods than oocytes at a more advanced stage of meiosis (e.g., Ml oocytes).

[0530] The cell culture medium for culturing oocytes may comprise any one of the cell culture medium formulations described herein (e.g., Section IB). In some embodiments, include LAG media (Medicult, COOPERSURGICAL®). For example, LAG media may be used for the incubation of oocytes and / or COCs post-retrieval from minimal stimulation protocol. For example, a modified-Medicult IVM media may be used as a baseline control during the culturing process. The cell culture medium may be supplemented with human serum albumin (HSA) (e.g., at about 5-15 mg / mL, e.g., 10 mg / mL), FSH (e.g., at about 70-80 mIU / mL, e.g., 75 mIU / mL), human chorionic gonadotropin (hCG) (e.g., at about 95-105 PATENT

[0531] Attorney Docket No.: 51763-009WO2 mIU / mL, e.g., 100 mIU / mL), androstenedione (e.g., at about 495-505 ng / mL, e.g., 500 ng / mL), doxycycline (e.g., 0.5-1 .5 pg / mL, e.g., 1 pg / mL) and other compounds such as hyaluronidase and / or Dulbecco’s phosphate buffered saline (dPBS). Media may be equilibrated for about 18 to 24 hours (e.g., about 18 hours, about 20 hours, about 22 hours, about 24 hours) pre-culture in a standard sterile 37°C incubator with 02 (e.g., having a 1 -10% 02 atmosphere, such as 4-8% 02 or 5-7% 02, e.g., 6% 02) and proper CO2 levels, which are known in the art.

[0532] Co-cultures and specialized granulosa cell cultures may be adherent cell cultures in cell culture dishes or flasks. Co-cultures and specialized granulosa cell cultures may be suspension cell cultures in cell culture flasks. Cell culture materials and methods include standard sterile cell culturing methods known in the art. Cell morphology and cell viability may be evaluated via one or more established methods known in the art. v. Oocyte scoring

[0533] At any stage of in vitro maturation or directly following in vitro maturation, an oocyte and / or granulosa cells may be appropriately frozen and stored for future analyses, experimentation, or for use in oocyte maturation. Oocytes may be scored with a scoring metric based on their morphology as determined by imagine analysis. In some embodiments, assignment of the scoring metric may include imaging the group cultures and analyzing the images of one or both of co-culture and no co-culture growth media-only control groups. In some embodiments, oocytes are scored and comparatively analyzed during any such stage of in vitro maturation. For example, group culture images may contain a pre-culture group COC image, a post-culture group COC image, and a post-culture denuded oocyte image. In some embodiments, oocytes subjected to scoring have never been frozen. In some embodiments oocytes subjected to scoring via image analysis may be thawed after storage by freezing. In some embodiments, oocytes subjected to scoring may be retrieved without in vitro maturation as described. In some embodiments, oocytes subjected to scoring may be cultured without described granulosa. In some embodiments, images may be sent to a qualified third party, such as an embryologist, developmental biologist, or other relevant skilled practitioner for scoring assignment.

[0534] In some embodiments of the methods described herein, oocytes may be assessed and subsequently classified by their maturation state according to the following criteria:

[0535] GV - presence of a germinal vesicle, typically containing a single nucleolus within the oocyte. Ml - absence of a germinal vesicle within the oocyte and absence of a polar body in the perivitelline space between the oocyte and the zona pellucida.

[0536] Mil - absence of a germinal vesicle within the oocyte and presence of a polar body in the perivitelline space between the oocyte and the zona pellucida.

[0537] In some embodiments of the method, the scoring metric may include total oocyte scoring (TOS) as a function of analyzing the imaged group cultures via relevant microscopy or imaging analysis software. Methods and approaches of TOS have been described in the art (Lazzaroni-Tealdi et al., PLoS One 10:e0143632, 2015). Oocyte scoring may include metrics such as shape, size, ooplasm characteristics, structure of the perivitelline space (PVS), zona pellucida (ZP), polar body (PB) morphology, among other possible qualifiers. Total oocyte scoring on both pre and post culture oocyte images for generation of the TOS metric may be based on a scale system of -6 to + 6. PATENT

[0538] Attorney Docket No.: 51763-009WO2

[0539] Regarding oocyte shape, if oocyte morphology is poor (dark general oocyte coloration and / or ovoid shape), it may be assigned a value of -1 ; if it is almost normal (less dark general oocyte coloration and less ovoid shape), it may be assigned a value of 0; if it is judged to be normal, it may be assigned a value of + 1 . Regarding oocyte size: if oocyte size is defined as abnormally small or large, it may be assigned a value -1 if size is below 120 pm or greater 160 pm. If the size is almost normal, i.e., does not deviate from normal by more than 10 pm, a value of 0 may be assigned, and a value of + 1 may be assigned if oocyte size is within normal range > 130 pm and <150 pm. Regarding ooplasm characteristics, if the ooplasm is very granular and / or very vacuolated and / or demonstrates several inclusions, a value of -1 may assigned. If it is only slightly granular and / or demonstrates only few inclusions, a value of 0 may be assigned. Absence of granularity and inclusions may result in a +1 value. Regarding structure of the perivitelline space (PVS), the PVS may defined as -1 with an abnormally large PVS, an absent PVS or a very granular PVS. It may be assigned a value of 0 with a moderately enlarged PVS and / or small PVS and / or a less granular PVS. A value of +1 may be assigned to a normal size PVS with no granules. Regarding, zona pellucida (ZP), if ZPs is very thin or thick (<10 pm or >20 pm) the oocyte may be assigned a -1 . If the ZP does not deviate from normal by more than 2 pm it may be assigned 0. A normal zona (> 12 pm and <18 pm) may be assigned a +1 . Regarding polar body (PB) morphology, PB morphology is defined as follows: Flat and / or multiple PBs or zero PBs, granular and / or either abnormally small or large PBs is designated as -1 . PBs, judged as fair but not excellent may be designated as 0, and a designation of +1 may be given to PBs of normal size and shape. In some embodiments, Mil oocytes PB score may not be aggregated into TOS.

[0540] In some embodiments of the method, the scoring metric may include performing an outcome analysis as a function of the TOS. Parametric or non-parametric tests may be applied to determine the significance of findings during the analysis. Outcome analysis may be used to determine GV-stage to Milstage oocyte maturation rate; GV-stage to Ml-stage oocyte maturation rate; Ml-stage to Mil-stage oocyte maturation rate; Average Total Oocyte Score; Average Oocyte Shape; Average Oocyte Size; Average Ooplasm quality; Average PVS quality; Average ZP quality; Average Polar Body quality, and the like. In some embodiments these outcomes may reported as a as mean, median, and deviation. vi. In vitro fertilization and embryo culture

[0541] In some embodiments of the methods, any one or more ova or oocytes as described herein may be evaluated for quality or maturation state, such as by the scoring metrics described herein, to determine their readiness for use in in vitro fertilization and embryo formation.

[0542] In some embodiments of the method, the ova or oocytes may be matured via in vitro maturation and subsequently utilized for IVF and / or ART as described herein. Any one or more oocytes may be utilized for intracytoplasmic sperm injection (ICSI). Following fertilization of the ovum by contact with one or more sperm cells, the subsequently formed zygote can be matured ex vivo so as to produce an embryo, such as a morula or blastula (e.g., a mammalian blastocyst), which can then be transferred to the uterus of a subject (e.g., a subject from which the oocyte was initially harvested) for implantation into the endometrium. Embryo transfers that can be performed using the methods described herein include fresh embryo transfers, in which the ovum or oocyte used for embryo generation is retrieved from the subject and the ensuing embryo is transferred to the subject during the same menstrual cycle. The PATENT

[0543] Attorney Docket No.: 51763-009WO2 embryo can alternatively be produced and cryopreserved for long-term storage prior to transfer to the subject.

[0544] III. Kits or articles of manufacture

[0545] The compositions or methods described herein can be provided in a kit for use in reprogramming iPSCs (e.g., hiPSCs) into a population of OSCs (e.g., theca cells, granulosa cells, stroma cells, and / or lutein cells). In some embodiments, the compositions and methods described herein can be provided in a kit for use in producing an implant for in vivo use. In some embodiments, the compositions and methods described herein can be provided in a kit for use in producing an organoid such as an ovaroid or a uteroid. In some embodiments, the compositions and methods described herein can be provided in a kit for use in co-culturing one or more oocytes with OSCs to produce one or more mature oocytes, optionally wherein the resulting mature oocytes are further fertilized to form an embryo in an ART or IVF procedure. In some embodiments, the kit may include a package insert that instructs a user of the kit to perform iPSC differentiation and / or assembly of an implant in a matrix. In some embodiments, the kit may include a package insert that instructs a user of the kit to perform any one of the methods of administering an OSC implant to a subject. In some embodiments, the kit may include a package insert that instructs a user of the kit to perform ovarian stimulation and / or oocyte retrieval described herein. The kit may optionally include a syringe or device for administering the compositions of the present disclosure or for retrieving an oocyte. In some embodiments, the kit may include one or more additional cell media or agents used for cell culture. In some embodiments, the kit includes one or more antibodies or binding molecules to detect the expression of one or more genes or biomarkers described herein.

[0546] EXAMPLES

[0547] Example 1. A method of producing ovarian support cells by reprogramming induced pluripotent stem cells

[0548] This example demonstrates methods of producing OSCs from iPSCs and confirming cellular identity by determining the expression of biomarkers that correspond to naturally occurring OSCs (e.g., OSCs in vivo). In addition to demonstrating that the produced OSCs share a gene expression profile to their naturally occurring cellular counterparts, this example also shows that the produced OSCs are functionally equivalent to naturally occurring OSCs.

[0549] Induced pluripotent stem cells (iPSCs) are derived from adult cells that have been reprogrammed into stem cells with the potential to differentiate into any cell type in the body and, as a result, have widespread applications in biomedicine. We have applied a combinatorial and technical platform to engineer iPSCs with inducible transcription factors that drive differentiation towards ovarian cell types. These cells, which are called ovarian support cells (OSCs), express protein markers similar to granulosa cells, the essential functional cells of the ovary, and exhibit similar transcriptomic and steroidogenic profiles. These OSCs provide a much more physiologically relevant, ovarian-like dynamic environment for IVM than media alone, leading to improved maturation outcomes and therefore creating significant potential for the engineered cells of the invention as an ART to improve fertility treatments in the clinic. Detailed in this example is the development and optimization of approaches for consistent manufacturing at scale under good manufacturing practice conditions with animal origin-free materials without impacting product purity, efficacy, or safety. Also provided is a case study and methodology for clinical translation of PATENT

[0550] Attorney Docket No.: 51763-009WO2 both iPSCs in cell therapy and ART for fertility treatments, showing that a strategic plan for scalable and controlled manufacturing ultimately creates a much more consistent and functional product.

[0551] / '. Transcription factor mediated differentiation consistently generated ovarian support cells in different stages of ovarian development and folliculogenesis

[0552] To evaluate the feasibility of utilizing a gene-modified hiPSC line harboring three inducible transcription factors (NR5A1 , RUNX2, and GATA4) as a source to generate consistent and functional OSCs, we compared 6 independent batches of hiPSC-derived OSCs differentiated across 8 months by multiple operators following a standard operating procedure. After 5 days of induction, hiPSCs multiplied 5.63±2.85 times, and acquired morphological features that resembled human granulosa cells, such as clusters of cells with spiky edges and granules observed in the cell body (FIG. 1A). Differentiated OSCs also expressed FOXL2 and CD82, two well-characterized markers of granulosa cell-fate, indicating successful differentiation into the desired cell type (FIG. 1 B).

[0553] To further characterize the molecular phenotype of the differentiated OSCs, as well as better understand differences and similarities among independent batches, we performed single cell RNA- sequencing (scRNA-seq) on cryopreserved samples from six batches of differentiation (FIG. 1C). We identified 15 initial Leiden clusters that were combined by molecular similarities, resulting in nine final clusters. Among the 15 initial Leiden clusters identified, 12 of them expressed markers that are differentially expressed in human granulosa cells (GJA1 , MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1 , COL4A2, LAMC1 , ITGAV, ITGB1) compared to other cell types in the developing ovary. These clusters were all identified as granulosa cells, and therefore assigned as the two major classes: Early GCs or GCs. The remaining three clusters were included in a third major class identified as Others, as expression of major granulosa markers was not evident in these groups of cells (FIG. ID).

[0554] Despite the expression of all the granulosa markers, the class assigned as Early GCs also shared transcriptional similarities to preGC-l and -lla / llb subclusters, including expression of the genes FOXO1 and CDH1 (FIG. 1 D). A subcluster of these cells, labeled as Early GC I expressed the aromatase gene, CYP19A1 , which has been described to be upregulated in preGC-ls in the ovarian medulla, as well as the gene for the chemotactic protein, RARRES2, which has been shown to reduce steroidogenesis and block oocyte meiotic progression in bovine models. The subcluster Early GC II also expressed the gene for RARRES2, similarly to the previous subcluster described, in addition to the receptor NOTCH2 (FIG. 1D). The NOTCH signaling pathway is involved in the oocyte-GC crosstalk during folliculogenesis, and high levels of expression of NOTCH2 and NOTCH3 in cumulus cells have been positively correlated with IVF response. Finally, in the subcluster Early GC III, RARRES2 expression was no longer observed, as in the previous subcluster, while NOTCH2 expression continued to be detected in significant levels. Together, these patterns of expression suggested that the clusters in the Early GC class shared transcriptional signature with both granulosa cells and preGC-l and -lia / lib, and differential expression of CYP19A1 , RARRES2, and NOTCH2, suggested a gradual developmental and functional progression from the subcluster Early GC I to Early GC III.

[0555] The class of GCs was marked by the expression of CDH2 in addition to all the other granulosa markers previously described, including the NOTCH2 / 3 receptors (FIG. 1D). The subcluster GC I was enriched for the genes NRG1 , BMPR1 B, and genes of the ERBB family of receptors (FIG. 1D). NRG1 has been identified to be differentially expressed in preGC-l la / llb and was found to be expressed and PATENT

[0556] Attorney Docket No.: 51763-009WO2 secreted by granulosa cells in response to ovulatory surge. BMPR1 B, EGFR (ERBB1), and ERBB4 are all receptors identified in granulosa cells and have counterpart ligands expressed in oocytes (BMP6, TGFA, and NRG4, respectively). These interactions have been proposed to mediate follicular assembly. The subcluster GO II was enriched by expression of the gene ID3, which is a target of the receptor BMPR2, also expressed by these cells. Interestingly, although BMPR2 was expressed by all Early GCs and GO clusters, the CG II subcluster was the subcluster with the strongest enrichment of this target gene (FIG. 1D), suggesting activation of the receptor BMPR2 in these cells. The last subcluster from the GO class, GO III, was composed of cells expressing both CDH2 and NOTCH2, but this subcluster was not enriched for any of the other genes previously described in the subclusters for this class. These data suggested that the three subclusters of GCs represented ovarian support cells in slightly different cell states that were mediated by a distinct combination of active signaling pathways.

[0557] The last three subclusters identified (Atresia / Luteolysis, Ribosomal enriched, and Mitochondrial enriched) were incorporated into a third class labeled as Others. These subclusters had overall lower expression of most markers including GJA1 and CDH2 (FIG. 1E). Lower expression levels of GJA1 and CDH2 have been described in GCs undergoing early stages of atresia. Interestingly, cells on the Atresia / Luteolysis subcluster also expressed genes involved in steroidogenesis, such as CYP11 A1 , CYP19A1 , and HSD17B1 , as well as CGA, which is an estrogen receptor alpha-responsive gene in human breast cancer cells. The other two clusters were also enriched for the GCA gene, but the top expressed genes in each of the clusters are either mitochondrial genes in the Mitochondrial enriched subcluster or ribosomal genes for the Ribosomal enriched subcluster. Generally, enrichment of mitochondrial and / or ribosomal genes in scRNA-seq analysis is associated with poor quality cells, further suggesting that these clusters were composed of dying cells.

[0558] After identifying that most of the cells in our analysis were classified as granulosa cells (Early GCs and GCs), we sought to understand whether our protocol gave rise to OSCs in different stages of fol liculogenesis or whether cells were overrepresented by a specific follicular stage. For that, we leveraged as a reference a published transcriptome landscape of human folliculogenesis to generate gene signature scores that were then applied to our samples. We did not observe a clear representation of either the Primary GC or Secondary GC stages within our samples, and most of the genes associated to these signature scores were not enriched in the analyzed cells. Conversely, the signature scores for Antral GC and Pre-ovulatory GC were more clearly represented within the clusters identified in our analysis, and multiple genes driving these signatures seemed to be enriched by multiple clusters (FIG. 1E).

[0559] Following characterization of the cellular outcome resultant from our differentiation process, we investigated the reproducibility and consistency of these methods across independent batches of hiPSC- derived OSCs. Overall, all batches analyzed consistently generated clusters from the three major classes previously described (Early GCs, GCs, and Others), five of which were very similar in terms of cluster distribution per batch (FIG. IF). These results demonstrated a consistent methodology of producing OSCs from reprogrammed hiPSCs for clinical use for ART or fertility treatments.

[0560] / ' / . In vitro maturation of human oocytes was robustly achieved by multiple batches of hiPSC- derived ovarian support cells

[0561] \Ne next examined whether the functional readouts of independent batches would have variable PATENT

[0562] Attorney Docket No.: 51763-009WO2 or correlated cellular outcomes. To assess the functional readout of OSCs, we leveraged IVM protocols, in which OSCs are co-cultured with immature oocytes retrieved from individuals undergoing abbreviated gonadotropin stimulation and Mil formation was recorded as an endpoint of oocyte maturation (FIG. 3A). To capture the different spectrum of cell composition variability among the six batches analyzed by scRNA-seq, we performed functional analysis on lot 6, which was overall more represented by GO clusters; lot 8, which contained a balanced representation of Early GO and GO clusters; and lot 56, which was more represented by Early GO and Other clusters, with a lower contribution of the GO clusters (FIG. 1F). We utilized state of the art Medicult IVM media that is typically used for IVM as the baseline control to assess positive functional readout of IVM induced by the addition of OSCs to the medium (FIG. 2A). For the initial analyses, oocytes retrieved from each donor were split into the two conditions (Control-IVM and OSC-IVM) prior to maturation and Mil formation rate was recorded among the different groups. We demonstrated that all three individual batches analyzed successfully led to higher Mil formation rates as compared to the control (p=0.021 , lot 6 / control: 1 .37, lot 8 / control: 1 .31 , lot 56 / control: 1 .28) (FIG. 2B). These results indicated that variable cellular outcomes robustly retained the ability to improve human oocyte maturation rate compared to control, suggesting that both GC and Early GC OSC clusters may contribute to oocyte maturation.

[0563] To gain insight into the potential mechanism of action associated with these IVM results, we analyzed the expression of key receptors, ligands, and target genes that have an important role in oocyte and ovarian support cell interactions and compared this expression among different classes of cells identified in our samples. Comparison of relative expression of multiple pairs of ligand and receptors indicated that GC and Early GC clusters expressed relatively higher levels of BMP4, EFNB2, TGFBR1 , BMPR2, NOTCH2, NOTCH3, and CD46, suggesting a potential involvement of one of these elements as part of the mechanism of action of these cells (FIG. 2C). Other receptors such as STRA6, ERBB4, RARRES2, and EGFR, were also detected particularly in the Early GC clusters, suggesting that these genes may not be the primary drivers of oocyte maturation (FIG. 2C). Additionally, we assessed the expression of growth factors that are modulated by oocyte-somatic cell interactions and play a role in oocyte maturation and folliculogenesis. Among the genes differentially expressed across clusters, TGFB1 and TGFB2 were more enriched in the GCI cluster, while VEGFA and VEGFB, in addition to BMP7 and PDGFA, were generally more abundant in the Early GC cluster (FIG. 2D). The Early GC and GC clusters were enriched for IGF2BP1 , IGF2BP2, and IGF2BP3 (FIG. 2D).

[0564] Hi. Translation of the protocol toward clinical manufacturing led to more reproducible cellular outcomes

[0565] As part of the strategy to translate the research manufacturing protocol towards clinical standards, we performed a risk assessment on our bill of materials, which led to the substitution of key components of the protocol with higher quality alternatives, including animal origin free reagents, GMP manufactured components, and cell-therapy grade raw materials (FIG. 3A). We further sought to understand additional factors and reagents as variables that influence OSC differentiation to drive greater optimization and ensure reproducibility at the manufacturing stage for improved efficacy and safety.

[0566] The combined effects of the inducible transcription factors with media supplements and cell substrates in the growth media environment was investigated. To systematically evaluate the effects of multiple variables simultaneously, we employed Design of Experiments (DOE) to create a custom design PATENT

[0567] Attorney Docket No.: 51763-009WO2 that included center points for each factor and was optimized for D-optimality criterion, which is an experimental design matrix that allowed for maximization of efficiency and accuracy and minimization of uncertainty in the response parameters. For responses in the design, FOXL2 expression and viability was chosen, as FOXL2 is a biomarker of OSCs and viability screens for factors that are essential for manufacturing.

[0568] We demonstrated that the cell substrate has a clear and strong influence on FOXL2 expression that was far greater than any other variable (FIG. 3B; p-value < 0.01). These results emphasized the importance of substrate matrix selection for iPSCs differentiation. The DOE study further demonstrated that the duration of doxycycline treatment was an important factor, while doxycycline concentration was not as significant. Controls that included doxycycline-free treatment groups demonstrated a positive correlation between doxycycline and FOXL2 expression, underscoring the necessity of transcription factor induction and providing further validation of our TF-mediated methodology. Additionally, chemical antioxidant supplementation in the medium negatively impacted cellular viability and was subsequently excluded from manufacturing. These results confirmed that transcription factors drive differentiation independent of added small-molecule components. Interestingly, these data regarding cell substrate influence led us to further optimize substrates for producing desired OSCs from iPSCs.

[0569] Among all the raw materials utilized during the generation of OSCs for research purposes, one of the reagents with the highest complexity was matrigel, which is derived from Engelbreth-Holm-Swarm mouse sarcoma cells and contains multiple extracellular matrix components of tissue basement membranes. Due to the source and inherent complexity of this reagent, as well as the nature of its production, matrigel has significant lot-to-lot variability, which can impact the overall reproducibility of the final differentiated cell product (e.g., OSCs).

[0570] We directly compared differentiation of hiPSCs that were cultured on either of the alternative substrates, human recombinant laminin-521 or vitronectin. Initial assessment of cellular morphology during the differentiation process indicated subtle differences between the experimental groups (FIG. 3C). Vitronectin-OSCs presented a larger cell body and organized themselves in more sparse clusters of cells, while laminin-OSCs were smaller and organized into compact groups of cells (FIG. 3C). Expression of CD82 was consistent between the two groups (FIG. 3D). To better understand the molecular profile of OSCs produced by these different matrix conditions and the reproducibility, we performed scRNA-seq in two batches of laminin-OSCs and two batches of vitronectin-OSCs and compared these data to our initial datasets (FIGS. 3E-3F). Laminin-OSCs were mostly distributed among subclusters GC I, GC II, GC III (FIGS. 3E-3F). In contrast, vitronectin-OSCs were primarily represented by Early GC II, Early GC III, and GC III subclusters (FIGS. 3E-3F). Notably, OSCs differentiated in media with vitronectin matrices had a higher percentage of cells in Mitochondrial enriched and Ribosomal enriched subclusters (FIGS. 3E-3F). Interestingly, expression of N-cadherin (CDH2), a hallmark of GC subclusters that was not present in the Early GC subclusters, has been described to protect granulosa cells from apoptosis associated with follicular atresia and luteolysis. Additionally, vitronectin has been demonstrated to be upregulated in porcine atretic follicles. Collectively, these data suggested that there was an association between vitronectin matrices and higher percentages of cells in the Others class (i.e., Mitochondrial enriched, Ribosomal enriched, atresia / luteolysis).

[0571] Despite the differences observed in cellular outcome generated from these two matrix conditions, major differences were not observed in cluster distribution among the two independent batches therein PATENT

[0572] Attorney Docket No.: 51763-009WO2

[0573] (FIGS. 3G-3H), suggesting that changes in the bill of materials to include higher quality reagents yielded consistent and reproducible cellular outcomes, independent of the matrix utilized. It is also important to highlight that each independent batch of differentiation was performed by a different operator, which strengthened the evidence of reproducibility. Importantly, these results demonstrated that the final OSC fate was impacted by not only the overexpression of the three transcription factors but was also significantly influenced by the matrix utilized as the substrate during differentiation (FIGS. 3G-3H). iv. Differentiation over laminin-521 led to a scalable, pure, and functional population of ovarian support cells

[0574] After ensuring that transition to an overall higher quality bill of materials did not negatively affect reproducibility or final cellular outcome, we investigated which of the two conditions (laminin-521 and vitronectin) yielded better clinical outcomes for optimal clinical manufacturing. As a measurement of successful clinical outcome, we considered a few parameters that would directly inform throughput, safety, and potency of each condition. For throughput, we compared the ratio of OSC:hiPSC for each batch that was analyzed per condition (Table 1). The condition that yielded more viable cells at the time of harvest, without changing the initial cell number or surface area of the cell culture, would be more scalable. Laminin-OSCs were harvested at 94.63±0.01% viability and during differentiation were multiplied at a ratio of 14.83±4.48 OSC:hiPSCs (Table 1). In contrast, vitronectin-OSCs were harvested at 87.00±0.08% viability and were multiplied at a ratio of 6.49±1 .43 OSC:hiPSC. (Table 1).

[0575] Table 1 : Ovarian support cell batch production and specifications

[0576] Abbreviations: RUO: research-use only; hiPSC: human induced pluripotent stem cells; M: Matrigel; V: vitronectin; L: laminin; OSC: ovarian support cells; CG: clinical-grade; XF: xeno-free; OP: operator

[0577] We next investigated whether the final cellular composition was functional and could promote maturation of human oocytes. We co-cultured both laminin-OSCs and vitronectin-OSCs independently PATENT

[0578] Attorney Docket No.: 51763-009WO2 with immature human oocytes following a similar approach discussed herein and scored the yield of oocyte maturation in each group. Co-culture with laminin-OSCs and vitronectin-OSCs led to a higher MH formation rate compared to the control (p=0.018, lot 41 / control:! .08, lot 49 / control: 1 .36, lot 86 / control: 1 .27) (FIGS. 4A-4B). Although both approaches seemed to have generated functional OSCs that contributed to successfully increasing oocyte maturation rate compared to control conditions, the vitronectin-OSC condition resulted in variable functional outputs (FIG. 4B). Overall, although MH formation rates were slightly higher in the laminin-OSCs group comparatively, both approaches generated functional OSCs that contributed to successfully increasing oocyte maturation rates as compared to control media-only conditions.

[0579] Despite the overall higher rates of Mil maturation in both laminin-OSC and vitronectin-OSC compared to control, it is clear that these two conditions are composed of cells with different phenotypic compositions (FIGS. 3C-3H) and may drive oocyte maturation through different mechanisms. To further investigate potential OSC-oocyte interactions and involvement of key signaling pathways associated with follicle assembly and oocyte meiotic progression, we utilized data from endogenous tissue to characterize how these genes were expressed in OSCs from each condition (FIGS. 4C-4D). Overall expression of ligand-receptor patterns was similar between vitronectin-OSC group and laminin-OSC group, with BMPR1 B slightly more enriched in the laminin-OSC group compared to the vitronectin-OSC group (FIG. 4C). This suggested that distinct subgroups of cells (i.e., Early GC and GC clusters) were likely equally receptive to paracrine and / or autocrine signaling. Comparison of expression pattern of growth factor genes among both groups indicated a few differences (FIG. 4D). For instance, VEGFA and VEGFB as well as PDGFA were more enriched in the Early GCs and therefore more enriched in the vitronectin-OSC samples. In contrast, BMP7 had greater expression in the Early GC II, GC I, and GC III clusters of the laminin-OSC samples (FIG. 4D). v. Generation of a clinical-grade hiPSC line with the ability to generate functional ovarian support cells

[0580] The preceding experiments in this Example were performed with the hiPSC line GC3, which is a cell line that is designated for research-use-only (RUO) and not a clinical-grade cell line. Thus, we next sought to generate a clinical-grade hiPSC line from an allogeneic female donor for commercial clinical- grade materials. To minimize discrepancies between the results from the clinical-grade hiPSC line and the original RUO hiPSC line, which provided the foundation for initial preclinical studies and potency tests, we applied the same manufacturing strategy as the RUO hiPSC line to generate the clinical-grade hiPSC line.

[0581] The clinical-grade hiPSC line (CG-hiPSC) was engineered to harbor inducible versions of the three transcription factors that drove differentiation into OSCs, namely NR5A1 , RUNX2, and GATA4. Individual clones were generated by limiting dilution of the pooled engineered population and then expanded into seed banks. Clones that were successfully expanded were initially screened by genotyping PCR to confirm the integration of the three transcription factors (FIG. 5A). Nine seed clones harboring all the transcription factors were selected to proceed with a more in-depth screening process, which included assessing their identity, potency, and safety. To this end, each clone was individually differentiated into OSCs (FIG. 5B), to identify lead candidate clones. To specifically assess clonal identity, we verified expression of the OSC markers, FOXL2 and CD82 after 5 days of differentiation and confirmed that PATENT

[0582] Attorney Docket No.: 51763-009WO2 despite minor expression level variability among clones, all clones were positive for both markers, indicating successful generation of OSCs (FIG. 5B). Moreover, we confirmed that the level of the hiPSC marker OCT4 was null or very low to verify efficient and pure OSC outcome (FIG. 5B).

[0583] As a functional readout of individual clones, cells were differentiated for 5 days and exposed to follicle stimulating hormone (FSH, #2), Androstenedione (A4, #3) or a combination of both (FSH+A4, #4) hormones for 48 hours (FIG. 5C). Functional OSCs generated estradiol (E2) in response to FSH, and A4 was used as a substrate by the OSCs to complete the reaction. We observed that individual clones had variable responses following treatment with FSH+A4. Clones that were more responsive generally exhibited improved performance while maturing immature human oocytes (FIG. 5C). Treatment with FSH or A4 (#2 and #3) alone enabled identification of clones that were intrinsically steroidogenic, which is an indication of an immature profile. To gain a more comprehensive overview of the molecular signature of the individual clones, we performed bulk RNA-sequencing of all clones individually and assessed expression of granulosa cell markers, as well as hiPSC cell markers (POU5F1 and NANOG) (FIG. 5D). All clones robustly expressed established granulosa cell markers (FOXL2, STAR, GJA5), including genes related to important signaling pathways (NOTCH3, HES1 , ID3, KITLG) (FIG. 5D), suggesting that despite the functional differences observed among clones (FIG. 5C), differences in marker gene signatures were less pronounced among CG-OSC clones. Based on the attributes previously described, in addition to the ratio OSC:hiPSC and viability at harvest, we identified the clone 2-D10 as the top lead candidate (hereafter referred to as CG-hiPSC) (FIG. 6E). This selection of the top candidate was primarily based on levels of FOXL2 and CD82 expression, as well as its responsiveness to FSH and A4 as measured by E2 production (FIG. 5E). vi. Clinical-grade hiPSC line generated for clinical manufacturing showed reproducible differentiation and comparable molecular profiling to the research-use only cell line To further characterize CG-hiPSC for clinical applications, we assessed and confirmed the presence of hiPSC markers, as well as confirmed cell identity and normal karyotype. We then generated two independent batches of differentiated CG-hiPSC, leveraging the protocol previously identified as the most appropriate to be transitioned into clinical manufacturing. More specifically CG-hiPSCs were differentiated on dishes coated with a laminin-521 matrix. As expected, cell morphology upon differentiation was characterized by small cells with granules in the cell body, tightly packed into clusters with spiky edges (FIG 6A). We also confirmed that viability at harvest remained high, averaging 96.90±0.00%, and that the ratio of OSC:hiPSC was similar to the ratio achieved with the RUG hiPSC line when differentiated over laminin-521 (14.83±4.48), averaging at 1 1 .41 ±2.19. Identity of OSCs was confirmed by FOXL2 and CD82 expression. Moreover, hiPSC markers POU5F1 and NANOG were not detected following differentiation, further confirming OSC cellular identity and indicating the cell populations were not contaminated with residual hiPSCs (FIGS. 6B-6C).

[0584] To further characterize the transcriptional signature of the differentiated OSCs, as well as assess reproducibility among independent lots, we performed scRNA-seq of two batches of differentiated CG- OSC-L (FIG. 6D). Strikingly, when compared with previous samples analyzed, the two batches were nearly identical in terms of cluster distribution, and they were composed primarily of GC class clusters, particularly subclusters GC I and GC III (FIGS. 6E-6G). Interestingly, the transcriptomic profile of the OSCs derived from CG-hiPSC resembled the batch of the RUO hiPSC line that was initially generated PATENT

[0585] Attorney Docket No.: 51763-009WO2

[0586] (RUO-OSC-M lot 6 (FIGS. 1C, 1F), as well as the two batches of laminin-OSCs that generated after the raw material optimization (FIG. 3H). These results demonstrated successful hiPSC reprogramming into OSCs among independent batches of cells, independent of genetic backgrounds (RUO and clinical-grade hiPSC lines), and independent of operators. These data collectively demonstrated promising clinical utility of these cells and this methodology of hiPSC differentiation for ART and IVF applications.

[0587] To expand our analysis beyond transcriptomics readouts, we performed proteomics of the bulk population of differentiated OSCs derived from both CG-hiPSC and RUO-hiPSC. We included in our analysis samples of undifferentiated hiPSCs from both genetic backgrounds. Despite the limited detection range of this assay compared with RNA sequencing, inclusion of these additional samples in the analysis could provide insight into the differentiation process and the mechanism of action. To assess proteins and pathways that were upregulated during differentiation, we calculated the ratio of expression of each detected entity in OSCs and hiPSCs for both genetic backgrounds. Among the top 200 proteins detected with a higher ratio of expression in OSCs compared to hiPSCs, 26 were overexpressed in both cell lines and had enrichment in functional profiling terms such as cell-cell adhesion mediator activity, cytoskeleton organization, and focal adhesion (FIG. 6H), suggesting that these processes were involved with OSC differentiation. Interestingly, terms related to cytoskeleton remodeling and cell adhesion were not just enriched on the shared 26 proteins by both cell lines, but also on the total top 200 proteins from each genetic background, emphasizing the importance of these processes throughout the differentiation into OSCs. Additionally, comparison of RUO-OSC and CG-OSC secretome had demonstrated high correlation between these samples, further supporting comparability between both cell lines, and suggesting potential functional similarities (FIG. 6I). v / 7. Ovarian support cells derived from clinical-grade hiPSCs consistently led to higher rates of oocyte maturation

[0588] To further assess the comparability between CG-OSC and RUO-OSC in terms of functional outcomes, we cultured three independent batches of CG-OSC-L with immature human oocytes and evaluated the rate of Mil formation relative to the control group (FIG. 7A). We observed that all three batches successfully led to higher rates of Mil maturation compared to the control (p=0.019, lot 88 / control:1 .24, lot 90 / control: 1 .22, lot 116 / control: 1 .29) in a very consistent manner (FIGS. 7A-7B). Notably, the relative values compared to control from these three individual batches were also very similar to the relative value of RUO-OSC-L indicating that di...

Claims

PATENTAttorney Docket No.: 51763-009WO2CLAIMS1 . A method of preparing a composition comprising one or more ovarian support cells (OSCs), the method comprising culturing one or more induced pluripotent stem cells (iPSCs) in vitro in a cell culture medium comprising an alkanethiol, a mitogen-activated protein kinase inhibitor, a bone morphogenetic protein (BMP) pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, a fibroblast growth factor (FGF), a keto acid, a WNT pathway activator, and / or a BMP.

2. A method of preparing a composition comprising one or more OSCs, the method comprising:(a) culturing one or more iPSCs in vitro;(b) differentiating the one or more iPSCs by:(i) inducing, in the one or more iPSCs, expression or overexpression of one or more transcription factors comprising NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, GATA4, or any combination thereof; and / or(ii) culturing the one or more iPSCs in a cell culture medium comprising an alkanethiol, a mitogen-activated protein kinase inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, and / or a BMP; and(c) determining that the one or more differentiated cells resulting from (b) exhibit a gene expression profile that is indicative of one or more OSCs, thereby confirming OSC identity.

3. A method of preparing a composition comprising one or more OSCs, the method comprising culturing one or more iPSCs in a cell culture medium comprising an alkanethiol, a mitogen-activated protein kinase inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, and / or a BMP, wherein the iPSCs were previously induced to express or overexpress one or more transcription factors comprising NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, GATA4, or any combination thereof, thereby producing the one or more OSCs.

4. A method of producing a mature oocyte for use in an ART procedure, the method comprising coculturing one or more oocytes that have been previously retrieved from a human subject with one or more OSCs that have been differentiated from one or more iPSCs, wherein the one or more iPSCs were differentiated in a cell medium comprising an alkanethiol, a mitogen-activated protein kinase inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, a BMP, or a combination thereof.

5. A method of inducing oocyte maturation in vitro, the method comprising co-culturing one or more oocytes with one or more OSCs that have been previously differentiated from one or more iPSCs, wherein the one or more iPSCs were differentiated in a cell medium comprising an alkanethiol, a mitogen- activated protein kinase inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, a BMP, or a combination thereof.

6. A method of preparing a composition comprising one or more OSCs, the method comprising:PATENTAttorney Docket No.: 51763-009WO2(a) culturing one or more iPSCs in vitro;(b) differentiating the one or more iPSCs by:(i) inducing, in the one or more iPSCs, expression or overexpression of one or more transcription factors comprising NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, GATA4, or any combination thereof; and / or(ii) culturing the one or more iPSCs in a cell culture medium comprising an alkanethiol, a mitogen-activated protein kinase inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, and / or a BMP;(c) determining that the one or more differentiated cells resulting from (b) exhibit a gene expression profile that is indicative of one or more OSCs; and(d) preparing an ovarian or uterine implant comprising the one or more OSCs and an extracellular matrix (ECM) component.

7. A method of preparing an ovarian or uterine implant comprising one or more OSCs and an ECM component, wherein the method comprises culturing one or more differentiated iPSCs in a cell culture medium comprising an alkanethiol, a mitogen-activated protein kinase inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, and / or a BMP, wherein the one or more iPSCs were previously induced to express or overexpress one or more transcription factors comprising NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, and / or GATA4, wherein the method further comprises determining that the one or more differentiated iPSCs exhibit a gene expression profile that is indicative of one or more OSCs.

8. A method of preparing a composition comprising one or more OSCs, the method comprising:(a) culturing one or more iPSCs in vitro;(b) differentiating the one or more iPSCs by:(i) inducing, in the one or more iPSCs, expression or overexpression of one or more transcription factors comprising NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, GATA4, or any combination thereof; and / or(ii) culturing the one or more iPSCs in a cell culture medium comprising an alkanethiol, a mitogen-activated protein kinase inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, and / or a BMP;(c) determining that the one or more differentiated cells resulting from (b) exhibit a gene expression profile that is indicative of one or more OSCs; and(d) preparing an organoid comprising the one or more OSCs.

9. A method of preparing an organoid comprising a population of OSCs, the method comprising: a) introducing, into one or more iPSCs, one or more nucleic acid molecules that encode NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, and / or GATA4; b) culturing the one or more iPSCs in a cell culture medium thereby differentiating the one or more iPSCs into a population of ovarian granulosa cells, ovarian stroma cells, ovarian lutein cells, and / or ovarian theca cells; andPATENTAttorney Docket No.: 51763-009WO2 c) contacting the cells resulting from (a) with an ECM component, optionally wherein the ECM component comprises one or more of collagen, an elastin, a fibronectin, a vitronectin, a laminin, a cell adhesion protein, or a plant-derived protein or protein polymer.

10. A method of determining whether a candidate pharmaceutical intervention is efficacious in treating a disease or condition of the human female reproductive system, the method comprising: a) contacting the candidate pharmaceutical intervention with an organoid that comprises a population of OSCs and an ECM substrate, wherein the population of OSCs were previously produced by culturing one or more iPSCs in a cell culture medium comprising an alkanethiol, a mitogen-activated protein kinase inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, and / or a BMP; b) determining that the organoid exhibits (i) an increase in one or more metrics of ovarian and / or uterine function relative to a measurement of the one or more metrics of ovarian and / or uterine function obtained prior to the contacting, and / or (ii) a decrease in one or more metrics of severity of the disease or condition relative to a measurement of the one or more metrics of severity of the disease or condition obtained prior to the contacting; and, optionally, c) releasing the candidate pharmaceutical intervention for treatment of the disease or condition in a subject in need thereof.11 . A method of preparing an organoid comprising a population of OSCs, wherein the method comprises culturing one or more differentiated iPSCs in a cell culture medium comprising an alkanethiol, a mitogen-activated protein kinase inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, and / or a BMP, wherein the one or more iPSCs were previously induced to express or overexpress one or more transcription factors comprising NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, and / or GATA4, wherein the method further comprises determining that the one or more differentiated iPSCs exhibit a gene expression profile that is indicative of one or more OSCs.

12. The method of any one of claims 1 , 4, 5, and 10, wherein the one or more iPSCs were previously induced to express or overexpress one or more transcription factors comprising NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, GATA4, or any combination thereof.

13. The method of any one of claims 1 -12, wherein the alkanethiol is beta-mercaptoethanol, optionally wherein the beta-mercaptoethanol is present in the cell culture medium at a concentration between about 0.1 mM to about 1 mM.

14. The method of any one of claims 1 -13, wherein BMP pathway inhibitor is LDN-193189, optionally wherein the LDN-193189 is present in the cell culture medium at a concentration between about 5 nM to about 10 nM.

15. The method of any one of claims 1 -14, wherein the mitogen-activated protein kinase inhibitor is PD0325901 , optionally wherein the PD0325901 is present in the cell culture medium at a concentration between about 1 nM to about 10 pM.PATENTAttorney Docket No.: 51763-009WG216. The method of any one of claims 1 -15, wherein the nonessential amino acid is one or more nonessential amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, and tyrosine, optionally wherein the nonessential amino acid is present in the cell culture medium at a concentration between about 0.1 mM to about 1 mM.

17. The method of any one of claims 1 -16, wherein the sonic hedgehog pathway activator is a smoothened agonist, optionally wherein the smoothened agonist is present in the cell culture medium at a concentration between about 0.1 pM to about 1 pM.

18. The method of any one of claims 1 -17, wherein the keto acid is sodium pyruvate, optionally wherein the sodium pyruvate is present in the cell culture medium at a concentration between about 1 mM to about 2 mM.

19. The method of any one of claims 1 -18, wherein Wnt pathway activator is CHIR99021 , optionally wherein CHIR99021 is present in the cell culture medium at a concentration between about 1 pM to about 7 pM.

20. The method of any one of claims 1 -19, wherein the FGF is selected from the group consisting of FGF1 , FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21 , FGF22, FGF23, or a combination thereof, optionally wherein the FGF is present in the cell culture medium at a concentration between about 1 ng / mL to about 100 ng / mL.21 . The method of claim 20, wherein the FGF is FGF9.

22. The method of any one of claims 1 -21 , wherein the BMP is selected from the group consisting of BMP1 , BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMP9, BMP10, BMP15, BMP16, or a combination thereof, optionally wherein the BMP is present in the cell culture medium at a concentration between about 1 ng / mL to about 10 ng / mL.

23. The method of claim 22, wherein the BMP is BMP4.

24. The method of any one of claims 1 -23, wherein the cell culture medium further comprises one or more morphogens and / or growth factors comprising an insulin-like growth factor (IGF), an insulin-like growth factor binding protein (IGFBP), an epidermal growth factor (EGF), and / or a transforming growth factor (TGF).

25. The method of any one of claims 20-24, wherein the IGF is IGF1 and / or IGF2.

26. The method of any one of claims 20-25, wherein the IGFBP is selected from the group consisting of IGFBP1 , IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6, IGFBP7, or a combination thereof.PATENTAttorney Docket No.: 51763-009WG227. The method of any one of claims 20-26, wherein the EGF is EGF and / or amphiregulin.

28. The method of any one of claims 20-27, wherein the TGF is a TGF-a or a TGF-p.

29. The method of claim 28, wherein the TGF is selected from the group consisting of TGF-a, TGF-p1 , TGF-p2, TGF-p3, or a combination thereof.

30. The method of any one of claims 1 -9 and 11 -29, wherein the one or more iPSCs are cultured on a matrix.31 . The method of claim 30, wherein the matrix comprises alginate, laminin, collagen, vitronectin, chitosan, hyaluronic acid, Poly-D-Lactone, or a mixture thereof, optionally wherein the laminin is selected from the group consisting of laminin-111 , laminin-211 , laminin-121 , laminin-221 , laminin-332, laminin-311 , laminin-321 , laminin-411 , laminin-421 , laminin-511 , laminin-521 , laminin-213, or a combination thereof.

32. The method of claim 31 , wherein matrix comprises laminin-521 .

33. The method of claim 31 or 32, wherein the matrix comprises vitronectin.

34. The method of any one of claims 1 -9 and 11 -33, wherein the one or more iPSCs are reprogrammed using a transposase method to carry one or more inducible transcription factors.

35. The method of any one of claims 1 -9 and 11 -33, wherein the one or more iPSCs are transformed via electroporation or liposome-mediated transformation.

36. The method of any one of claims 1 -9 and 11 -33, wherein the one or more iPSCs are transformed via viral-mediated gene transfer.

37. The method of any one of claims 2, 3, 6-8, 11 -35, wherein the expression or overexpression of the one or more transcription factors is induced by way of a doxycycline-responsive transcription regulatory element.

38. The method of any one of claims 1 -37, wherein the one or more iPSCs are human iPSCs.

39. The method of claim 38, wherein the one or more iPSCs were previously retrieved from a subject.

40. The method of claim 39, wherein the subject has or is at risk of developing a decline in ovarian function.41 . The method of claim 40, wherein the decline in ovarian function is a decline in one or more of follicular development, oocyte release, and oocyte maturation.PATENTAttorney Docket No.: 51763-009WO242. The method of any one of claims 2, 6-8, and 1 1 -41 , wherein the gene expression profile comprises one or more genes selected from NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, GATA4, or a combination thereof.

43. The method of any one of claims 1 -42, wherein the one or more OSCs or population of OSCs comprise one or more ovarian theca cells.

44. The method of claim 43, wherein the one or more ovarian theca cells express one or more genes selected from NR2F2 and GATA4.

45. The method of claim 43 or 44, wherein the one or more ovarian theca cells express a cytochrome P450 enzyme, 17p-hydroxysteroid dehydrogenase (HSD17B), luteinizing hormone receptor (LHR), smooth muscle actin (SMA), platelet-derived growth factor receptor beta (PDGFRp), or a combination thereof.

46. The method of claim 45, wherein the cytochrome P450 enzyme is CYP1 1 A1 (P450scc), CYP17A1 (P450c17), and / or CYP19A1 (aromatase).

47. The method of any one of claims 43-46, wherein the one or more ovarian theca cells express one or more hormones.

48. The method of claim 47, wherein the one or more ovarian theca cells express one or more hormones in the presence of LH.

49. The method of claim 47 or 48, wherein the one or more hormones is progesterone and / or an androgen.

50. The method of claim 49, wherein the androgen comprises androstenedione, testosterone, dihydrotestosterone, or a combination thereof.51 . The method of any one of claims 43-46, wherein the one or more ovarian theca cells is cocultured with a population of cells comprising one or more cell types, wherein the one or more cell types comprise a granulosa cell, an ovarian stroma cell, an ovarian lutein cell, one or more germ cells, one or more uterine cells, and / or an iPSC.

52. The method of claim 51 , wherein one or more germ cells comprise a primordial germ cell-like cell (PGCLC), an oogonium, an oocyte, or a combination thereof.

53. The method of claim 51 or 52, wherein the one or more uterine cells comprise a uterine endometrial cell, a uterine myometrial cell, a uterine perimetrial cell, or a combination thereof.PATENTAttorney Docket No.: 51763-009WO254. The method of any one of claims 43-53, wherein the one or more ovarian theca cells is cocultured with one or more cells that express one or more genes comprising:(a) FOXL2, AMHR2, CD82, FSHR, IGFBP7, KRT19, STAR, and / or WNT4;(b) NR2F2;(c) KRT19, CYP19A1 , STAR, CYP17A1 , and / or PGR;(d) NANOS3, CD, ITGA6, EpCAM, BLIMP1 , TFAP2C, and / or SOX17;(e) DDX4, DAZL, and / or STRA8; and / or(f) SYCP1 , ZP1 , ZP2, REC8, LHX8, and / or SOHLH1 .

55. A method of selecting an OSC, wherein the method comprises:(a) contacting a pluripotent progenitor cell with one or more differentiation agents, optionally comprising an alkanethiol, a mitogen-activated protein kinase inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, and / or a BMP in a cell culture medium, thereby differentiating the pluripotent progenitor cell into the OSC;(b) determining the OSC from (a) exhibits a gene expression profile comprising expression of one or more of a cytochrome P450 enzyme, HSD17B, LHR, SMA, and PDGFRp; and(c) selecting the OSC for use in an ART procedure, preparation of an ovarian or uterine implant comprising the OSC, or preparation of an organoid comprising the OSC.

56. A method of selecting an OSC for use in an ART procedure, wherein the method comprises:(a) contacting a pluripotent progenitor cell with one or more differentiation agents, optionally comprising an alkanethiol, a mitogen-activated protein kinase inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, and / or a BMP in a cell culture medium, thereby differentiating the pluripotent progenitor cell into the OSC;(b) determining the OSC from (a) exhibits a gene expression profile comprising expression of one or more of a cytochrome P450 enzyme, HSD17B, LHR, SMA, and PDGFRp; and(c) selecting the OSC for co-culture with an oocyte.

57. A method of selecting an OSC for preparation of an ovarian or uterine implant, wherein the method comprises:(a) contacting a pluripotent progenitor cell with one or more differentiation agents, optionally comprising an alkanethiol, a mitogen-activated protein kinase inhibitor, a bone morphogenetic protein BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, and / or a BMP in a cell culture medium, thereby differentiating the pluripotent progenitor cell into the OSC;(b) determining the OSC from (a) exhibits a gene expression profile comprising expression of one or more of a cytochrome P450 enzyme, HSD17B, LHR, SMA, and PDGFRp; and(c) selecting the OSC for contact with an extracellular matrix component.

58. A method of selecting an OSC for preparation of an organoid, wherein the method comprises:PATENTAttorney Docket No.: 51763-009WO2(a) contacting a pluripotent progenitor cell with one or more differentiation agents, optionally comprising an alkanethiol, a mitogen-activated protein kinase inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, and / or a BMP in a cell culture medium, thereby differentiating the pluripotent progenitor cell into the OSC;(b) determining the OSC from (a) exhibits a gene expression profile comprising expression of one or more of a cytochrome P450 enzyme, HSD17B, LHR, SMA, and PDGFRp; and(c) selecting the OSC for contact with an extracellular matrix component.

59. The method of any one of claims 55-58, wherein the pluripotent progenitor cell was previously induced to express or overexpress one or more transcription factors comprising NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, GATA4, or any combination thereof.

60. The method of any one of claims 55-59, wherein the pluripotent progenitor cell is an iPSC.61 . The method of any one of claims 55-60, wherein the pluripotent progenitor cell is a human cell.

62. The method of claim 61 , wherein the pluripotent progenitor cell was previously retrieved from a subject.

63. The method of claim 62, wherein the subject has or is at risk of developing a decline in ovarian function.

64. The method of claim 63, wherein the decline in ovarian function is a decline in one or more of follicular development, oocyte release, and oocyte maturation.

65. The method of any one of claims 55-64, wherein the alkanethiol is beta-mercaptoethanol, optionally wherein the beta-mercaptoethanol is present in the cell culture medium at a concentration between about 0.1 mM to about 1 mM.

66. The method of any one of claims 55-65, wherein BMP pathway inhibitor is LDN-193189, optionally wherein the LDN-193189 is present in the cell culture medium at a concentration between about 5 nM to about 10 nM.

67. The method of any one of claims 55-66, wherein the mitogen-activated protein kinase inhibitor is PD0325901 , optionally wherein the PD0325901 is present in the cell culture medium at a concentration between about 1 nM to about 10 pM.

68. The method of any one of claims 55-67, wherein the nonessential amino acid is one or more nonessential amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, and tyrosine, optionally wherein thePATENTAttorney Docket No.: 51763-009WO2 nonessential amino acid is present in the cell culture medium at a concentration between about 0.1 mM to about 1 mM.

69. The method of any one of claims 55-68, wherein the sonic hedgehog pathway activator is a smoothened agonist, optionally wherein the smoothened agonist is present in the cell culture medium at a concentration between about 0.1 pM to about 1 pM.

70. The method of any one of claims 55-69, wherein the keto acid is sodium pyruvate, optionally wherein the sodium pyruvate is present in the cell culture medium at a concentration between about 1 mM to about 2 mM.71 . The method of any one of claims 55-70, wherein Wnt pathway activator is CHIR99021 , optionally wherein CHIR99021 is present in the cell culture medium at a concentration between about 1 pM to about 7 pM.

72. The method of any one of claims 55-71 , wherein the FGF is selected from the group consisting of FGF1 , FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21 , FGF22, FGF23, or a combination thereof, optionally wherein the FGF is present in the cell culture medium at a concentration between about 1 ng / mL to about 100 ng / mL.

73. The method of claim 72, wherein the FGF is FGF9.

74. The method of any one of claims 55-73, wherein the BMP is selected from the group consisting of BMP1 , BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMP9, BMP10, BMP15, BMP16, or a combination thereof, optionally wherein the BMP is present in the cell culture medium at a concentration between about 1 ng / mL to about 10 ng / mL.

75. The method of claim 74, wherein the BMP is BMP4.

76. The method of any one of claims 55-75, wherein the cell culture medium further comprises one or more morphogens and / or growth factors comprising an IGF, an IGFBP, an EGF, and / or a TGF.

77. The method of any one of claims 72-76, wherein the IGF is IGF1 and / or IGF2.

78. The method of any one of claims 72-77, wherein the IGFBP is selected from the group consisting of IGFBP1 , IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6, IGFBP7, or a combination thereof.

79. The method of any one of claims 72-78, wherein the EGF is EGF and / or amphiregulin.

80. The method of any one of claims 72-79, wherein the TGF is a TGF-a or a TGF-p.PATENTAttorney Docket No.: 51763-009WG281 . The method of claim 80, wherein the TGF is selected from the group consisting of TGF-a, TGF-p1 , TGF-p2, TGF-p3, or a combination thereof.

82. The method of any one of claims 57-81 , wherein the extracellular matrix component is a collagen, an elastin, a fibronectin, a vitronectin, a laminin, a cell adhesion protein, a hyaluronic acid, a plant-derived protein or protein polymer, or a combination thereof.

83. The method of any one of claims 55-82, wherein the OSC exhibits a gene expression profile that is indicative of an ovarian theca cell.

84. The method of any one of claims 55-83, wherein the gene expression profile comprises genes NR2F2 and GATA4.

85. The method of any one of claims 55-84, wherein the OSC expresses a cytochrome P450 enzyme, HSD17B, LHR, SMA, and PDGFRp.

86. The method of any one of claims 55-85, wherein the cytochrome P450 enzyme is CYP11 A1 (P450scc), CYP17A1 (P450c17), and / or CYP19A1 (aromatase).

87. The method of any one of claims 55-86, wherein the OSC produces one or more growth factors and / or steroids.

88. The method of claim 87, wherein the one or more growth factors and / or steroids comprises progesterone and / or an androgen.

89. The method of claim 88, wherein the androgen comprises androstenedione, testosterone, dihydrotestosterone, or a combination thereof.

90. The method of any one of claims 87-89, wherein the one or more growth factors and / or steroids are produced following contact of the OSC and LH.91 . The method of any one of claims 55-87, wherein the OSC has low or undetectable expression of one or more genes associated with pluripotency relative to an iPSC.

92. The method of claim 91 , wherein the one or more genes associated with pluripotency comprises NANOG and / or POU5F1 .

93. A composition comprising one or more OSCs, wherein the one or more OSCs were previously produced by a method of differentiating one or more iPSCs in an in vitro cell culture comprising a cell culture medium comprising an alkanethiol, a mitogen-activated protein kinase inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, a BMP, or a combination thereof, optionally wherein the one or more iPSCs werePATENTAttorney Docket No.: 51763-009WO2 induced to express or overexpress one or more transcription factors comprising NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, GATA4, or any combination thereof, thereby producing the one or more OSCs.

94. The composition of claim 93, wherein the alkanethiol is beta-mercaptoethanol.

95. The composition of claim 93 or 94, wherein the BMP pathway inhibitor is LDN-193189.

96. The composition of any one of claims 93-95, wherein the mitogen-activated protein kinase inhibitor is PD0325901 .

97. The composition of any one of claims 93-96, wherein the nonessential amino acid is one or more nonessential amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, and tyrosine.

98. The composition of any one of claims 93-97, wherein the sonic hedgehog pathway activator is a smoothened agonist.

99. The composition of any one of claims 93-98, wherein the keto acid is sodium pyruvate.

100. The composition of any one of claims 93-99, wherein Wnt pathway activator is CHIR99021 .101 . The composition of any one of claims 93-100, wherein the FGF is selected from the group consisting of FGF1 , FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21 , FGF22, FGF23, or a combination thereof.

102. The composition of claim 101 , wherein the FGF is FGF9.

103. The composition of any one of claims 93-102, wherein the BMP is selected from the group consisting of BMP1 , BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMP9, BMP10, BMP15, BMP16, or a combination thereof.

104. The composition of claim 103, wherein the BMP is BMP4.

105. The composition of any one of claims 93-104, wherein the composition further comprises an extracellular matrix component.

106. The composition of claim 105, wherein the extracellular matrix component is a collagen, an elastin, a fibronectin, a vitronectin, a laminin, a cell adhesion protein, a hyaluronic acid, a plant-derived protein or protein polymer, or a combination thereof.

107. The composition of any one of claims 93-106, wherein the one or more OSCs comprises one or more ovarian theca cells.PATENTAttorney Docket No.: 51763-009WG2108. The composition of any one of claims 93-107, wherein the one or more OSCs express a cytochrome P450 enzyme, HSD17B, LHR, SMA, PDGFRp, or a combination thereof.

109. The composition of claim 108, wherein the cytochrome P450 enzyme is CYP1 1 A1 (P450scc), CYP17A1 (P450c17), and / or CYP19A1 (aromatase).

110. The composition of any one of claims 93-109, wherein the one or more OSCs produce one or more growth factors and / or steroids.

111. The composition of claim 110, wherein the one or more growth factors and / or steroids comprises progesterone and / or an androgen.

112. The composition of claim 1 11 , wherein the androgen comprises androstenedione, testosterone, dihydrotestosterone, or a combination thereof.

113. The composition of any one of claims 1 10-112, wherein the one or more growth factors and / or steroids are produced following contact of the OSC and LH.

114. The composition of any one of claims 93-113, wherein the OSC has low or undetectable expression of one or more genes associated with pluripotency relative to an iPSC.

115. The composition of claim 1 14, wherein the one or more genes associated with pluripotency comprises NANOG and / or POU5F1 .

116. The composition of any one of claims 93-115, wherein the composition further comprises or is suitable for combination with a granulosa cell, an ovarian stroma cell, an ovarian lutein cell, one or more germ cells, one or more uterine cells, and / or an iPSC.

117. The composition of claim 1 16, wherein one or more germ cells comprise a primordial germ cell-like cell (PGCLC), an oogonium, an oocyte, or a combination thereof.

118. The composition of claim 1 16 or 117, wherein the one or more uterine cells comprise a uterine endometrial cell, a uterine myometrial cell, a uterine perimetrial cell, or a combination thereof.

119. The composition of any one of claims 93-118, wherein the iPSC is a human iPSC.

120. The composition of claim 1 19, wherein the iPSC was previously retrieved from a subject.

121. The composition of claim 120, wherein the subject has or is at risk of developing a decline in ovarian function, optionally wherein the decline in ovarian function is a decline in one or more of follicular development, oocyte release, and oocyte maturation.PATENTAttorney Docket No.: 51763-009WO2122. The composition of claim 120 or 121 , wherein the subject has or is at risk of developing primary ovarian insufficiency (POI) , polycystic ovarian syndrome (POOS), ovarian cancer, ovarian hyperstimulation syndrome, endometriosis, uterine fibroids, adenomyosis, a gynecological cancer, pelvic inflammatory disease (PI D) , cervical dysplasia, or pelvic floor prolapse.

123. The composition of any one of claims 93-122, wherein the composition further comprises a pharmaceutically acceptable excipient and is suitable for administration to a subject.

124. The composition of claim 123, wherein the subject has or is at risk of developing a decline in ovarian function, optionally wherein the decline in ovarian function is a decline in one or more of follicular development, oocyte release, and oocyte maturation.

125. The composition of claim 123 or 124, wherein the subject has or is at risk of developing POI, POOS, ovarian cancer, ovarian hyperstimulation syndrome, endometriosis, uterine fibroids, adenomyosis, a gynecological cancer, PID, cervical dysplasia, or pelvic floor prolapse.

126. An ovarian or uterine implant comprising a population of OSCs and an extracellular matrix substrate, wherein the OSCs were produced by differentiating one or more iPSCs in an in vitro cell culture comprising a cell culture medium comprising an alkanethiol, a mitogen-activated protein kinase inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, a BMP, or a combination thereof.

127. An implant for ovarian decline, the implant comprising at least an engineered OSC, an extracellular matrix substrate, and a delivery apparatus, wherein the engineered OSC was produced by differentiating one or more iPSCs in an in vitro cell culture comprising a cell culture medium comprising an alkanethiol, a mitogen-activated protein kinase inhibitor, a BMP pathway inhibitor, a nonessential amino acid, a sonic hedgehog pathway activator, an FGF, a keto acid, a WNT pathway activator, a BMP, or a combination thereof.

128. The implant of any one of claims 126 or 127, wherein the alkanethiol is betamercaptoethanol.

129. The implant of any one of claims 126-128, wherein the BMP pathway inhibitor is LDN- 193189.

130. The implant of any one of claims 126-129, wherein the mitogen-activated protein kinase inhibitor is PD0325901 .131 . The implant of any one of claims 126-130, wherein the nonessential amino acid is one or more nonessential amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, and tyrosine.PATENTAttorney Docket No.: 51763-009WO2132. The implant of any one of claims 126-131 , wherein the sonic hedgehog pathway activator is a smoothened agonist.

133. The implant of any one of claims 126-132, wherein the keto acid is sodium pyruvate.

134. The implant of any one of claims 126-133, wherein Wnt pathway activator is CHIR99021 .

135. The implant of any one of claims 126-134, wherein the FGF is selected from the group consisting of FGF1 , FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF16, FGF17,FGF18, FGF19, FGF20, FGF21 , FGF22, FGF23, or a combination thereof.

136. The implant of claim 135, wherein the FGF is FGF9.

137. The implant of any one of claims 126-136, wherein the BMP is selected from the group consisting of BMP1 , BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMP9, BMP10, BMP15, BMP16, or a combination thereof.

138. The implant of claim 137, wherein the BMP is BMP4.

139. The implant of any one of claims 126-138, wherein the extracellular matrix substrate is a collagen, an elastin, a fibronectin, a vitronectin, a laminin, a cell adhesion protein, a hyaluronic acid, a plant-derived protein or protein polymer, or a combination thereof.

140. The implant of any one of claims 126 and 128-139, wherein the population of OSCs comprises ovarian theca cells.

141. The implant of any one of claims 127-139, wherein the engineered OSC is an ovarian theca cell.

142. The implant of any one of claims 126 and 128-140, wherein the population of OSCs has been determined to express one or more of genes NR2F2 and GATA4.

143. The implant of any one of claims 126, 128-140, and 142, wherein the population of OSCs has been determined to express a cytochrome P450 enzyme, HSD17B, LHR, SMA, PDGFRp, or a combination thereof.

144. The implant of any one of claims 127-139 and 141 , wherein the engineered OSC has been determined to express one or more genes comprising NR2F2, FOXL2, NR5A1 , RUNX1 , RUNX2, GATA4, or a combination thereof.PATENTAttorney Docket No.: 51763-009WO2145. The implant of any one of claims 127-139, 141 , and 144, wherein the engineered OSC has been determined to express a cytochrome P450 enzyme, HSD17B, LHR, SMA, PDGFRp, or a combination thereof.

146. The implant of claim 143 or 145, wherein the cytochrome P450 enzyme is CYP11 A1 (P450scc), CYP17A1 (P450c17), and / or CYP19A1 (aromatase).

147. The implant of any one of claims 126-146, wherein the implant expresses a growth factor and / or a hormone.

148. The implant of claim 147, wherein the growth factor and / or hormone comprises progesterone and / or an androgen.

149. The implant of claim 148, wherein the androgen comprises androstenedione, testosterone, dihydrotestosterone, or a combination thereof.

150. The implant of any one of claims 147-149, wherein the growth factor and / or hormone are expressed following contact of the implant and LH.151 . The implant of any one of claims 126-150, wherein the implant is further assembled as a plurality of microparticles.

152. Use of implant of any one of claims 126-151 for treating a subject in need thereof, wherein the treatment comprises administering the implant to the subject.

153. The use of claim 152, wherein the subject has or is at risk of developing ovarian decline.

154. The use of claim 153, wherein the ovarian decline is a result of menopause or premature menopause.

155. The use of any one of claims 152-154, wherein the subject has or is at risk of developing POI, POOS, ovarian cancer, ovarian hyperstimulation syndrome, endometriosis, uterine fibroids, adenomyosis, a gynecological cancer, PID, cervical dysplasia, or pelvic floor prolapse.

156. The use of any one of claims 152-155, wherein the implant is administered to the subject as a semi-permeable rod, a semi-permeable pouch, an omental pouch, a patch, or an intrauterine device.

157. The use of claim 156, wherein the semi-permeable rod is administered subdermally to the arm of the subject.

158. The use of claim 156, wherein the semi-permeable pouch, omental pouch, or patch is administered to the abdomen of the subject.PATENTAttorney Docket No.: 51763-009WO2159. The use of claim 156, wherein the patch is delivered onto or adjacent to the ovary or fallopian tube of the subject.

160. An implant comprising (i) a population of OSCs and (ii) an extracellular matrix substrate, wherein the OSCs and extracellular matrix substrate are assembled as a plurality of microparticles.161 . The implant of claim 160, wherein the population of OSCs comprises one or more ovarian granulosa cells, ovarian lutein cells, and / or ovarian theca cells.

162. The implant of claim 161 , wherein the population of OSCs comprises one or more ovarian granulosa cells.

163. The implant of claim 162, wherein the one or more ovarian granulosa cells express one or more, or all, of proteins FOXL2, CD82, follicle-stimulating hormone receptor (FSHR), FOXL2, NR5A1 , GATA4, RUNX1 , and RUNX2.

164. The implant of claim 162 or 163, wherein the implant comprises from about 1 x 106and to about 1 x 107ovarian granulosa cells.

165. The implant of claim 164, wherein the implant comprises from about 1 x 106to about 2 x 106ovarian granulosa cells, from about 2 x 106to about 3 x 106ovarian granulosa cells, from about 3 x 106to about 4 x 106ovarian granulosa cells, from about 4 x 106to about 5 x 106ovarian granulosa cells, from about 5 x 106to about 6 x 106ovarian granulosa cells, from about 6 x 106to about 7 x 106ovarian granulosa cells, from about 7 x 106to about 8 x 106ovarian granulosa cells, from about 8 x 106to about 9 x 106ovarian granulosa cells, or from about 9 x 106to about 1 x 107ovarian granulosa cells.

166. The implant of claim 164, wherein the implant comprises about 1 x 106ovarian granulosa cells, about 2 x 106ovarian granulosa cells, about 3 x 106ovarian granulosa cells, about 4 x 106ovarian granulosa cells, about 5 x 106ovarian granulosa cells, about 6 x 106ovarian granulosa cells, about 7 x106ovarian granulosa cells, about 8 x 106ovarian granulosa cells, about 9 x 106ovarian granulosa cells, or about 1 x 107ovarian granulosa cells.

167. The implant of any one of claims 162-166, wherein the one or more ovarian granulosa cells secrete an estrogen.

168. The implant of claim 167, wherein the estrogen is estradiol.

169. The implant of any one of claims 161 -168, wherein the population of OSCs comprises one or more ovarian lutein cells.

170. The implant of claim 169, wherein the one or more ovarian lutein cells express one or more, or all, of proteins KRT19, CYP19A1 , STAR, CYP17A1 , and PGR.PATENTAttorney Docket No.: 51763-009WO2171 . The implant of claim 169 or 170, wherein the implant comprises from about 1 x 106and to about 1 x 107ovarian lutein cells.

172. The implant of claim 171 , wherein the implant comprises from about 1 x 106to about 2 x 106ovarian lutein cells, from about 2 x 106to about 3 x 106ovarian lutein cells, from about 3 x 106to about 4 x 106ovarian lutein cells, from about 4 x 106to about 5 x 106ovarian lutein cells, from about 5 x 106to about 6 x 106ovarian lutein cells, from about 6 x 106to about 7 x 106ovarian lutein cells, from about 7 x106to about 8 x 106ovarian lutein cells, from about 8 x 106to about 9 x 106ovarian lutein cells, or from about 9 x 106to about 1 x 107ovarian lutein cells.

173. The implant of claim 171 , wherein the implant comprises about 1 x 106ovarian lutein cells, about 2 x 106ovarian lutein cells, about 3 x 106ovarian lutein cells, about 4 x 106ovarian lutein cells, about 5 x 106ovarian lutein cells, about 6 x 106ovarian lutein cells, about 7 x 106ovarian lutein cells, about 8 x 106ovarian lutein cells, about 9 x 106ovarian lutein cells, or about 1 x 107ovarian lutein cells.

174. The implant of any one of claims 169-173, wherein the one or more ovarian lutein cells secrete a progestogen.

175. The implant of claim 174, wherein the progestogen is progesterone.

176. The implant of any one of claims 161 -175, wherein the population of OSCs comprises one or more ovarian theca cells.

177. The implant of claim 176, wherein the one or more ovarian theca cells express one or both of proteins NR2F2 and GATA4.

178. The implant of claim 176 or 177, wherein the implant comprises from about 1 x 106and to about 1 x 107ovarian theca cells.

179. The implant of claim 178, wherein the implant comprises from about 1 x 106to about 2 x 106ovarian theca cells, from about 2 x 106to about 3 x 106ovarian theca cells, from about 3 x 106to about 4 x 106ovarian theca cells, from about 4 x 106to about 5 x 106ovarian theca cells, from about 5 x 106to about 6 x 106ovarian theca cells, from about 6 x 106to about 7 x 106ovarian theca cells, from about 7 x106to about 8 x 106ovarian theca cells, from about 8 x 106to about 9 x 106ovarian theca cells, or from about 9 x 106to about 1 x 107ovarian theca cells.

180. The implant of claim 178, wherein the implant comprises about 1 x 106ovarian theca cells, about 2 x 106ovarian theca cells, about 3 x 106ovarian theca cells, about 4 x 106ovarian theca cells, about 5 x 106ovarian theca cells, about 6 x 106ovarian theca cells, about 7 x 106ovarian theca cells, about 8 x 106ovarian theca cells, about 9 x 106ovarian theca cells, or about 1 x 107ovarian theca cells.PATENTAttorney Docket No.: 51763-009WO2181 . The implant of any one of claims 176-180, wherein the one or more ovarian theca cells secrete an androgen.

182. The implant of claim 181 , wherein the androgen is androstenedione.

183. The implant of any one of claims 161 -182, wherein the OSCs comprise one or more ovarian granulosa cells, ovarian lutein cells, and ovarian theca cells, and wherein the ratio of ovarian granulosa cells to ovarian lutein cells to ovarian theca cells in the implant is from about 10:1 :1 to about 1 ,000:1 :1 .

184. The implant of claim 183, wherein the ratio of ovarian granulosa cells to ovarian lutein cells to ovarian theca cells in the implant is about 100:1 :1 .

185. The implant of any one of claims 160-184, wherein the one or more OSCs are obtained by differentiating a population of induced pluripotent stem cells (iPSCs) into one or more ovarian granulosa cells, ovarian lutein cells, and / or ovarian theca cells.

186. The implant of any one of claims 160-185, wherein the extracellular matrix substrate comprises one or more of collagen, an epidermal growth factor (EGF), an elastin, a fibronectin, a vitronectin, a laminin, a cell adhesion protein, or a plant-derived protein or protein polymer.

187. The implant of claim 186, wherein the collagen is fibrillar collagen.

188. The implant of claim 186 or 187, wherein the collagen is collagen type I, type II, type III, type V, type XI.

189. The implant of any one of claims 186-188, wherein the elastin is tropoelastin or mature elastin.

190. The implant of any one of claims 186-189, wherein the plant-derived protein polymer is alginate.191 . A method of increasing secretion of an estrogen, progestogen, and / or androgen in a human subject, the method comprising administering to the subject the implant of any one of claims 160-190.

192. The method of claim 191 , wherein the subject is a pre-menopausal human subject, optionally wherein administration of the implant restores secretion of an estrogen, progestogen, and / or androgen in the subject.

193. The method of claim 191 or 192, wherein the subject is diagnosed as having ovarian dysfunction.PATENTAttorney Docket No.: 51763-009WO2194. The method of any one of claims 191 -193, wherein, prior to administration of the implant to the subject, the subject exhibits consistent, circulating estradiol levels of about 20 pg / ml or less, optionally wherein the subject exhibits consistent, circulating estradiol levels of from about 10 to about 20 pg / ml.

195. The method of any one of claims 191 -194, wherein, prior to administration of the implant to the subject, the subject exhibits consistent, circulating progesterone levels of about 0.5 ng / ml or less, optionally wherein the subject exhibits consistent, circulating progesterone levels of from about 0.01 ng / ml to about 0.5 ng / ml.

196. The method of any one of claims 191 -195, wherein the subject has previously undergone an ovariectomy.

197. The method of any one of claims 191 -196, wherein prior to administration of the implant to the subject, the subject exhibits ovarian decline.

198. The method of claim 197, wherein the ovarian decline is a result of menopause.

199. The method of claim 198, wherein the ovarian decline is not a result of menopause, optionally wherein the ovarian decline is a result of premature menopause.

200. The method of any one of claims 197-199, wherein the ovarian decline is a result of a disease or condition selected from primary ovarian insufficiency (POI) , polycystic ovarian syndrome (POOS), endometriosis, uterine fibroids, gynecological cancer, interstitial cystitis, pelvic inflammatory disease (PID) , vaginitis, cervical dysplasia, and pelvic floor prolapse.201 . The method of any one of claims 191 -200, wherein the population of OSCs secretes estradiol during an early- to mid-follicular phase of the subject’s menstrual cycle at a serum concentration of from about 20 pg / ml to about 80 pg / ml.

202. The method of claim 201 , wherein the population of OSCs secretes estradiol during an early- to mid-follicular phase of the subject’s menstrual cycle at a serum concentration of from about 20 pg / ml to about to 30 pg / ml, from about 30 pg / ml to about 40 pg / ml, from about 40 pg / ml to about 50 pg / ml, from about 50 pg / ml to about 60 pg / ml, from about 60 pg / ml to about 70 pg / ml, or from about 70 pg / ml to about 80 pg / ml.

203. The method of any one of claims 191 -202, wherein the population of OSCs secretes estradiol during a mid- to late-follicular phase of the subject’s menstrual cycle at a serum concentration of from about 20 pg / ml to about 200 pg / ml.

204. The method of claim 203, wherein the population of OSCs secretes estradiol during a mid- to late-follicular phase of the subject’s menstrual cycle at a serum concentration of from about 20 pg / ml to about to 50 pg / ml, from about 50 pg / ml to about 70 pg / ml, from about 70 pg / ml to about 90 pg / ml, fromPATENTAttorney Docket No.: 51763-009WO2 about 90 pg / ml to about 110 pg / ml, from about 110 pg / ml to about 130 pg / ml, from about 130 pg / ml to about 150 pg / ml, from about 150 pg / ml to about t170 pg / ml, or from about 170 pg / ml to about 200 pg / ml.

205. The method of any one of claims 191 -204, wherein the population of OSCs secretes estradiol during a pre-ovulation phase of the subject’s menstrual cycle at a serum concentration of from about 100 pg / ml to about 400 pg / ml.

206. The method of claim 205, wherein the population of OSCs secretes estradiol during a pre- ovulation phase of the subject’s menstrual cycle at a serum concentration of from about 100 pg / ml to about to 150 pg / ml, from about 150 pg / ml to about 200 pg / ml, from about 200 pg / ml to about 250 pg / ml, from about 250 pg / ml to about 300 pg / ml, from about 300 pg / ml to about 350 pg / ml, or from about 350 pg / ml to about 400 pg / ml.

207. The method of any one of claims 191 -206, wherein the population of OSCs secretes estradiol during an ovulation phase of the subject’s menstrual cycle at a serum concentration of from about 250 pg / ml to about 500 pg / ml.

208. The method of claim 207, wherein the population of OSCs secretes estradiol during an ovulation phase of the subject’s menstrual cycle at a serum concentration of from about 250 pg / ml to about to 300 pg / ml, from about 300 pg / ml to about 350 pg / ml, from about 350 pg / ml to about 400 pg / ml, from about 400 pg / ml to about 450 pg / ml, or from about 450 pg / ml to about 500 pg / ml.

209. The method of any one of claims 191 -208, wherein the implant is administered to the subject in the form of a semi-permeable rod, a semi-permeable pouch, an omental pouch, a patch, or an intrauterine device, optionally wherein (a) the semi-permeable rod is administered subdermally to the arm of the subject, (b) the semi-permeable pouch, omental pouch, or patch is administered to the abdomen of the subject, or (c) the patch is delivered onto or adjacent to the ovary or fallopian tube of the subject.

210. The method of any one of claims 191 -209, wherein the implant is administered to the subject subdermally, optionally wherein the implant is inserted beneath a segment of skin on the subject’s arm.

211. The method of any one of claims 191 -209, wherein the implant is administered onto one, or both, of the subject’s ovaries.

212. The method of any one of claims 191 -209, wherein the implant is administered into the subject’s uterus, optionally wherein the implant is embedded into the subject’s uterine lining.

213. The method of any one of claims 191 -212, wherein the implant is administered to the subject at a frequency of no greater than once every 6, 7, 8, 9, 10, 11 , or 12 months.

214. The method of any one of claims 191 -212, wherein the implant is administered to the subject at a frequency of no greater than once every 24, 36, 48, or 52 weeks.PATENTAttorney Docket No.: 51763-009WO2215. The method of any one of claims 191 -212, wherein the implant is administered to the subject at a frequency of no greater than once every one, two, or three years.

216. A kit comprising the implant of any one of claims 160-190, a delivery apparatus, and a package insert.

217. The kit of claim 216, wherein the package insert instructs a user of the kit to perform the method of any one of claims 191 -215.

Images