Artificial testis cells and method for their production

Abstract

The present invention relates to in vitro methods for production of testis cells (e.g., Sertoli and / or Leydig cells) and related organoids. The testis cells and testis like organoids may be used for therapeutic purposes including facilitation of the production of spermatogonia from pro-spermatogonia stem cells.

Description

[0001] ARTIFICIAL TESTIS CELLS AND METHOD FOR THEIR PRODUCTION

[0002] Cross-Reference to Related Applications

[0003] The present application claims priority to U.S. Provisional Application No. 63 / 533,148, filed August 17, 2023, which is incorporated herein by reference in its entirety.

[0004] Field of the Invention

[0005] The present invention relates to in vitro methods for production of testis cells (e.g., Sertoli and / or Leydig cells) and related organoids.

[0006] Background of the Invention

[0007] Infertility is a rapidly rising crisis worldwide, and in roughly 50% of cases the cause is male factor infertility. One percent of all reproductive age men (ages 20-50) globally suffer from non-obstructive azoospermia (i.e., lacking any germ or mature sperm cells). In the US, this accounts for - 300,000 men and most are of unknown genetic origin. Traditional treatment options, such as in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), require sperm, and leave these men with no therapies. In addition to adult infertility, each year in the US -10,000 prepubertal boys develop cancers and require gonadotoxic treatments, such as chemotherapy and radiation (1). Furthermore, -1,000 pediatric patients with blood and immune deficiencies and autoimmune disorders receive myeloablative conditioning prior to bone marrow transplantation, which is also gonadotoxic (2). These treatments - with alkylating chemotherapeutic agents, total body irradiation (3), and gonadal radiation (4) - put patients at a significant risk of infertility due to damage to both somatic and germ cell populations in the testis. However, with the advancements in medicine 85% of these children will be cured and, upon reaching adulthood they desire to have their own children (5). Since gametes are needed for any reproductive therapy, developing methods to reconstitute germ cell development in vitro or in vivo for these patients remains a clinical challenge, and will continue to be the focus of the reproductive biology community for the coming decades. Successful generation of patient-derived, testis-like somatic cells in vitro will provide a novel cell-based therapy for known and unknown genetic and iatrogenic (i.e., induced by treatment) forms of male infertility. Such a treatment is also applicable to restore endocrine function in aging men, those with hypogonadism, or patients with gender dysphoria. Further, if in vitro derived cells or cell communities (sometimes called organoids) can recapitulate the natural spermatogenesis processes, they can be used as a new experimental system to screen for new classes of male contraceptives or environmental reproductive toxicants.

[0008] The current race for cell-based treatment has centered on the development of germ cell-like precursors known as primordial germ cell-like cells (PGCLCs). While PGCLCs have been derived in vitro from mouse and human embryonic stem cells (ESCs), they cannot be maintained in vitro (6). Only when transplanted into mouse neonatal testis can these mouse PGCLCs develop into sperm, which can then be used to make live born pups. Furthermore, when female and male mouse PGCLCs were reconstituted with fetal gonadal tissue in vitro, successful reconstitution of the oogenesis and spermatogenesis program was possible, but the frequency of live born pups obtained from the in vitro derived germ cells is roughly ~ 1-3% (6-9). These preliminary findings are exciting and underscore the importance of somatic cells for the execution of the gametogenesis program. In similar experiments using human or rhesus PGCLCs with mouse embryonic gonadal tissue from three research groups it was shown that when the female and male human hPGCLCs or male rhesus PGCLCs were combined with mouse fetal ovarian tissue or testis tissue, respectively, the PGCLC initiated differentiation, but failed to initiate meiosis (9-11). These observations indicate that somatic and germ cell sources must be compatible (i.e., from the same species) to initiate meiosis.

[0009] Utilizing somatic cells from human fetal testis to aid the progression of in vitro derived human PGCLCs has ethical and technical limitations related to fetal tissue-based research. Therefore, to generate testis-like somatic cells without relying on fetal tissue is of clinical significance. This has previously been attempted by reprogramming human fibroblasts into Sertoli- or Leydig-like cells using a combination of well-chosen transcription factors. Despite expressing a handful of known markers for Sertoli and Leydig cells, the extent to which the in vitro derived cells resemble in vivo cells or recapitulate endogenous functions remain difficult to assess. Furthermore, given the requirement for transgenesis for efficient induction, and for the continuous expression of transcription factors for maintenance of Sertoli and Leydig cell fates, the clinical utility of such cells is unclear.

[0010] Summary of the Invention

[0011] The present invention relates to in vitro methods for production of testis cells and related organoids.

[0012] In a first aspect, the present invention provides in vitro methods for production of artificial testis cells from vertebrate pluripotent stem cells comprising: deriving genital ridge cells from pluripotent stem cells; and treating the genital ridge cells with a base medium comprising fibroblast growth factor 9 (FGF9), insulin and / or Insulin-like Growth Factor 1 (IGFl),and Epidermal Growth Factor (EGF) so that the genital ridge cells differentiate into testis cells. In some preferred embodiments, the base medium further comprises Prostaglandin D2 (PGD2) and / or retinoic acid (RA). In some preferred embodiments, the base medium further comprises Follicle Stimulating Hormone (FSH) and / or luteinizing hormone (LH) or Human Chorionic Gonadotropin (HCG). In some preferred embodiments, base medium further comprises testosterone (T). In some preferred embodiments, the base medium further comprises IWR1.

[0013] In some preferred embodiments, the step of deriving genital ridge cells further comprises: providing vertebrate pluripotent stem cells in a maintenance medium comprising a ROCK inhibitor; at day 0, removing the maintenance medium comprising a ROCK inhibitor and culturing the vertebrate pluripotent stem cells with the base medium comprising CHIR99021 so that the vertebrate pluripotent stem cells differentiate into presomitic mesoderm cells; on about day 4, removing the base medium comprising CHIR99021 and culturing the presomitic mesoderm cells in base medium comprising fibroblast growth factor 9 (FGF9) and heparin so that the presomitic mesoderm cells differentiate into intermediate mesoderm cells; and on about day 7 or 8, removing the medium comprising FGF9 and heparin and culturing the cells in a base medium comprising FGF9, insulin and / or IGF1, EGF, RA,PGD2, LH, FSH and / or T or a base medium comprising insulin and / r IGF1, FGF9, RA, and PDG2 so that the cells differentiate into genital ridge cells.

[0014] In some preferred embodiments, the genital ridge cells are treated with the base medium comprising FGF9, insulin and / or IGF1, EGF, LH, FSH and / or T on about day 8 or 9 to provide gonadogenesis-induced cells.

[0015] In some preferred embodiments, the gonadogenesis-induced cells are cultured to induce the formation of organoids.

[0016] In some preferred embodiments, the genital ridge cells are dissociated into single cells on about day 8.

[0017] In some preferred embodiments, the methods further comprise, preferably on about day 12, removing organoids from the culture and culturing the organoids at an air-liquid interphase to allow maturation of testis organoids. In some preferred embodiments, the testis organoids comprise one or more of artificial Sertoli cells, artificial Leydig cells, artificial myoid cells and artificial stromal cells. In some preferred embodiments, the testis organoids comprise two or more of artificial Sertoli cells, artificial Leydig cells, artificial myoid cells and artificial stromal cells. In some preferred embodiments, the testis organoids comprise three or more of artificial Sertoli cells, artificial Leydig cells, artificial myoid cells and artificial stromal cells. In some preferred embodiments, the testis organoids comprise artificial Sertoli cells, artificial Leydig cells, artificial myoid cells and artificial stromal cells. In some preferred embodiments, the organoids demonstrate upregulation or expression of one or more markers selected from the group consisting of LHX9, PDGRA, COUPTFII, TCF21, SOX9, GATA4, SF1, SMA, DHH, STAR and 3BHSD.

[0018] In some preferred embodiments, the vertebrate pluripotent stem cells are human stem cells. In some preferred embodiments, the human stem cells are human embryonic stem cells. In some preferred embodiments, the human stem cells are induced pluripotent stem cells.

[0019] In some preferred embodiments, the methods further comprise the step of isolating 1) the artificial testis cells from the organoids or 2) the testis organoid. In some preferred embodiments, the artificial testis cells are Sertoli-like cells. In some preferred embodiments, the artificial testis cells are Leydig-like cells. In some preferred embodiments, the artificial testis cells are Myoid-like cells. In some preferred embodiments, the artificial testis cells are stromal progenitor cells.

[0020] In some preferred embodiments, the methods further comprise transplanting the isolated artificial testis cells or artificial testis organoid into a mammal.

[0021] In some preferred embodiments, the methods further comprise contacting the artificial testis cells or artificial testis cell organoid with a test reagent and assaying the effect of the test reagent on the artificial testis cells or artificial testis cell organoid.

[0022] In some preferred embodiments, the methods further comprise obtaining stem cells or tissue comprising stem cells from a patient and culturing the stem cells or tissue comprising stem cells from the patient with the artificial testis cells or artificial testis cell organoid to provide differentiated patient stem cells. In some preferred embodiments, the stem cells are primordial germ cells, pro-spermatogonia or spermatogonial stem cells. In some preferred embodiments, the primordial germ cell like cells and pro-spermatogonia stem cells differentiate into spermatogonia.

[0023] In some preferred embodiments, the methods further comprise transferring the stem cells or differentiated spermatogonia back to a patient in need thereof. In some preferred embodiments, the patient is diagnosed with nonobstructive azoospermia or severe oligospermia or has previously undergone a gonadotoxic treatment. In some preferred embodiments, the stem cells or tissue comprising stem cells are obtained from the patient prior to a gonadotoxic treatment. In some preferred embodiments, the stem cells or tissue comprising stem cells are obtained from the patient from NOA. In some preferred embodiments, the methods further comprise about day 8 coculturing primordial germ cells with the genital ridge cells. In some preferred embodiments, the primordial germ cells are derived from an embryo. In some preferred embodiments, the primordial germ cells are primordial germ cell-like cells. In some preferred embodiments, the primordial germ cells differentiate into spermatogonia.

[0024] In some preferred embodiments, the methods further comprise isolating the spermatogonia.

[0025] In a second aspect, the present invention provides a cell culture comprising artificial testis cells produced by the foregoing methods.

[0026] In a third aspect, the present invention provides isolated artificial testis cells produced by the foregoing methods, preferably isolated artificial Sertoli cells, isolated artificial Leydig cells, isolated artificial Myoid cells and / or isolated artificial stromal progenitor cells.

[0027] In a fourth aspect, the present invention provides an artificial testis organoid produced by the foregoing methods.

[0028] In a fifth aspect, the present invention provides spermatogonia produced by the foregoing methods.

[0029] In a sixth aspect, the present invention provides differentiated patient stem cells produced by the foregoing methods.

[0030] In a seventh aspect, the present invention provides in vitro methods for production of artificial Leydig-like cells from vertebrate pluripotent stem cells comprising: deriving genital ridge cells from pluripotent stem cells; and treating the genital ridge cells with a base medium comprising SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), bFGF2 (Basic fibroblast growth factor), DAPT and LiCh so that the genital ridge cells differentiate into Leydig-like cells.

[0031] In some preferred embodiments, the step of deriving genital ridge cells further comprises: providing vertebrate pluripotent stem cells in a maintenance medium comprising a ROCK inhibitor; at day 0, removing the maintenance medium comprising a ROCK inhibitor and culturing the vertebrate pluripotent stem cells with the base medium comprising CHIR99021 so that the vertebrate pluripotent stem cells differentiate into presomatic mesoderm cells; on about day 4, removing the base medium comprising CHIR99021 and culturing the presomatic mesoderm cells in base medium comprising fibroblast growth factor 9 (FGF9) and heparin so that the presomatic mesoderm cells differentiate into intermediate mesoderm cells; and on about day 7 or 8, removing the medium comprising FGF9 and heparin and culturing the cells in a base medium comprising FGF9, insulin and / or IGF1, EGF, RA,PGD2, LH, FSH and / or T or a base medium comprising insulin and / r IGF1, FGF9, RA, and PDG2 so that the cells differentiate into genital ridge cells.

[0032] In some preferred embodiments, the genital ridge cells are treated with the base medium comprising SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), bFGF2 (Basic fibroblast growth factor), DAPT and LiCh on about day 8. In some preferred embodiments, the genital ridge cells are dissociated into single cells.

[0033] In some preferred embodiments, the Leydig-like cells are produced in the culture by about day 16. In some preferred embodiments, the Leydig-like cells demonstrate upregulation or expression of one or more markers selected from the group consisting of STAR and 3BHSD.

[0034] In some preferred embodiments, the vertebrate pluripotent stem cells are human stem cells. In some preferred embodiments, the human stem cells are human embryonic stem cells. In some preferred embodiments, the human stem cells are induced pluripotent stem cells.

[0035] In some preferred embodiments, the methods further comprise the step of isolating the Leydig-like cells.

[0036] In some preferred embodiments, the methods further comprise the step of transplanting the isolated Leydig-like cells into a mammal.

[0037] In some preferred embodiments, the methods further comprise contacting the Leydig- like cells with a test reagent and assaying the effect of the test reagent on the cells.

[0038] In some preferred embodiments, the methods further comprise obtaining stem cells or tissue comprising stem cells from a patient and culturing the stem cells or tissue comprising stem cells from the patient with the Leydig-like cells. In some preferred embodiments, the methods further comprise transferring the stem cells or cells differentiated from the stem cells back to a patient in need thereof.

[0039] In ninth aspect, the present invention provides artificial Leydig cells produced by the foregoing methods.

[0040] In a tenth aspect, the present invention provides in vitro method for production of artificial Myoid-like cells from vertebrate pluripotent stem cells comprising: deriving genital ridge cells from pluripotent stem cells; and treating the genital ridge cells with a base medium comprising SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), valproic acid, BMP2 (Bone Morphogenetic Protein 2), BMP4 (Bone Morphogenetic Protein 4), and Activin A so that the genital ridge cells differentiate into Myoid-like cells.

[0041] In some preferred embodiments, the step of deriving genital ridge cells further comprises: providing vertebrate pluripotent stem cells in a maintenance medium comprising a ROCK inhibitor; at day 0, removing the maintenance medium comprising a ROCK inhibitor and culturing the vertebrate pluripotent stem cells with the base medium comprising CHIR99021 so that the vertebrate pluripotent stem cells differentiate into presomatic mesoderm cells; on about day 4, removing the base medium comprising CHIR99021 and culturing the presomatic mesoderm cells in base medium comprising fibroblast growth factor 9 (FGF9) and heparin so that the presomatic mesoderm cells differentiate into intermediate mesoderm cells; and on about day 7 or 8, removing the medium comprising FGF9 and heparin and culturing the cells in a base medium comprising FGF9, insulin and / or IGF1, EGF, RA,PGD2, LH, FSH and / or T or a base medium comprising insulin and / r IGF1, FGF9, RA, and PDG2 so that the cells differentiate into genital ridge cells.

[0042] In some preferred embodiments, the genital ridge cells are treated with the base medium comprising SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), valproic acid, BMP2 (Bone Morphogenetic Protein 2), BMP4 (Bone Morphogenetic Protein 4), and Activin A on about day 8. In some preferred embodiments, the genital ridge cells are dissociated into single cells.

[0043] In some preferred embodiments, the Myoid-like cells are produced in the culture by about day 16. In some preferred embodiments, the Myoid- like cells demonstrate upregulation or expression of one or more Myoid cell markers.

[0044] In some preferred embodiments, the vertebrate pluripotent stem cells are human stem cells. In some preferred embodiments, the human stem cells are human embryonic stem cells. In some preferred embodiments, the human stem cells are induced pluripotent stem cells.

[0045] In some preferred embodiments, the methods further comprise the step of isolating the Myoid-like cells. In some preferred embodiments, the methods further comprise transplanting the isolated Myoid-like cells into a mammal.

[0046] In some preferred embodiments, the methods further comprise contacting the Myoidlike cells with a test reagent and assaying the effect of the test reagent on the cells.

[0047] In some preferred embodiments, the methods further comprise obtaining stem cells or tissue comprising stem cells from a patient and culturing the stem cells or tissue comprising stem cells from the patient with the Leydig-like cells. In some preferred embodiments, the stem cells or cells differentiated from the stem cells back to a patient in need thereof. In an eleventh aspect, the present invention provides artificial Myoid cells produced by the foregoing methods.

[0048] In a twelfth aspect, the present invention provides in vitro methods for production of artificial testis cells from mouse pluripotent stem cells comprising: deriving intermediate mesoderm cells from mouse pluripotent stem cells; and treating the intermediate mesoderm cells with a base medium comprising FGF9, insulin and / or IGF1, PGD2, RA, BMP4 (Bone Morphogenetic Protein 4), and EGF or a base medium comprising FGF9, insulin and / or IGF1, PGD2, RA, BMP4 (Bone Morphogenetic Protein 4), EGF, FSH and LH or Human Chorionic Gonadotropin (HCG) so that the intermediate ridge cells differentiate into testis cells.

[0049] In some preferred embodiments, the step of deriving intermediate mesoderm cells further comprises: providing mouse pluripotent stem cells; at day 0, culturing the mouse pluripotent stem cells with the base medium comprising Activin A (A A) and bFGF (Basic Fibroblast Growth Factor); and on about day 2, removing the base medium comprising AA and bFGF and culturing the cells in base medium comprising AA, RA, and BMP4. In some preferred embodiments, the cells are treated with the base medium comprising FGF9, insulin and / or IGF1, PGD2, RA, BMP4, and EGF on about day 6 or FGF9, insulin and / or IGF1, PGD2, RA, BMP4, EGF, FSH, and LH or HCG on about day 6 or 7. In some preferred embodiments, on about day 7 the culture medium is replaced with a Leydig cell differentiation medium comprising a base medium supplemented with SAG (Smoothened Agonist), PDGF- AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), bFGF2 (Basic fibroblast growth factor), DAPT and LiCh. In some preferred embodiments, on about day 7 the culture medium is replaced with a Myoid cell differentiation medium comprising a base medium supplemented with SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), valproic acid, BMP2 (Bone Morphogenetic Protein 2), BMP4 (Bone Morphogenetic Protein 4), and Activin A.

[0050] In some preferred embodiments, testis organoids are formed in the culture by about day 8. In some preferred embodiments, the testis organoids comprise one or more of artificial Sertoli cells, artificial Leydig cells, artificial myoid cells and artificial stromal cells. In some preferred embodiments, the testis organoids comprise two or more of artificial Sertoli cells, artificial Leydig cells, artificial myoid cells and artificial stromal cells. In some preferred embodiments, the testis organoids comprise three or more of artificial Sertoli cells, artificial Leydig cells, artificial myoid cells and artificial stromal cells. In some preferred embodiments, the testis organoids comprise artificial Sertoli cells, artificial Leydig cells, artificial myoid cells and artificial stromal cells. In some preferred embodiments, the organoids demonstrate upregulation or expression of one or more markers selected from the group consisting of LHX9, PDGRA, COUPTFII, TCF21, SOX9, GATA4, SF1, SMA, DHH, STAR and 3BHSD.

[0051] In some preferred embodiments, the mouse stem cells are mouse embryonic stem cells. In some preferred embodiments, the mouse stem cells are induced pluripotent stem cells.

[0052] In some preferred embodiments, the methods further comprise the step of isolating 1) the artificial testis cells from the organoids or 2) the testis organoid. In some preferred embodiments, the artificial testis cells are Sertoli-like cells. In some preferred embodiments, the artificial testis cells are Leydig-like cells. In some preferred embodiments, the artificial testis cells are Myoid-like cells. In some preferred embodiments, the artificial testis cells are stromal progenitor cells.

[0053] In some preferred embodiments, the methods further comprise transplanting the isolated artificial testis cells or artificial testis organoid into a mammal.

[0054] In some preferred embodiments, the methods further comprise contacting the artificial testis cells or artificial testis cell organoid with a test reagent and assaying the effect of the test reagent on the artificial testis cells or artificial testis cell organoid.

[0055] In a thirteenth aspect, the present invention provides testis organoid produced by the foregoing methods.

[0056] In a fourteenth aspect, the present invention provides artificial Leydig cells produced by the foregoing methods.

[0057] In a fifteenth aspect, the present invention provides artificial Sertoli cells produced by the foregoing methods.

[0058] In a sixteenth aspect, the present invention provides artificial Myoid cells produced by the foregoing methods.

[0059] In a seventeenth aspect, the present invention provides artificial stromal progenitor cells produced by the foregoing methods.

[0060] In an eighteenth aspect, the present invention provides methods comprising: contacting artificial testis cells or organoids as described in any aspect above with a test reagent; and assaying the effect of the test reagent on the artificial testis cells or organoids.

[0061] In a nineteenth aspect, the present invention provides methods comprising: transplanting the artificial testis cells or organoids according to any aspect above into a subject. In a twentieth aspect, the present invention provides methods comprising: obtaining stem cells or tissue comprising stem cells from a patient; and co-culturing the stem cells or tissue comprising stem cells from the patient with artificial testis cells or organoids according to any aspect described above. In some preferred embodiments, the stem cells are selected from the group consisting of primordial germ cell like cells (PGCLC), prospermatogonia, and spermatogonial stem / progenitor cells (SSC / SPCs). In some preferred embodiments, the prospermatogonia stem cells differentiate into spermatogonia.

[0062] In some preferred embodiments, the methods further comprise transferring the spermatogonia back to a patient in need thereof.

[0063] In some preferred embodiments, the patient has previously undergone a gonadotoxic treatment and / or has nonobstructive azoospermia patient or severe oligospermy. In some preferred embodiments, the gonadotoxic treatment is selected from the group consisting of chemotherapy and radiation. In some preferred embodiments, the stem cells or tissue comprising stem cells are obtained from the patient prior to a gonadotoxic treatment.

[0064] In a twenty -first aspect, the present invention provides methods of expanding patient derived or in vitro derived germ cells comprising: co-culturing patient or in vitro derived germ cells with artificial testis cells or organoids according to any aspect described above. In some preferred embodiments, the primordial germ cells are primordial germ cell-like cells (PGCLCs), prospermatogonia or spermatogonia. In some preferred embodiments, co- culturing results in licensure of the primordial germ cell like cells or pro- spermatogonia or pro-spermatogonia-like cells to form spermatogonia.

[0065] Brief Description of the Figures

[0066] FIG. 1. Schematic depiction of mouse ESC differentiation protocol.

[0067] FIG. 2. Fluorescence micrographs showing improved expression of many gonadal (WT1, GATA4, SF1, CoupTFII) and Sertoli cell markers (SOX9, GAT A3) in the improved mouse protocol as compared to an older protocol.

[0068] FIG. 3. Fluorescence micrographs demonstrating generation of Leydig-like cells after 15 days in culture. Cells are co-stained by SF1 and 3BHSD.

[0069] FIG. 4A-B. Graphs demonstrating improved expression of multiple testicular cell type markers. (A) Markers of Sertoli cells (blue= old; yellow = new). (B) peritubular myoid cells. FIG. 5. Schematic depiction of human ESC differentiation protocol.

[0070] FIG. 6. Schematic depiction of human iPSC differentiation protocol. FIG. 7. Schematic depiction of protocol for combining testis like cells derived from human ESCs with primordial germ cells.

[0071] FIG. 8. Schematic depiction of protocol for deriving peritubular myoid cells from human ESCs.

[0072] FIG. 9. Schematic depiction of protocol for deriving Leydig cells from human ESCs.

[0073] FIG. 10. Differentiation efficiency of human somatic-like cells in the presence of hormone using the protocol described in PCT US2023 / 13608. A) Revised differentiation schema. B) qPCR expression of gonadal, Sertoli, Leydig, and off-target markers.

[0074] FIG. 11. Revised testis differentiation protocol of the present invention. (A) Schematic and media composition. (B) Various gonadal, Sertoli and Leydig cell markers. D16 Soma+PGC -(- / -Hormone is the most efficient and refined condition.

[0075] FIG. 12. Schematic depiction of the revised mouse ESC differentiation protocol. Briefly Day 4 cells are dissociated and 25 K cells are placed in a 96 well u-bottom plate. The 3D aggregates are maintained for 3 days before being transplanted to ThinCert® and collected at Dayl7.

[0076] FIG. 13A-E. Graphical data demonstrating that the differentiation schema depicted in FIG. 12 effectively induces testis progenitor markers. Additionally, it promotes the expression of markers for Sertoli cells, Leydig cells, and myoid cells, with a few endothelial cell markers also beginning to peak. Two different color represents two different medium composition to grow organoids on ThinCert® cell culture inserts. Note: testis progenitor markers include Tcf21, Pdgfr-alfa and Nr2f2 (A), Sertoli cell markers are Sox9, Wtl, Gata4 and Inhbb (B), Leydig cell markers Cypl lal, Cypl7al, Hsd3b6, Nr5al, Cyp21al and Cyl lbl (C), Myoid markers Cnnl, Sma-Alfa and Myhl l (D) and Endothelial cell markers Pecaml, Esam and Cdh5 (E). The fold change of gene expression was calculated in compression with day 0 undifferentiated ESCs. Gapdh is used as housekeeping gene to normalize Ct values in qPCR analysis.

[0077] FIG. 14A-D. Fluorescence micrographs demonstrating complete reconstitution of the testis microenvironment and tubular structure in organoids made by the schema depicted in FIG. 12. A) Co-staining for Leydig (SF1), peritubular myoid (SMA) and Sertoli cells (SOX9). B) Sertoli (GATA4+ SOX9) cells form a blood testis barrier (ZO-1). C) Our invitro derived Sertoli cells (SOX9+) make anti-Mullerian hormone (AMH). D) The Leydig cells present in the organoids are mature Leydig cells as they express (SF1, HSD3B and StAR).

[0078] FIG. 15. The in vitro derived organoids produce testosterone in response to LH or HCG induction. FIG. 16. Schema for Mixing of Postnatal Day 2 germ cells with our in vitro derived somatic cells. Aggregation occurs at day 4, following the differentiation schema depicted in FIG. 12. FIG. 17A-D. Graphs showing germ cell counts and percentage at day 1 and day 3 organoids in 96 well plates. The total number of germ cell number is determined by counting the total number of DDX4 or DAZL+ cells at day 1 and 3-day (A). Percentage and total number of OCT4-GFP positive Pro-spermatogonia (B), Percentage and total number of PLZF+ undifferentiated spermatogonia (C) Percentage and total number of Differentiating Stra8+ spermatogonia.

[0079] FIG. 18. Experimental schema for in vitro testis organoid development from human stem cells.

[0080] FIG. 19. Graphical data showing that expression levels of Sertoli cell markers are improved by day 22 by increasing EGF1 concentration to 50ng / ml.

[0081] FIG. 20A-B. Immunofluorescence micrographs of testis organoids collected after 22 days in culture. (A) WT1 a marker for interstitial cells of the testis - progenitors for Leydig and myoid cells. (B) Formation of S0X9+ and Gata4+ tubules.

[0082] Definitions

[0083] As used herein the term "stem cell" ("SC") refers to cells that can self-renew and differentiate into multiple lineages. A stem cell is a developmentally pluripotent or multipotent cell. A stem cell can divide to produce two daughter stem cells, or one daughter stem cell and one progenitor ("transit") cell, which then proliferates into the tissue's mature, fully formed cells. Stem cells may be derived, for example, from embryonic sources ("embryonic stem cells") or derived from adult sources. For example, U.S. Pat. No. 5,843,780 to Thompson describes the production of stem cell lines from human embryos. PCT publications WO 00 / 52145 and WO 01 / 00650 describe the use of cells from adult humans in a nuclear transfer procedure to produce stem cell lines. The term “stem cell” as used herein thus encompasses embryonic stem cells, adult stem cells and induced pluripotent stem cells.

[0084] Examples of adult stem cells include, but are not limited to, hematopoietic stem cells, neural stem cells, mesenchymal stem cells, and bone marrow stromal cells. These stem cells have demonstrated the ability to differentiate into a variety of cell types including adipocytes, chondrocytes, osteocytes, myocytes, bone marrow stromal cells, and thymic stroma (mesenchymal stem cells); hepatocytes, vascular cells, and muscle cells (hematopoietic stem cells); myocytes, hepatocytes, and glial cells (bone marrow stromal cells) and, indeed, cells from all three germ layers (adult neural stem cells). As used herein, the term "totipotent cell" refers to a cell that is able to form a complete embryo (e.g., a blastocyst).

[0085] As used herein, the term "pluripotent cell" or "pluripotent stem cell" refers to a cell that has complete differentiation versatility, e.g., the capacity to grow into any of the mammalian body's approximately 260 cell types. A pluripotent cell can be self-renewing and can remain dormant or quiescent within a tissue. Unlike a totipotent cell (e.g., a fertilized, diploid egg cell), a pluripotent cell, even a pluripotent embryonic stem cell, cannot usually form a new blastocyst.

[0086] As used herein, the term "induced pluripotent stem cells" ("iPSCs") refers to a stem cell induced from a somatic cell, e.g., a differentiated somatic cell, and that has a higher potency than said somatic cell. iPS cells are capable of self-renewal and differentiation into mature cells.

[0087] As used herein, the term "multipotent cell" refers to a cell that has the capacity to grow into a subset of the mammalian body's approximately 260 cell types. Unlike a pluripotent cell, a multipotent cell does not have the capacity to form all of the cell types.

[0088] As used herein, the term "progenitor cell" refers to a cell that is committed to differentiate into a specific type of cell or to form a specific type of tissue.

[0089] As used herein, the term "embryonic stem cell" ("ES cell" or ESC") refers to a pluripotent cell that is derived from the inner cell mass of a blastocyst (e.g., a 4- to 5 -day-old human embryo) and has the ability to yield many or all of the cell types present in a mature animal.

[0090] As used herein the term "feeder cells" refers to cells used as a growth support in some tissue culture systems. Feeder cells may be embryonic striatum cells or stromal cells. As used herein, the term "chemically defined media" refers to culture media of known or essentially- known chemical composition, both quantitatively and qualitatively. Chemically defined media is free of all animal products, including serum or serum-derived components (e.g., albumin).

[0091] As used herein, the term "serum-free media" refers to culture media that is devoid of serum, but not necessarily of other undefined components.

[0092] Detailed Description of the Invention

[0093] Methods, kits, compositions, and systems are provided for culturing pluripotent stem cells to produce populations of cells comprising artificial testis cells, such as Sertoli and Leydig cells. In particular, culture conditions are provided that result in the generation of artificial testis cells from a starting culture of human pluripotent stem cells. Methods of using the cells, for example in various therapies, are also provided.

[0094] These methods overcome the limitations noted in the Background and leverage genetic, evolutionary, and molecular insights gained from scRNAseq data that the inventors collected across developmental stages and multiple species to develop a new, highly efficient, directed somatic cell differentiation protocol. The inventors have tuned many parameters of the protocols on a mouse ESC and two human ESC lines and analysed scRNAseq data to confirm the progression through expected cell states along the developmental trajectories. Benchmarking and classification of the in vitro derived cell states has relied on the wealth of in vivo markers that have been previously described during gonadal differentiation.

[0095] Somatic cells of the testis are central to testis tissue homeostasis and men’s reproductive and overall health. The somatic cells provide a series of unknown growth factors and cytokines that are necessary for guiding germ cell development in vivo and required for the complete reconstitution of germline development for females in vitro or promoting the differentiation of male primordial germ cell like cells (PGCLC) to spermatogonia.

[0096] When working with mice, co-culturing of in vitro derived PGCLC with fetal somatic cells isolated from littermates or allogenic embryonic gonads is feasible, but this is costly in non-human primates and ethically inconceivable in human. Thus, an improved understanding of the somatic cell specification program and generation of alternative somatic cell sources will prove critical for reconstituting somatic cells in a petri-dish, replacement of damaged cells in vivo in response to inherited / natural occurring genetic mutations, iatrogenic agents because of cancer therapy, to rejuvenate the aging gonads, or indeed to utilize the organoids to make germ cells in vitro. To address this gap, the present inventors provide improved methods for the generation of human artificial testis cells, such as Sertoli and / or Leydig cells) from stem cells (e.g., embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) using the differentiation schema described below.

[0097] Pluripotent stem cells

[0098] The methods systems, systems and kit of the present invention find use with a variety of pluripotent cells. Suitable pluripotent stem cells include, but are not limited to, embryonic stem cells, adult stem cells, and induced pluripotent stem cells. In some preferred embodiments, the pluripotent stem cells are vertebrate pluripotent stem cells. In some particularly preferred embodiments, the pluripotent stem cells are human embryonic stem cells (hESCs). In other particularly preferred embodiments, the pluripotent stem cells are mouse embryonic stem cells (mESCs). In still other particularly preferred embodiments, the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

[0099] In some preferred embodiments, the pluripotent stem cells may be genetically modified by methods known in the art so that they comprise and express one or more exogenous genes.

[0100] Base media

[0101] A concern in the culture of human ES cells is to remove, to the extent possible, undefined constituents and constituents of animal origin from ES cell culture conditions. Standardizing culture conditions minimizes the normal variations in biological materials to which the cells are exposed. Further, by avoiding the use of materials, cells, exudates or constituents of animal origin, one can avoid possible cross-species viral transmission through the culture system. Thus, utilization of chemically defined media (CDM) that avoid the use of animal products provides a baseline culture condition upon which differentiation factors may be added with predictable effects.

[0102] CDM (e.g., for hESCs) may include maintenance or basal media containing salts, vitamins, glucose and amino acids. For maintenance of human stem cells prior to the differentiation protocol, a mTeSR medium such as mTeSRl™ from StemCell Technologies may be utilized. In some embodiments, the maintenance medium preferably comprises a ROCK inhibitor such as Y27632. Maintenance medium for mouse stem cells may preferably be GMEM from ThermoFisher Scientific, preferably supplemented with LIF (Leukemia Inhibitory Factor) and optionally knockout serum. The basal differentiation medium can be any of a number of commercially available media. In some preferred embodiments, a combination of Dulbecco's Modified Eagle Medium and Hams F12 medium, sold as a combination (DMEM / F12; Invitrogen) may be utilized. In other preferred embodiments, an APEL medium may be utilized, for example, STEMdiff™ APEL™ medium from StemCell Technologies. STEMdiff ™ APEL ™ Medium is a serum-free and animal component-free medium specifically developed to support hPSC differentiation. It was first described for the induction of hemato-endothelial cells, when supplemented with VEGF, BMP-4, SCF, and Activin A, but it has also been proven to be an effective basal medium for hPSC differentiation to other lineages, including cardiomyocytes. In other preferred embodiments, an mTeSR medium may be utilized for maintenance of stem cells.

[0103] Differentiation into artificial testis cells The present invention provides methods and reagents for producing artificial testis cells (e.g., Sertoli cells, Leydig cells, Myoid cells, and / or stromal cells) from pluripotent stems cells. The present invention is not limited to the use of any particular pluripotent stem cells or chemically defined media. The methods described herein for the production of artificial testis cells are described in relation to events occurring at various time points. It will be recognized that the methods may be varied by making alterations to the described time schedules. “Day 0” as used herein refers to the day and time that the pluripotent stem cells are removed from a maintenance medium and exposed to a differentiation medium. The differentiation timeline is then defined from the Day 0 starting point. When the term “about” X days is utilized, it refers to the number of days from the Day 0 starting time point plus or minus 12 hours. For example, “on about Day 4” means 96 hours (i.e., 4 days) from the Day 0 starting point plus or minus 12 hours. If the Day 0 stating time was 9:00 AM, “about Day 4” then refers to 96 hours from that time point plus or minus 12 hours.

[0104] The first step in a method for producing human artificial testis cells according to the invention comprises providing pluripotent hESC or iPSCs as described above. In some preferred embodiments, the pluripotent stem cells are provided in a stem cell maintenance medium. In some preferred embodiments, the stem cell maintenance medium is a chemically defined medium such as an mTeSR medium. In some preferred embodiments, the stem cell maintenance medium comprises a ROCK inhibitor. In some preferred embodiments, the ROCK inhibitor is Y27632.

[0105] The second step of the method of the present invention comprises removing the pluripotent stem cells from the maintenance medium and culturing the pluripotent stem cells in a basal medium supplemented with agents suitable for directing the pluripotent stem cells to a presomatic mesoderm lineage. In some preferred embodiments, the basal medium is a chemically defined medium. In some particularly preferred embodiments, the basal medium is an APEL medium such as STEMdiff™ APEL™ medium from StemCell Technologies. In some particularly preferred embodiments, the basal medium is supplemented with from between 0.5 to 15 pM (e.g., 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0. 12.0, 13.0, and 14.0 pM and values and ranges therein) CHIR99021. This step defines Day 0 of the process. The base medium with supplements is preferably changed daily.

[0106] The third step of the method of the present invention comprises on about or at day 4 culturing the presomitic mesoderm cells produced in step 2 in a basal medium supplemented with agents for directing the presomitic mesoderm cells to form intermediate mesoderm. In some preferred embodiments, the basal medium is a chemically defined medium. In some particularly preferred embodiments, the basal medium is an APEL medium such as STEMdiff™ APEL™ medium from StemCell Technologies. In some particularly preferred embodiments, the basal medium is supplemented with from between 20 and 500 ng / ml (50, 100, 150, 200, 250, 300, 350, 400, 450 ng / ml and values and ranges therein) FGF 9. In some particularly preferred embodiments, the basal medium is further supplemented with from between 0.1 and 10 pg / ml (e.g., 0.4, 0.8, 1.0. 1.5, 2.0, 3.0. 5.0. 6.0, 7.0, 8.0, 9.0 pg / ml and values and ranges therein) heparin. The base medium with supplements may preferably be changed every two days.

[0107] The fourth step of the method of the present invention comprises on about or at day 7 culturing the intermediate mesoderm cells produced in step 3 in a basal medium to direct the intermediate mesoderm cells to form genital ridge cells. In some preferred embodiments, the basal medium is a chemically defined medium. In some particularly preferred embodiments, the basal medium is an APEL medium such as STEMdiff™ APEL™ medium from StemCell Technologies. The base medium may preferably be changed every two days. In some particularly preferred embodiments, the basal medium is supplemented with from between 5 and 100 nM (e.g., 10, 17, 20, 30, 40, 50, 60, 70, 80, 90 nM and values and ranges therein) IGF1. In some particularly preferred embodiments, the basal medium is further supplemented with from between 10 and 500 nM (e.g., 20, 50, 100, 200, 300, 400 nM and values and ranges therein) insulin. In some embodiments, the base medium may be further supplemented with from between 0.01 and 10 pM (e.g., 0.05, 0.1, 1.0, 5.0, 8.0 pM and values and ranges therein) retinoic acid (RA). In some embodiments, the base medium may be further supplemented with from between 50 and 1000 ng / ml (e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900 ng / ml and values and ranges therein) PGD2. In some embodiments, the base medium may be further supplemented with from between 50 and 500 ng / ml (e.g., 100, 200, 300, 400 ng / ml and values and ranges therein) FGF9. In some further preferred embodiments, the base medium used in step 5 may be further be supplemented with from 5 to 100 ng / ml (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90 ng / ml and values and ranges therein) bone morphogenetic protein 4 (BMP4). In some further preferred embodiments, the base medium used in step 4 may be further be supplemented with from 5 to 200 ng / ml (e.g., 10, 20, 30, 50, 70, 100, 150 ng / ml and values and ranges therein) epidermal growth factor (EGF). In some still further preferred embodiments, the base medium used in step 5 may be further be supplemented with from 0.5 to 10 pM (e.g., 1.0, 2.0, 3.0, 4.0 5.0, 6.0, 7.0, 8.0, 9.0 pM and values and ranges therein) IWR1. In some preferred embodiments, the base medium used in step 4 is further supplemented with supplemented with from 5 to 100 ng / ml (10, 20, 30, 40, 50, 60, 70, 80, 90 ng / ml and values and ranges therein) luteinizing hormone (LH). In an alternative embodiment, human chorionic gonadotropin (HCG) is used instead of LH at an amount of 1 to 10 units (1, 2, 3, 5 or 10 units and ranges and values therein). In still another embodiment, the cells are treated first with HCG at the recited range for until about day 15 and then with LH at the recited range after about day 15. In some preferred embodiments, the base medium used in step 4 is further supplemented with from 5 to 300 ng / ml (10, 20, 30, 40, 50, 100, 150, 200, 250 ng / ml and values and ranges therein) follicle stimulating hormone (FSH). In some preferred embodiments, a combination of insulin and IGF1 in the above ranges is utilized. In some preferred embodiments, a combination of insulin, IGF1, and FGF9 in the above ranges is utilized. In some preferred embodiments, a combination of insulin, IGF1, FGF9, RA, EGF, and PG Di in the above ranges is utilized.

[0108] The fifth step of the method of the present invention comprises on about or at day 8 culturing the genital ridge cells produced in step 4 in a basal medium supplemented with agents for directing the genital ridge cells to form artificial testis cells. In some preferred embodiments, the cells are dissociated and plated into an Aggrewell® plate or a u-bottom plate. In some preferred embodiments, from 10000 to about 50000, and most preferably about 30000 dissociated cells are transferred into u-bottom plates. In some preferred embodiments, the basal medium is a chemically defined medium. In some particularly preferred embodiments, the basal medium is an APEL medium such as STEMdiff® APEL® medium from StemCell Technologies. In some particularly preferred embodiments, the basal medium is supplemented with from between 5 and 100 nM (e.g., 10, 17, 20, 30, 40, 50, 60, 70, 80, 90 nM and values and ranges therein) IGF1. In some particularly preferred embodiments, the basal medium is further supplemented with from between 10 and 500 nM (e.g., 20, 50, 100, 200, 300, 400 nM and values and ranges therein) insulin. In some embodiments, the base medium may be further supplemented with from between 0.01 and 10 pM (e.g., 0.05, 0.1, 1.0, 5.0, 8.0 pM and values and ranges therein) retinoic acid (RA). In some embodiments, the base medium may be further supplemented with from between 50 and 1000 ng / ml (e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900 ng / ml and values and ranges therein) PGD2. In some embodiments, the base medium may be further supplemented with from between 50 and 500 ng / ml (e.g., 100, 200, 300, 400 ng / ml and values and ranges therein) FGF9. In some further preferred embodiments, the base medium used in step 5 may be further be supplemented with from 5 to 100 ng / ml (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90 ng / ml and values and ranges therein) bone morphogenetic protein 4 (BMP4). In some further preferred embodiments, the base medium used in step 5 may be further be supplemented with from 1 to 200 ng / ml (e.g., 5, 10, 20, 30, 50, 70, 100, 150 ng / ml and values and ranges therein) epidermal growth factor (EGF). In some still further preferred embodiments, the base medium used in step 5 may be further be supplemented with from 0.1 to 10 pM (e.g., 0.5, 1.0, 2.0, 3.0, 4.0 5.0, 6.0, 7.0, 8.0, 9.0 pM and values and ranges therein) IWR1. In some preferred embodiments, a combination of insulin and IGF1 in the above ranges is utilized. In some preferred embodiments, a combination of insulin, IGF1, and FGF9 in the above ranges is utilized. In some preferred embodiments, a combination of insulin, IGF1, FGF9, RA and PGD2 in the above ranges is utilized. In some preferred embodiments, a combination of insulin, IGF1, FGF9, EGF and IWR1 in the above ranges is utilized. In some preferred embodiments, a combination of insulin, IGF1, FGF9, EGF, RA, IWR1, and PGD2 in the above ranges is utilized. The base medium with supplements may preferably be changed every two days.

[0109] In some further preferred embodiments, the base medium used in step 5 is further supplemented with one or more hormones (FSH, LH or HCG). In some further preferred embodiments, the one or more hormones are added on or at about day 9. In some preferred embodiments, the base medium (which may preferably comprise one or more of insulin, IGF1, FGF9, EGF, RA, IWR1 and PGD2 in the above ranges) is further supplemented with from 5 to 100 ng / ml (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90 ng / ml and values and ranges therein) luteinizing hormone (LH). ). In an alternative embodiment, human chorionic gonadotropin (HCG) is used instead of LH at an amount of 1 to 10 units (1, 2, 3, 5 or 10 units and ranges and values therein). In some preferred embodiments, the base medium (which may preferably comprise one or more of insulin, IGF1, FGF9, EGF, RA, IWR1 and PGD2 in the above ranges) is further supplemented with from 20 to 300 ng / ml (e.g., 50, 100, 150, 200, 250 ng / ml and values and ranges therein) follicle stimulating hormone (FSH). In some preferred embodiments, the base medium (which may preferably comprise one or more of insulin, IGF1, FGF9, BMP4, EGF, RA, IWR1, and PGD2 in the above ranges) is further supplemented with from between 0.01 and 10 pM (e.g., 0.05, 0.1, L0, 5.0, 8.0 pM and values and ranges therein) testosterone. In some preferred embodiments, the base medium (which may preferably comprise one or more of insulin, IGF1, FGF9, EGF, RA, IWR1 and PGD2 in the above ranges) with LH, FSH and testosterone in the ranges described herein.

[0110] In some preferred embodiments, in a sixth step the cultures of step 5 for hESC are maintained until about day 12 so that organoids are formed and the organoids are then transferred to a culture system for culture at an air liquid interface. The base medium is preferably supplemented with the same factors as described for step 5. In some preferred embodiments, the air- liquid interface culture system utilizes a ThinCert® cell culture insert in a Transwell® plate. In some preferred embodiments, organoids comprising one or more of Sertoli cells, Leydig cells, Myoid cells and stromal cells are produced by about day 22. At this point, the organoids may be collected for use or dissociated for isolation of, for example, Leydig, Sertoli, Myoid or stromal cells.

[0111] In some embodiments, the present invention provides methods for producing artificial Leydig cells. In these embodiments, the steps described above for deriving testis cells are followed up until on or about day 8 (i.e., steps 1 to 4). On or about day 8, the genital ridge cells are preferably dissociated and cultured in a basal medium supplemented with agents for directing the genital ridge cells to form artificial Leydig cells. In some preferred embodiments, the basal media is supplemented with one or more of SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), bFGF2 (Basic fibroblast growth factor), DAPT and LiCh. In some preferred embodiments, the basal media is supplemented with SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), bFGF2 (Basic fibroblast growth factor), DAPT and LiCh. In some preferred embodiments, SAG is included in the basal medium at from 0.1 to 5.0 pM (e.g., 0.2, 0.5, 1.0, 2.0, 2.0, 4.0 pM and ranges and values therein). In some preferred embodiments, PDGF-AA is included in the basal medium at from 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml and ranges and values therein. In some preferred embodiments, PDGF-BB is included in the basal medium at from 1 to 50 ng / ml (1, 5, 10, 15, 20, 30, 40 ng / ml and ranges and values therein. In some preferred embodiments, bFGF is included in the basal medium at from 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml and ranges and values therein. In some preferred embodiments, DAPT is included in the basal medium at from 1.0 to 20.0 pM (e.g., 2.0, 5.0. 10.0, 15.0 pM and ranges and values therein). In some preferred embodiments, LiCh is included in the basal medium at from 1.0 to 20.0 mM (e.g., 2.0, 5.0. 10.0, 15.0 mM and ranges and values therein). In some preferred embodiments, Leydig cells characterized by the expression of Leydig cell markers as described in the Examples are produced and collected on or about on day 16.

[0112] In some embodiments, the present invention provides methods for producing artificial Myoid cells. In these embodiments, the steps described above for deriving testis cells are followed up until on or about day 8 (i.e., steps 1 to 4). On or about day 8, the genital ridge cells are preferably dissociated and cultured in a basal medium supplemented with agents for directing the genital ridge cells to form artificial Myoid cells. In some preferred embodiments, the basal medium is supplemented with one or more of SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), valproic acid, BMP2 (Bone Morphogenetic Protein 2), BMP4 (Bone Morphogenetic Protein 4), and Activin A so that the genital ridge cells differentiate into Myoid-like cells. In some preferred embodiments, the basal medium is supplemented with SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), valproic acid, BMP2 (Bone Morphogenetic Protein 2), BMP4 (Bone Morphogenetic Protein 4), and Activin A. In some preferred embodiments, SAG is included in the basal medium at from 0.1 to 5.0 M (e.g., 0.2, 0.5, 1.0, 2.0, 2.0, 4.0 M and ranges and values therein). In some preferred embodiments, PDGF-AA is included in the basal medium at from 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml and ranges and values therein. In some preferred embodiments, PDGF-BB is included in the basal medium at from 1 to 50 ng / ml (1, 5, 10, 15, 20, 30, 40 ng / ml and ranges and values therein. In some preferred embodiments, BMP2 is included in the basal medium at from 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml and ranges and values therein. In some preferred embodiments, BMP4 is included in the basal medium at from 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml and ranges and values therein. In some preferred embodiments, Activin A is included in the basal medium at from 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml and ranges and values therein. In some preferred embodiments, valproic is included in the basal medium at from 1.0 to 50.0 nM (e.g., 2.0, 10.0, 20.0, 30.0, 40.0 nM and ranges and values therein). In some preferred embodiments, Myoid cells (most preferably peritubular myoid cells) characterized by the expression of Myoid cell markers as described in the Examples are produced and collected on or about on day 16.

[0113] In some embodiments, the present invention provides methods for expanding primordial germ cells, such as primordial germ cell-like cells (PGCLCs). In these embodiments, the steps described above for deriving testis cells are followed up until on or about day 8 (i.e., steps 1 to 4). On or about on day 8, the genital ridge cells are dissociated and combined with primordial germs cells. In some preferred embodiments, the basal medium is supplemented with the same agents in the same concentration ranges as described for production of the artificial testis cells and organoids described above. In some preferred embodiments, the dissociated genital ridge cells are combined with the primordial germ cells in a ratio of about 9: 1. On or about on day 9, the medium is replaced with base medium comprising insulin, FGF9, RA, PDG2, EGF, FSH, LH and testosterone in the same concentration ranges as described above for derivation of artificial testis cells and organoids. As will be recognized, IWR1 is not included in this culture medium. As with the artificial testis cell protocol, in some preferred embodiments, the organoids form by about day 12 and on or about day 12 the organoids are preferably cultured at an air-liquid interface as described above. Organoids may preferably be collected on about day 16 for further use.

[0114] In some embodiments, the present invention provides methods for producing artificial testis cells and organoids from pluripotent mouse cells, such as mESC. In some preferred embodiments, the pluripotent stem cells are provided in a stem cell maintenance medium. In some preferred embodiments, the stem cell maintenance medium is GMEM supplemented with LIF and serum.

[0115] The second step of the method of the present invention comprises removing the pluripotent stem cells from the maintenance medium and culturing the pluripotent stem cells in a basal medium supplemented with agents suitable for directing the pluripotent stem cells to epiblast. This is defined as Day 0. In some preferred embodiments, the basal medium is DMEM / F12 and Neurobasal Medium (both from ThermoFisher Scientific) in a ratio of from 2:1 to 1 :2 and most preferably at a ratio of about 1 :1. In some preferred embodiments, the basal medium is supplemented with N2 supplement, B-27 supplement, and Knockout Serum Replacer (KSR; all from ThermoFisher Scientific), Activin A and bFGF. The supplemented basal medium is referred to as priming medium. In some particularly preferred embodiments, the basal medium is supplemented with from 1 to 10 pl / ml (e.g., 1.0. 2.0, 3.0, 4.0, 5.0, 6.0. 7.0, 8.0, 9.0, 10.0 pl / ml and ranges and values therein) N2 supplement. In some particularly preferred embodiments, the basal medium is supplemented with from 1 to 20 pl / ml (e.g., 1.0. 5.0, 10.0, 20.0 pl / ml and ranges and values therein) B-27 supplement. In some particularly preferred embodiments, the basal medium is supplemented with from 1 to 20 pl / ml (e.g., 1.0. 5.0, 10.0, 20.0 pl / ml and ranges and values therein) KSR. In some particularly preferred embodiments, the basal medium is supplemented with from 1 to 20 ng / ml (e.g., 1.0. 5.0, 10.0, 20.0 ng / ml and ranges and values therein) Activin A. In some particularly preferred embodiments, the basal medium is supplemented with from 1 to 20 ng / ml (e.g., 1.0. 5.0, 10.0, 20.0 ng / ml and ranges and values therein) bFGF.

[0116] The third step of the method of the present invention comprises removing the epiblasts from the priming medium used in step 2 at about day 2 and culturing the epiblasts in a basal medium supplemented with agents suitable for directing the epiblast cells to form anterior intermediate mesoderm. In some preferred embodiments, the basal medium is DMEM / F12. In some preferred embodiments, the basal medium is supplemented with BMP4, Activin A and Retinoic Acid (RA). The supplemented basal medium is referred to as differentiation medium. In some particularly preferred embodiments, the basal medium is supplemented with 1 to 20 ng / ml (e.g., 1.0. 5.0, 10.0, 20.0 ng / ml and ranges and values therein) Activin A. In some particularly preferred embodiments, the basal medium is supplemented with from 10 to 200 nM (e.g., 10.0, 50.0. 100.0, 200.0 nM) and ranges and values therein) RA. In some particularly preferred embodiments, the basal medium is supplemented with 1 to 20 ng / ml (1.0. 2.5, 5.0, 10.0, 20.0 ng / ml and ranges and values therein) BMP4.

[0117] In some preferred embodiments, in a fourth step the anterior intermediate mesoderm cells are dissociated on about day 4 and transferred into a u-bottom 96-well plate. In some embodiments, from 10,000 to 50,000, and most preferably about 25,000 dissociated cells are transferred to the plates. In an alternative embodiment, about 100000 to 500000, and most preferably about 300000 dissociated cells are plated into Aggrewell® plates.

[0118] In some embodiments, the intermediate mesoderm cells on day 4 are dissociated and reaggregated in Aggrewell or u-bottom dishes and cultured in a progenitor medium comprising RA, IGF1, insulin, FGF9, PDG2, BMP4, EGF1 and / or y 27632. In some particularly preferred embodiments, the basal medium is supplemented with from between 5 and 100 nM (e.g., 10, 17, 20, 30, 40, 50, 60, 70, 80, 90 nM and values and ranges therein) IGF1. In some particularly preferred embodiments, the basal medium is further supplemented with from between 10 and 500 nM (e.g., 20, 50, 100, 200, 300, 400 nm and values and ranges therein) insulin. In some embodiments, the base medium may be further supplemented with from between 10.0 and 200 nM (e.g., 10.0, 20.0, 50.0, 100.0 and 200.0 nM and values and ranges therein) RA. In some embodiments, the base medium may be further supplemented with from between 50 and 1000 ng / ml (e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900 ng / ml and values and ranges therein) PGD2. In some embodiments, the base medium may be further supplemented with from between 5 and 40 ng / ml (10, 20, 30 ng / ml and values and ranges therein) FGF9. In some preferred embodiments, the base medium may be further supplemented with from between 1 and 10 pM (e.g., 1.0. 2.0, 3.0, 4.0, 5.0, 6.0. 7.0, 8.0, 9.0, 10.0 pM and ranges and values therein) Y-27632. In some further preferred embodiments, the base medium used in step 5 may be further be supplemented with from 5 to 100 ng / ml (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90 ng / ml and values and ranges therein) bone morphogenetic protein 4 (BMP4). In some further preferred embodiments, the base medium used in step 5 may be further be supplemented with from 1 to 200 ng / ml (e.g., 2, 5, 10, 20, 30, 50, 70, 100, 150 and values and ranges therein) epidermal growth factor (EGF). In some preferred embodiments, the base medium used in step 4 is further supplemented with 5 to 100 ng / ml (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90 ng / ml and values and ranges therein) luteinizing hormone (LH). In an alternative embodiment, human chorionic gonadotropin (HCG) is used instead of LH at an amount of 1 to 10 units (1, 2, 3, 5 or 10 units and ranges and values therein). In some preferred embodiments, the base medium used in step 4 is further supplemented with from 5 to 300 ng / ml (e.g., 10, 20, 30, 40, 50, 100, 150, 200, 250 ng / ml and values and ranges therein) follicle stimulating hormone (FSH).

[0119] In some embodiments, the cells are transferred to a ThinCert® cell culture inserts on about day 7 or an about day 3 days after culture in the Aggrewell® or u-bottom plates.

[0120] The base medium with supplements may preferably be changed every two days. When the culture is maintained with progenitor up to on or about day 8, testis organoids are formed.

[0121] In other preferred embodiments, the progenitor medium is replaced after about 24 hours with Myoid differentiation medium to enrich production of myoid cells in the organoids. In some preferred embodiments, the basal medium is supplemented with one or more of SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF- BB (Platelet-derived growth factor BB), valproic acid, BMP2 (Bone Morphogenetic Protein 2), BMP4 (Bone Morphogenetic Protein 4), and Activin A so that the genital ridge cells differentiate into Myoid-like cells. In some preferred embodiments, the basal medium is supplemented with SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), valproic acid, BMP2 (Bone Morphogenetic Protein 2), BMP4 (Bone Morphogenetic Protein 4), and Activin A. In some preferred embodiments, SAG is included in the basal medium at from 0.1 to 5.0 pM (e.g., 0.2, 0.5, 1.0, 2.0, 2.0, 4.0 pM and ranges and values therein). In some preferred embodiments, PDGF-AA is included in the basal medium at from 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml and ranges and values therein. In some preferred embodiments, PDGF-BB is included in the basal medium at from 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml and ranges and values therein. In some preferred embodiments, BMP2 is included in the basal medium at from 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml and ranges and values therein. In some preferred embodiments, BMP4 is included in the basal medium at from 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml and ranges and values therein. In some preferred embodiments, Activin A is included in the basal medium at from 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml and ranges and values therein. In some preferred embodiments, valproic is included in the basal medium at from 1.0 to 50.0 nM (e.g., 2.0, 10.0, 20.0, 30.0, 40.0 nM and ranges and values therein). In some preferred embodiments, Myoid cells (most preferably peritubular myoid cells) characterized by the expression of Myoid cell markers as described in the Examples are produced and collected on or about on day 8. In other preferred embodiments, the progenitor medium is replaced after about 24 hours with Leydig differentiation medium to enrich production of myoid cells in the organoids. In some preferred embodiments, the basal media is supplemented with one or more of SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), bFGF2 (Basic fibroblast growth factor), DAPT and LiCh. In some preferred embodiments, the basal media is supplemented with SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet- derived growth factor BB), bFGF2 (Basic fibroblast growth factor), DAPT and LiCh. In some preferred embodiments, SAG is included in the basal medium at from 0.1 to 5.0 pM (e.g., 0.2, 0.5, 1.0, 2.0, 2.0, 4.0 pM and ranges and values therein). In some preferred embodiments, PDGF-AA is included in the basal medium at from 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml and ranges and values therein. In some preferred embodiments, PDGF-BB is included in the basal medium at from 1 to 50 ng / ml (e.g., 1 , 5, 10, 15, 20, 30, 40 ng / ml and ranges and values therein. In some preferred embodiments, bFGF is included in the basal medium at from 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml and ranges and values therein. In some preferred embodiments, DAPT is included in the basal medium at from 1.0 to 20.0 pM (e.g., 2.0, 5.0. 10.0, 15.0 pM and ranges and values therein). In some preferred embodiments, LiCh is included in the basal medium at from 1.0 to 20.0 mM (e.g., 2.0, 5.0. 10.0, 15.0 mM and ranges and values therein). In some preferred embodiments, Leydig cells characterized by the expression of Leydig cell markers as described in the Examples are produced and collected on or about on day 8.

[0122] In some preferred embodiments, the artificial testis cells express one or more of the following markers: LHX9, PDGRA, COUPTFII, TCF21, SOX9, GATA4, SF1, SMA, DHH, STAR and 3BHSD. In some preferred embodiments, the artificial testis cells produced by the methods described above express at least one of the markers selected from the group consisting of EMX2, WT, SOX9, LHX9, DHH, STAR and 3BHSD. In some preferred embodiments, the artificial testis cells express at least two of the markers selected from the group consisting of EMX2, WT, SOX9, LHX9, DHH, STAR and 3BHSD. In some preferred embodiments, the artificial testis cells express at least three of the markers selected from the group consisting of EMX2, WT, SOX9, LHX9, DHH, STAR and 3BHSD. In some preferred embodiments, the artificial testis cells express the markers EMX2, WT, SOX9, LHX9, DHH, STAR and 3BHSD.

[0123] In some preferred embodiments, the cultured are maintained until organoids are formed. The organoids preferably are three dimensional organoids having a roughly spherical shape. In some preferred embodiments, the organoids are characterized by comprising tubule structures. In some preferred embodiments, the organoids are characterized by comprising smooth muscle actin.

[0124] In some preferred embodiments, the artificial testis cells or organoids may be harvested or isolated from the cultures for further use.

[0125] Uses of artificial testis cells and organoids

[0126] In some preferred embodiments, methods, reagents, and kits described herein, as well as the artificial testis cells and organoids generated therewith, find use in various research, diagnostic, clinical, and therapeutic applications. In some embodiments, artificial testis cells or organoids are used for direct transplantation into a subject. In some preferred embodiments, the artificial testis cells or organoids are used for somatic cell replacement therapy in a subject in need thereof. In some embodiments, artificial testis cells generated by methods herein are useful for diagnostic, prognostic, and / or therapeutic uses.

[0127] In some embodiments, the isolated artificial testis cells or organoids may be directly transplanted in a subject. If appropriate, cells are co-administered with one or more pharmaceutical agents or bioactives that facilitate the survival and function of the transplanted cells.

[0128] In some embodiments, human organoids are transplanted into mice for additional differentiation and / or maturation of the cells in the organoids. In other embodiments, the organoids are preferably combined with in vivo or in vitro derived germ cells to achieve human germline stem cell expansion and further promote differentiation. It is contemplated that these methods will result in production of haploid round or elongating spermatids which find use in assisted reproductive technologies such as IVF / ICSI.

[0129] In some embodiments, the testis cells or organoids of the invention may be used to coculture gamete stem cells such as primordial germ cell like cells, prospermatogonia or spermatogonial stem / progenitor cells (SSC / SPCs) from a patient. In some preferred embodiments, the cells are cultured so that the stem cells from the patient differentiate into spermatogonia. In some preferred embodiments, the gamete stem cells or cells derived from the gamete stem cells such as spermatogonia are transplanted back into the patient or a patient in need thereof. In some preferred embodiments, the patient suffers from non-obstructive azoospermia. In some preferred embodiments, the patient has previously undergone a gonadotoxic treatment, including but not limited to chemotherapy and / or radiation. In some preferred embodiments, the stem cells or tissue comprising stem cells are obtained from the patient prior to a gonado toxic treatment. In some preferred embodiments, prior to gonadotoxic treatment (chemotherapy and radiation), the testicular tissue or somatic cells of the subject are preserved so that germline stem cells may be produced or isolated in the future. In some preferred embodiments, the patient has been born with a genetic disorder that affects fertility or renders them infertile. In some preferred embodiments, the germ line stem cells obtained from the tissue can be expanded using the in vitro derived cells of the present invention. In some preferred embodiments, the methods described above further comprise obtaining fibroblast tissue to reprogram to induced pluripotent stem cells from a patient which a can be used to make autologous artificial testis cells. In some preferred embodiments, these differentiated cells can be combined with germline stem cells: either primordial germ cell like cells (PGCLC), prospermatogonia (proSSC), or neonatal / adult spermatogonial stem / progenitor cells (SSC / SPCs). In some preferred embodiments, the stem cells can either proliferate or differentiate into spermatogonia or later germ cell stages. In some preferred embodiments, the methods further comprise transferring the expanded stem cells or differentiated spermatogonia back to a patient in need thereof.

[0130] In some embodiments, the artificial testis cells or organoids may be provided on a support material. Support materials suitable for use for purposes of the present invention include tissue templates, conduits, barriers, and reservoirs useful for tissue repair. In particular, synthetic and natural materials in the form of foams, sponges, gels, hydrogels, textiles, and nonwoven structures, which have been used in vitro and in vivo to reconstruct or regenerate biological tissue, as well as to deliver chemotactic agents for inducing tissue growth, are suitable for use in practicing the methods of the present invention. See, for example, the materials disclosed in U.S. Pat. No. 5,770,417, U.S. Pat. No. 6,022,743, U.S. Pat. No. 5,567,612, U.S. Pat. No. 5,759,830, U.S. Pat. No. 6,626,950, U.S. Pat. No. 6,534,084, U.S. Pat. No. 6,306,424, U.S. Pat. No. 6,365,149, U.S. Pat. No. 6,599,323, U.S. Pat. No. 6,656,488, U.S. Published Application 2004 / 0062753 Al, U.S. Pat. No. 4,557,264 and U.S. Pat. No. 6,333,029.

[0131] Cells generated with methods and reagents herein may be implanted as dispersed cells or formed into implantable clusters. In some embodiments, cells are provided in biocompatible degradable polymeric supports; porous, permeable, or semi-permeable non- degradable devices; or encapsulated (e.g., to protect implanted cells from host immune response, etc.). Cells may be implanted into an appropriate site in a recipient. Suitable implantation sites may include, for example, the testis or subcutaneously. In some embodiments, cells or cell clusters are encapsulated for transplantation into a subject. Encapsulation techniques are generally classified as microencapsulation, involving small spherical vehicles, and macroencapsulation, involving larger flat-sheet and hollow-fiber membranes (Uludag, H. et al. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64, herein incorporated by reference in its entirety). Methods of preparing microcapsules include those disclosed by Lu M Z, et al. Biotechnol Bioeng. 2000, 70: 479-83; Chang T M and Prakash S, Mol Biotechnol. 2001, 17: 249-60; and Lu M Z, et al, J. Microencapsul. 2000, 17: 245-51.; herein incorporated by reference in their entireties. For example, microcapsules may be prepared by complexing modified collagen with a terpolymer shell of 2 -hydroxy ethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 pm. Such microcapsules can be further encapsulated with additional 2-5 pt | ter-polymer shells in order to impart a negatively charged smooth surface and to minimize plasma protein absorption (Chia, S. M. et al. Multi-layered microcapsules for cell encapsulation Biomaterials. 2002 23: 849-56; herein incorporated by reference in its entirety). In some embodiments, microcapsules are based on alginate, a marine polysaccharide (Sambanis, Diabetes Technol. Ther. 2003, 5: 665-8; herein incorporated by reference in its entirety) or its derivatives. For example, microcapsules can be prepared by the poly electrolyte complex ation between the poly anions sodium alginate and sodium cellulose sulphate with the polycation poly(methylene-co-guanidine) hydrochloride in the presence of calcium chloride.

[0132] In some embodiments, cells generated using methods and reagents described herein are microencapsulated for transplantation into a subject (e.g., to prevent immune destruction of the cells). Microencapsulation of cells (e.g., pancreatic lineage cells, beta-like cells, etc.) provides local protection of implanted / transplanted cells from immune attack (e.g., along with or without the use of systemic immune suppressive drugs). In some embodiments, cells and / or cell clusters are microencapsulated in a polymeric, hydrogel, or other suitable material, including but not limited to: poly(orthoesters), poly (anhydrides), poly(phosphoesters), poly (phosphazenes), polysaccharides, polyesters, poly(lactic acid), poly (L-ly sine), poly(glycolic acid), poly(lactic-co-glycolic acid), poly(lactic acid-co-lysine), poly(lactic acid- graft-lysine), polyanhydrides, poly(fatty acid dimer), poly(fumaric acid), poly(sebacic acid), poly(carboxyphenoxy propane), poly(carboxyphenoxy hexane), poly (anhydride-co-imi des), poly(amides), poly(ortho esters), poly(iminocarbonates), poly (urethanes), poly(organophasphazenes), poly (phosphates), poly(ethylene vinyl acetate), poly (caprolactone), poly(carbonates), poly(amino acids), poly(acrylates), polyacetals, poly (cyanoacrylates), poly(styrenes), poly(vinyl chloride), poly (vinyl fluoride), poly (vinyl imidazole), chlorosulfonated polyolefins, polyethylene oxide, polystyrene, polysaccharides, alginate, hydroxypropyl cellulose (HPC), N-isopropylacrylamide (NIP A), polyethylene glycol, polyvinyl alcohol (PVA), polyethylenimine, chitosan (CS), chitin, dextran sulfate, heparin, chondroitin sulfate, gelatin, etc., and their derivatives, co-polymers, and mixtures thereof. In some embodiments, cell are microencapsulated in an encapsulant comprising or consisting of alginate. Cells may be embedded in a material or within a particle (e.g., nanoparticle, microparticle, etc.) or other structure (e.g., matrix, nanotube, vesicle, globule, etc.). In some embodiments, microencapsulating structures are modified with immune- modulating or immunosuppressive compounds to reduce or prevent immune response to encapsulated cells. For example, pancreatic lineage cells are encapsulated within an encapsulant material (e.g., alginate hydrogel) that has been modified by attachment of an immune-modulating agent (e.g., the immune modulating chemokine, CXCL12 (also known as SDF-1). In some embodiments, such an immune modulating agent is a T-cell chemorepellent and / or a pro-survival factor.

[0133] In some embodiments, cells generated using methods and reagents described herein are macroencapsulated for transplantation into a subject. Macroencapsulation of cells, for example, within a permeable or semipermeable chamber, provides local protection of implanted / transplanted cells from immune attack (e.g., along with or without the use of systemic immune suppressive drugs), prevents spread of cells to other tissues or areas of the body, and / or allows for efficient removal of cells. Suitable devices for macroencapsulation include those described in, for example, U.S. Pat. No. 5,914,262; Uludag, et al, Advanced Drug Delivery Reviews, 2000, pp. 29-64, vol. 42, herein incorporated by reference in their entireties.

[0134] Other encapsulation (micro or macro) devices and methods may find use in embodiments described herein. For example, methods and devices described in U.S. Pub No. 20130209421, U.S. Pat. No. 8,785,185, each of which are herein incorporated by reference in their entireties, are within the scope of embodiments described herein.

[0135] In some embodiments, the testis cells or organoids of the present invention may be used for hormone therapy. In some preferred embodiments, the organoids are encapsulated and subcutaneously implanted in the subject.

[0136] In some embodiments, the testis cells or organoids of the present invention may be used for fertility restoration. In some preferred embodiments, endogenous defective somatic cells in the testis are combined or replaced by the testis cells or organoids of the present invention. In other preferred embodiments, the organoids are transplanted into a subject to allow spermatogenesis to occur at an ectopic location (e.g., a subcutaneous location) other than the testis.

[0137] In further embodiments, populations of artificial testis cells and organoids may be used to prepare antibodies and cDNA libraries that are specific for the differentiated phenotype. General techniques used in raising, purifying and modifying antibodies, and their use in immunoassays and immunoisolation methods are described in Handbook of Experimental Immunology (Weir & Blackwell, eds.); Current Protocols in Immunology (Coligan et al, eds.); and Methods of Immunological Analysis (Masseyeff et al, eds., Weinheim: VCH Verlags GmbH). General techniques involved in preparation of mRNA and cDNA libraries are described in R A Methodologies: A Laboratory Guide for Isolation and Characterization (R. E. Farrell, Academic Press, 1998); cDNA Library Protocols (Cowell & Austin, eds., Humana Press); and Functional Genomics (Hunt & Livesey, eds., 2000). Relatively homogeneous cell populations are particularly suited for use in drug screening and therapeutic applications.

[0138] In some embodiments, the artificial testis cells and organoids generated by methods provided herein are used to screen for agents (e.g., small molecule drugs, peptides, polynucleotides, and the like) or environmental conditions (such as culture conditions or manipulation) that affect the cells. Particular screening applications relate to the testing of pharmaceutical compounds in drug research and to agents for use in cryopreservation of gametes including sperm. Assessment of the activity of candidate pharmaceutical compounds generally involves combining the cells with the candidate compound, determining any change in the morphology, marker phenotype, or metabolic activity of the cells that is attributable to the compound (compared with untreated cells or cells treated with an inert compound), and then correlating the effect of the compound with the observed change. Any suitable assays for detecting changes associated with test agents may find use in such embodiments. The screening may be done, for example, either because the compound is designed to have a pharmacological effect on testis cell types, because a compound designed to have effects elsewhere may have unintended side effects, or because the compound is part of a library screen for a desired effect. Two or more drugs can be tested in combination (by combining with the cells either simultaneously or sequentially), to detect possible drug-drug interaction effects. In some applications, compounds are screened for cytotoxicity.

[0139] In some embodiments, methods and systems are provided for assessing the safety and efficacy of drugs that act upon testis cells, or drugs that might be used for another purpose but may have unintended effects upon testis cells. In some embodiments, cells described herein find use in high throughput screening (HTS) applications. In some embodiments, a HTS screening platform is provided (e.g., cells and plates) that allows for the rapid testing of large number (e.g., 1 x 103, 1 x 104, 1 x 105, 1 x 106or more) of agents (e.g., small molecule compounds, peptides, etc.). In some embodiments, artificial testis cells or organoids generated using methods and reagents described herein are utilized for therapeutic delivery to a subject. Cells may be placed directly in contact with subject tissue or may be otherwise sealed or encapsulated (e.g., to avoid direct contact). In embodiments in which cells are encapsulated, exchange of nutrients, gases, etc. between the encapsulated cells and the subject tissue is allowed. In some embodiments, cells are implanted / transplanted on a matrix or other delivery platform.

[0140] In some embodiments, the methods and kits described herein are useful for identifying additional factors, reagents, and methods for the generation of artificial testis cells or other cell types. The methods used herein may be used to screen factors, reagents and / or conditions for their effect of differentiation. In some embodiments, any screening performed in this or other embodiments discussed herein may be high-throughput screening.

[0141] EXPERIMENTAL

[0142] Example 1

[0143] The following example provides a description of the reagents and protocols for producing artificial testis cells according to the present invention from mouse Embryonic Stem Cells (mESC). mESC Culture

[0144] Reagents / Materials

[0145] 1- Mouse embryonic stem cells (TG2A)

[0146] 2- GMEM (ThermoFisher scientific Cat # 11710035)

[0147] 3- Gelatin solution Type B (Sigma Aldrich Cat # G1393-100ML)

[0148] 4- MEM Non-Essential Amino Acids (ThermoFisher scientific Cat # 1 1140050)

[0149] 5- 2-Mercaptoethanol (ThermoFisher scientific Cat # 21985023)

[0150] 6- Sodium pyruvate solution (Sigma Aldrich Cat # S8636-100ML)

[0151] 7- Embryonic stem-cell Fetal bovine serum, qualified (ThermoFisher scientific Cat #16141079)

[0152] 8- TrypLE Express Enzyme (ThermoFisher scientific Cat # 12604013) 9- Leukemia Inhibitory Factor (Sigma Aldrich Cat # L5283-10UG)

[0153] 10- Penicillin-Streptomycin (5,000 U / mL) (ThermoFisher scientific Cat # 15070063)

[0154] 11- PBS - / - (ThermoFisher scientific Cat # 10010023)

[0155] 12- Dimethyl Sulfoxide (Sigma- Aldrich Cat # D8418- 100ML)

[0156] 13- 6 well cell culture plates (ThermoFisher scientific Cat # 140675)

[0157] Growth medium-

[0158] A small amount of growth medium can be prepared and stored in 4°C for up to one month. Growth medium composition for 100 ml - Glasgow's MEM (GMEM) 92 ml, 10 ml fetal bovine serum ES cell qualified, 1 ml of Nonessential amino acid, 1ml of sodium pyruvate, 1 ml of Penicillin-Streptomycin, 100 pl of 2-mercaptoethanol, 100 pl LIF.

[0159] 2X Freezing buffer-

[0160] 20% Dimethyl Sulfoxide, 80% fetal bovine serum ES cell qualified was prepared and stored in -20° C freezer.

[0161] Growing the cells:

[0162] 1) Add 2 ml / well of 0.1% gelatin solution and incubate at room temperature for overnight before seeding the cells.

[0163] 2) Thaw TG2A cells by taking out 1 ml vial of the frozen cells (frozen in 1 : 1 freezing buffer and growth media) from liquid nitrogen and immediately thaw in 37° C water bath.

[0164] 3) Wash the cells by adding 2 ml of growth media in thawed cells and centrifuge the cells at 1000 RPM for 5 minutes.

[0165] 4) Aspirate the media and dilute pelleted cells in 2 ml of growth medium.

[0166] 5) Aspirate the gelatin from plate and seed - 500,000 cells in 2 ml of growth medium per well in 6 well plate.

[0167] 6) Perform a complete media change every 24 hours.

[0168] 7) Once cells reach ~ 80% confluency - passage cells into new plates. a) For passaging of cells, aspirate media and rinse cells with 1 ml of PBS per well. b) Aspirate PBS. c) Add 300 pl of TrypLE express enzyme per well and incubate for four minutes in 37°C, CO2 incubator. d) Take cells out of incubator and quench TrypLE express enzyme by adding 2 mL of growth medium. e) Centrifuge cells at 1000 RPM for 5 minutes. Aspirate the liquid and dilute cells in 2 ml of growth medium. f) Transfer ~ 500,000 cells per well in gelatin coated 6 well cell culture plate in 2 ml of growth media. g) Perform a complete media change every 24 hours. h) After reaching the confluency about 80% repeat the same process of point 7 again. Differentiation of TG2A cells into anterior intermediate mesoderm like cells Reagents / Materials

[0169] 1- Neurobasal Medium (ThermoFisher scientific Cat # 21103049)

[0170] 2- DMEM-F12 Medium (ThermoFisher scientific Cat #1 1320033)

[0171] 3- 2-Mercaptoethanol (ThermoFisher scientific Cat # 21985023)

[0172] 4- Sodium pyruvate solution (Sigma Aldrich Cat # S8636-100ML)

[0173] 5- MEM Non-Essential Amino Acids (ThermoFisher scientific Cat # 11140050)

[0174] 6- N-2 Supplement (ThermoFisher scientific Cat # 17502048)

[0175] 7- B-27 Supplement (ThermoFisher scientific Cat # 17504044)

[0176] 8- Glutamax (100X) (ThermoFisher scientific Cat # 35050061)

[0177] 9- Knockout serum replacer (ThermoFisher scientific Cat # 10828010)

[0178] 10- Penicillin-Streptomycin (5,000 U / mL) (ThermoFisher scientific Cat # 15070063)

[0179] 11- TrypLE Express Enzyme (ThermoFisher scientific Cat # 12604013)

[0180] 12- Activin A (R&D systems Cat # 338-AC-010)

[0181] 13- bFGF (Proteintech Cat #HZ- 1 85)

[0182] 14- Retinoic Acid (Sigma-Aldrich Cat # R2625)

[0183] 15- BMP4 (R and D systems Cat # 314-BP-500)

[0184] 16- 6 well cell culture plates (ThermoFisher scientific Cat # 140675)

[0185] 17- ROCK inhibitor Y-27632 (Enzo Life Sciences Cat # ALX-270-333)

[0186] 18- PBS - / - (ThermoFisher scientific Cat # 10010023)

[0187] Priming medium

[0188] A small amount of basal priming media can be prepared and stored in 4°C for up to one month.

[0189] Priming medium composition for 100 ml medium: add 46.9 ml of Neurobasal Medium, 46.9 ml of DMEM-F12 Medium, 500 pl of N2 supplement, 1 ml B27 supplement, 500 l of Glutamax, 100 pl 2-Mercaptoethanol, 1 ml Sodium pyruvate, 1 ml knockout serum replacer, 1 ml of non-essential amino acids, 1 ml of Penicillin-Streptomycin. The additional growth factors, 10 ng / ml Activin A and 10 ng / ml bFGF are mixed in medium just before the addition of medium into the cells.

[0190] AIM differentiation medium A small amount of basal AIM differentiation media can be prepared and stored in 4°C.

[0191] AIM differentiation medium composition for 100 ml of medium: add 91.9 ml of DMEM- F12 medium, 1 ml of Sodium pyruvate, 4 ml of knockout serum replacer, 1 ml of non- essential amino acids, 100 pl of 2-Mercaptoethanol, 1 ml of Penicillin-Streptomycin. Addition of growth factors 10 ng / ml Activin A, 100 nM RA, 2.5 ng / ml BMP4 are mixed in medium right before the addition of medium into the cells.

[0192] 1) Preparing starting cells for differentiation

[0193] To start differentiation, the starting cells need to be in undifferentiated and growth phase. To make starting cells in same growth phase consistently between experiments, Harvest cells from 80% confluent plate (at least one passage after thawing) a- Aspirate growth media and wash cells with 1 ml of PBS- / - of 6 well plate. b- Aspirate PBS- / - and incubate cells with 300 pl of TrypLE express per well of 6 well plate for 5 minutes in CO2 incubator. c- Take out cells from incubator and quench TrypLE express enzyme by adding 2 mL of growth medium. d- Centrifuge the cells at 1000 RPM for 5 minutes, aspirate the liquid and dilute the pelleted cells in growth medium. e- Seed ~ 300,000 cells per well of gelatin coated 6 well plate in 2 ml of growth medium. f- Change the complete medium with fresh growth medium every 24 hours. g- After 48 hours of seeding, the cells are ready as starting cells for differentiation.

[0194] 2) Start differentiation. a- Aspirate the growth medium from 48 hours grown cells and wash with 1 ml of PBS- / -per well of six well plate. b- Aspirate PBS- / - and incubating cells with 300 pl of TrypLE express per well of 6 well plate for 5 minutes in CO2 incubator. c- Take out cells from incubator and quench TrypLE express enzyme by adding 2 mL of growth medium. d- Centrifuge the cells at 1000 RPM for 5 minutes, aspirate the liquid and dilute the palleted cells in priming media. e- Seed ~ 30,000 cells per well of gelatin coated 6 well plate in 2 ml of priming medium and incubate the cells in CO2 incubator. f- Change complete priming media after 24 hours of incubation. Since cells loosely attached to the surface the medium was exchanged very carefully without disturbing the plate much. g- After 48 hours of total incubation in priming medium, change complete media with 2 ml of AIM differentiation media per well of 6 well plate. Since cells loosely attached to the surface the medium was exchanged very carefully without disturbing the plate much. h- After 72 hours of total incubation, change complete media with 3 ml of AIM differentiation media per well of 6 well plate with 5 M Y -27632. i- After 96 hours of total incubation cells are ready as starting cells for organoid differentiation.

[0195] The cells samples were collected every day after the second day of differentiation for RNA isolation. qPCR was done for multiple AIM markers, gonadal markers, pluripotency marker Oct4, posterior intermediate mesoderm marker HoxDl 1. The data was confirmed by immunostaining of AIM as well as gonadal markers Wtl, gonadal marker Sox9. Differentiating peritubular Myoid cells from 4 day differentiated AIM cells.

[0196] Materials

[0197] 1- DMEM-F12 Medium (ThermoFisher scientific Cat #1 1320033)

[0198] 2- 2-Mercaptoethanol (ThermoFisher scientific Cat # 21985023)

[0199] 3- Sodium pyruvate solution (Sigma Aldrich Cat # S8636-100ML)

[0200] 4- MEM Non-Essential Amino Acids (ThermoFisher scientific Cat # 11140050)

[0201] 5- Glutamax (ThermoFisher scientific Cat # 35050061)

[0202] 6- Knockout serum replacer (ThermoFisher scientific Cat # 10828010)

[0203] 7- Penicillin-Streptomycin (5,000 U / mL) (ThermoFisher scientific Cat # 15070063)

[0204] 8- FGF9 (R&D systems Cat # 273-F9-025)

[0205] 9- BMP4 (R and D systems Cat # 314-BP-500)

[0206] 10- EGF1

[0207] 11- PDGFAA (Sigma Aldrich Cat # SRP3228)

[0208] 12- PDGFBB (Sigma Aldrich Cat # S P3229)

[0209] 13- Smoothened agonist (SAG) (Millipore Cat # 566661)

[0210] 14- Valproic acid (Sigma Aldrich Cat # P4543-10G)

[0211] 15- BMP2 (R&D systems Cat # 355-BM-010)

[0212] 16- Activin A (R&D systems Cat # 338-AC-010)

[0213] 17- Retinoic Acid (Sigma-Aldrich Cat # #R2625) 18- IGF1 (Sigma- Aldrich cat # 13769)

[0214] 19- Insulin (Sigma- Aldrich cat #19278)

[0215] 20- Prostaglandin D2 (Cayman Chemical Cat # 12010)

[0216] 21- ROCK inhibitor Y-27632 (Enzo Life Sciences Cat # ALX-270-333)

[0217] 22- Insulin Transferrin Selenium (ITS) (100X) (ThermoFisher Scientific Cat # 41400045)

[0218] 23- Matrigel basement membrane matrix (Corning Cat # 354234)

[0219] 24- Bovine Serum Albumin (BS A) solution 10% in DPBS (Sigma Aldrich Cat # A 1595)

[0220] 25- TrypLE Express Enzyme (ThermoFisher scientific Cat # 12604013)

[0221] Two different types of medium are prepared.

[0222] 1. Progenitor Medium- Progenitor medium composition for 100 ml of medium: add 91.9 ml of DMEM-F12 medium, 1 ml of Sodium pyruvate, 5 ml of knockout serum replacer, 1 ml of non-essential amino acids, 100 pl of 2-Mercaptoethanol, 1 ml of Penicillin-Streptomycin. Additional factors, 1% Matrigel, lOOnM Retinoic Acid, 17nM IGF1, lOOnM Insulin, 10 ng / ml FGF9, 500ng / ml PGD2, 20 ng / ml BMP4, 50 ng / ml EGF1, 5 M Y-27632 was mixed right before adding the media in the cells.

[0223] 2. Myoid differentiation medium- Myoid differentiation medium for 100 ml of medium: add 98.9 ml of DMEM-F12 medium, 100 pl of ITS, 1 ml BSA. Additional factors, SAG 0.5 pM, PDGFRAA 10 ng / ml, PDGFRBB 10 ng / ml, Valproic Acid 10 nm, BMP2 10 ng / ml, BMP4 10 ng / ml, Activin A 10 ng / ml was mixed right before adding the media in the cells.

[0224] 1. Preparing plates for Myoid cell differentiation- Before 6 hours of seeding the cells into the plate, plate needs to be coated with Matrigel. Add 56 pL Matrigel into 12 ml DMEM / F12 medium and Add 1 ml per well in 12 well plate. Incubate plate in the CO2 incubator for about 6 hours.

[0225] 2. Seeding the day-4 AIM cells on Matrigel coated plates. a) Aspirate the AIM differentiation media and wash the cells thrice with 2 ml of PBS- / - per well of six well plate from 4 day differentiated AIM cells. b) Add 500 pl of TrypLE Express Enzyme per well of the six well plate and incubate for 5 minutes in CO2 incubator. c) Take out cells from incubator and quench TrypLE express enzyme by adding 2 mL of growth medium per well of six well plate. d) Centrifuge the cells at 1000 RPM for 5 minutes, aspirate the liquid and dilute the palleted cells in Progenitor Medium. e) About 50,000 cells were mixed in 1 ml of Progenitor Medium for one well of 12 well plate. f) Aspirate Matrigel coating solution from plate, which was added to plate about 6 hours. g) Seed the cells into plate and incubate in CO2 incubator for 24 hrs. h) Aspirate Progenitor Medium and add 1 ml of Myoid Differentiation medium and incubate plate in CO2 incubator. i) Change Myoid Differentiation medium for every day. j) Collect samples on day 3 and day 5 after seeding the cells.

[0226] Differentiating peritubular Leydig cells from 4 day differentiated AIM cells.

[0227] Materials

[0228] 1- DMEM-F12 Medium (ThermoFisher scientific Cat #11320033)

[0229] 2- 2-Mercaptoethanol (ThermoFisher scientific Cat # 21985023)

[0230] 3- Sodium pyruvate solution (Sigma Aldrich Cat # S8636-100ML)

[0231] 4- MEM Non-Essential Amino Acids (ThermoFisher scientific Cat # 11140050)

[0232] 5- Glutamax (ThermoFisher scientific Cat # 35050061)

[0233] 6- Knockout serum replacer (ThermoFisher scientific Cat # 10828010)

[0234] 7- Penicillin-Streptomycin (5,000 U / mL) (ThermoFisher scientific Cat # 15070063)

[0235] 8- FGF9 (R&D systems Cat # 273-F9-025)

[0236] 9- BMP4 (R and D systems Cat # 314-BP-500)

[0237] 10- EGF1

[0238] 11- PDGFAA (Sigma Aldrich Cat # SRP3228)

[0239] 12- PDGFBB (Sigma Aldrich Cat # SRP3229)

[0240] 13- Smoothened agonist (SAG) (Millipore Cat # 566661)

[0241] 14- DAPT (Stem cells Cat # 72082)

[0242] 15- Retinoic Acid (Sigma-Aldrich Cat # #R2625)

[0243] 16- IGF1 (Sigma- Aldrich cat # 13769)

[0244] 17- Insulin (Sigma- Aldrich cat #19278)

[0245] 18- Prostaglandin D2 (Cayman Chemical Cat # 12010)

[0246] 19- ROCK inhibitor Y-27632 (Enzo Life Sciences Cat # ALX-270-333) 20- insulin Transferrin Selenium (ITS) (100X) (ThermoFisher Scientific Cat # 41400045)

[0247] 21- Matrigel basement membrane matrix (Corning Cat # 354234)

[0248] 22- Bovine Serum Albumin (BSA) solution 10% in DPBS (Sigma Aldrich Cat # A1595)

[0249] 23- TrypLE Express Enzyme (ThermoFisher scientific Cat # 12604013)

[0250] 24-

[0251] Three different types of media are prepared.

[0252] 1. Progenitor Medium- Progenitor medium composition for 100 ml of medium: add 91.9 ml of DMEM-F12 medium, 1 ml of Sodium pyruvate, 5 ml of knockout serum replacer, 1 ml of non-essential amino acids, 100 pl of 2-Mercaptoethanol, 1 ml of Penicillin-Streptomycin. Additional factors, 1% Matrigel, lOOnM Retinoic Acid, 17nM IGF1, lOOnM Insulin, 10 ng / ml FGF9, 500ng / ml PGD2, 20 ng / ml BMP4, 50 ng / ml EGF1, 5 M Y-27632 was mixed right before adding the media in the cells.

[0253] 2. Leydig cell basal medium- Leydig cell basal medium composition for 100 ml of medium: add 98.9 ml of DMEM-F12 medium, 100 pl of ITS, 1 ml BSA. Additional factors, SAG 0.5 pM, PDGFRAA 10 ng / ml, PDGFRBB 10 ng / ml, FGF2 10 ng / ml was mixed right before adding the media in the cells.

[0254] 3. Leydig cell differentiation medium- Myoid differentiation medium for 100 ml of medium: add 98.9 ml of DMEM-F12 medium, 100 pl of ITS, 1 ml BSA. Additional factors, SAG 0.5 pM, PDGFRAA 10 ng / ml, PDGFRBB 10 ng / ml, LiCh 5 mM, FGF2 10 ng / ml, DAPT 10 pM was mixed right before adding the media in the cells.

[0255] 1. Preparing plates for Leydig cell differentiation- Before 2 hours of seeding the cells onto the plate, plate needs to be coated with Matrigel. Add 56 pL Matrigel into 12 ml DMEM / F12 medium and Add 1 ml per well in 12 well plate. Incubate plate in the CO2 incubator for about 6 hours.

[0256] 2. Seeding the day-4 AIM cells on Matrigel coated plates k) Aspirate the AIM differentiation media and wash the cells thrice with 2 ml of PBS- / - per well of six well plate from 4 day differentiated AIM cells. l) Add 500 pl of TrypLE Express Enzyme per well of the six well plate and incubate for 5 minutes in CO2 incubator. m) Take out cells from incubator and quench TrypLE express enzyme by adding 2 mL of growth medium per well of six well plate. n) Centrifuge the cells at 1000 RPM for 5 minutes, aspirate the liquid and dilute the palleted cells in Progenitor Medium. o) About 50,000 cells were mixed in 1 ml of Progenitor Medium for one well of 12 well plate. p) Aspirate Matrigel coating solution from plate, which was added to plate about 6 hours. q) Seed the cells into plate and incubate in CO2 incubator for 24 hrs. r) Aspirate Progenitor Medium and add 1 ml of Leydig cell basal medium and incubate plate in CO2 incubator. s) Change Leydig cell basal medium for every day up to three days. t) After 3 days incubation in the Leydig cell basal medium, change Leydig cell basal medium to Leydig cell differentiation medium. u) Change Leydig cell differentiation medium every day. v) Collect samples on day 4, day 8 and day 15 after seeding the cells.

[0257] Developing testis organoids from 4 day differentiated AIM cells.

[0258] Materials

[0259] 1. DMEM-F12 Medium (ThermoFisher scientific Cat #11320033)

[0260] 2. 2-Mercaptoethanol (ThermoFisher scientific Cat # 21985023)

[0261] 3. Sodium pyruvate solution (Sigma Aldrich Cat # S8636-100ML)

[0262] 4. MEM Non-Essential Amino Acids (ThermoFisher scientific Cat # 11140050)

[0263] 5. Glutamax (ThermoFisher scientific Cat # 35050061)

[0264] 6. Knockout serum replacer (ThermoFisher scientific Cat # 10828010)

[0265] 7. Penicillin-Streptomycin (5,000 U / mL) (ThermoFisher scientific Cat # 15070063)

[0266] 8. FGF9 (R&D systems Cat # 273-F9-025)

[0267] 9. BMP4 (R and D systems Cat # 314-BP-500)

[0268] 10. EGF1

[0269] 11. Retinoic Acid (Sigma-Aldrich Cat # #R2625)

[0270] 12. IGF1 (Sigma- Aldrich cat # 13769)

[0271] 13. Insulin (Sigma- Aldrich cat #19278)

[0272] 14. Prostaglandin D2 (Cayman Chemical Cat # 12010)

[0273] 15. ROCK inhibitor Y-27632 (Enzo Life Sciences Cat # ALX-270-333)

[0274] 16. Matrigel basement membrane matrix (Coming 354234)

[0275] 17. TrypLE Express Enzyme (ThermoFisher scientific Cat # 12604013)

[0276] 18. Aggrewell® 400, 24 well plate (Stemcell Technologies Cat # 34421)

[0277] 19. Anti-Adherence Rinsing Solution (Stemcell Technologies Cat # 07010) Organoid differentiation medium

[0278] A small amount of basal organoid differentiation media can be prepared and stored in 4°C for up to one month.

[0279] Organoid differentiation medium composition for 100 ml of medium: add 91.9 ml of DMEM-F12 medium, 1 ml of Sodium pyruvate, 5 ml of knockout serum replacer, 1 ml of non-essential amino acids, 100 pl of 2-Mercaptoethanol, 1 ml of Penicillin-Streptomycin. Additional factors, 1% Matrigel, lOOnM Retinoic Acid, 17nM IGF1, lOOnM Insulin, 10 ng / ml FGF9, 500ng / ml PGD2, 20 ng / ml BMP4, 50 ng / ml EGF1, 5 pM Y-27632 was mixed right before adding the media in the cells.

[0280] 1- Preparing plates for organoid differentiation

[0281] Before starting the organoid differentiation, the Aggrewell® 400-24 plate or a 96 u- bottom well plates are prepared as follow. a- Add 500 pl of anti- adherence rinsing solution per well of Aggrewell® 400-24 well plate and incubate for 30 minutes at room temperature. b- Aspirate anti-adherence rinsing solution and wash well with 1 ml of PBS- / - per well. c- Aspirate PBS- / - and add 500 pl of organoid differentiation medium per well of an Aggrewell® 400-24 well plate. Add 100 pl of organoid differentiation media into a 96 well u-bottom plate. d- Centrifuge the plate for 5 minutes at 500 RCF to remove the trapped air in the well. Now plates are ready for seeding of the cells.

[0282] 2- Organoid differentiation a- Aspirate the AIM differentiation media and wash the cells thrice with 2 ml of PBS- / - per well of six well plate from 4 day differentiated AIM cells. b- Add 500 pl of TrypLE Express Enzyme per well of the six well plate and incubate for 5 minutes in CO2 incubator. c- Take out cells from incubator and quench TrypLE express enzyme by adding 2 mL of growth medium per well of six well plate. d- Centrifuge the cells at 1000 RPM for 5 minutes, aspirate the liquid and dilute the palleted cells in organoid differentiation medium. e- About 300,000 cells was mixed in 500 pl of organoid differentiation media and added to preprepared Aggrewell® plate per well. If using a 96 well plate -25,000 cells in lOOpl of organoid differentiation media are transferred into a nonstick 96 u-bottom well plate. f- Incubate plate for 10 minutes at CO2 incubator to let cells settle down in the wells. g- Centrifuge Aggrewell® plate or 96 well plate again for 5 minutes at 500 RCF. h- Incubate cells in CO2 incubator. i- The forming organoids were undisturbed for 2 days and then after half of medium was exchanged with fresh organoid differentiation media every day. j- The organoids from the Aggrewell® plate were collected on a total of 6 days and 8 days of differentiation for RNA isolation and qPCR analysis of terminal differentiation markers. The organoids from the 96 well plates are collected or transferred at Day 7. The 96 well plate is the better method! k- Data was confirmed by whole mount immunostaining of progenitor marker CoupTf2, Sertoli cell markers Gata4, Sox9 and Wtl, Leydig cell marker 3BHSD, STAR, Cypl7al.

[0283] Extended culture of organoids into trans-wells

[0284] Materials

[0285] 1- DMEM-F12 Medium (ThermoFisher scientific Cat #11320033)

[0286] 2- 2-Mercaptoethanol (ThermoFisher scientific Cat # 21985023)

[0287] 3- Sodium pyruvate solution (Sigma Aldrich Cat # S8636-100ML)

[0288] 4- MEM Non-Essential Amino Acids (ThermoFisher scientific Cat # 11140050)

[0289] 5- Glutamax (ThermoFisher scientific Cat # 35050061)

[0290] 6- Knockout serum replacer (ThermoFisher scientific Cat # 10828010)

[0291] 7- Penicillin-Streptomycin (5,000 U / mL) (ThermoFisher scientific Cat # 15070063)

[0292] 8- FGF9 (R&D systems Cat # 273-F9-025)

[0293] 9- BMP4 (R and D systems Cat # 314-BP-500)

[0294] 10- EGF1

[0295] 11 - Retinoic Acid (Sigma- Aldrich Cat # R2625)

[0296] 12- IGF1 (Sigma-Aldrich cat # 13769)

[0297] 13 - Insulin (Sigma- Aldrich cat #19278)

[0298] 14- Prostaglandin D2 (Cayman Chemical Cat # 12010)

[0299] 15- ROCK inhibitor Y-27632 (Enzo Life Sciences Cat # ALX-270-333)

[0300] 16- Matrigel basement membrane matrix (Corning 354234)

[0301] 17- Follicle Stimulating Hormone (FSH) (Sigma- Aldrich Cat # F4021)

[0302] 18- Luteinizing Hormone (LH) (Sigma- Aldrich Cat # L6220)

[0303] 19- Testosterone

[0304] 20- TrypLE Express Enzyme (ThermoFisher scientific Cat # 12604013) ThinCert® Tissue culture insert (Greiner Bio Cat # 10443845) 2 Extended culture involves transferring 96 well or Aggrewell® plate organoids onto trans-well a) Before implanting the organoids on trans-wells, the ThinCert® cell culture inserts were soaked in 700 pl of the organoid reaggregation medium overnight in 24 well plate. b) The organoids were pipetted out using large orifice 200 pl pipette tip with about 10 pl medium and implanted in the meddle of trans well. c) The plate was incubated in a CO2 incubator. d) Half of the medium was exchanged with new reaggregation medium every other day. e) The implant was collected on day 7, 14 and 17 and fixed for 10 minutes in 4% PFA. f) The implant was imbedded in OCT and cryosection for immunostaining.

[0305] RESULTS AND DISCUSSION

[0306] An overview of the differentiation protocol is provided in FIG. 1. This protocol provides improvements to the protocol provided in PCT US2023 / 13608, the contents of which are incorporated herein by reference in their entirety. Modification to the protocol include addition of BMP4 between Days 2-4 as well reducing FGF9 concentration in ALLGF and adding BMP4 and EGF1. These modifications resulted in the improvement the expression of many gonadal and Sertoli cell markers (See. FIG. 2).

[0307] Additionally, extending the culture of day 8 organoids on an air-liquid interface results in improved production of Leydig cells. Specifically, at day 17 Leydig cells are detected that are double positive for SF1 and 3BHSD. These Leydig cells enrich in clusters like what is observed in mouse and human testis (See FIG. 3). In addition to specifying Leydig cells in the improved culture conditions, further maturation of Sertoli cells (Figure 4A) was detected as well as detection of peritubular myoid cell markers (Figure 4B).

[0308] Pure populations of Leydig and Peritubular myoid cells are obtained by following the protocols described above to day 8 and then isolating the cells and continuing the culture of the gonadal progenitor cells on Matrigel for an addition 3-8 using established Leydig and Myoid cell differentiation. Applying the existing Leydig and Myoid media to ESC is not sufficient to generate myoid and Leydig cells.

[0309] Finally, by transferring the Day 4 Anterior intermediate mesoderm cells into a nonstick 96 u-bottom plate and transferring the organoids afterwards to Transwell® plates, then adding FSH + HCG or FSH+LH) testis somatic cell differentiation and tubule formation in vitro has been significantly improved (Figures 12-17). Indeed, these organoids make testosterone and corticosterone.

[0310] Example 2

[0311] The following example provides a description of the reagents and protocols for producing artificial testis cells according to the present invention from human Embryonic Stem Cells (hESC) or iPSCs.

[0312] Directed differentiation of human ESC into multiple testis-like somatic cells.

[0313] A schematic of this protocol is provided as FIG. 5.

[0314] Reagents

[0315] Media and supplements

[0316] 1. STEMdiff APEL Medium (Stem Cell Technologies, cat. no. 05270 or 05275)

[0317] 2. DMEM / F-12 (Thermo Fisher Scientific, cat. no. 11320-082)

[0318] 3. DMSO (Sigma Aldrich, cat. no. D5879)

[0319] 4. Dulbecco's phosphate-buffered saline (DPBS) (Thermo Fisher Scientific, Cat. no.14190- 144)

[0320] 5. Accutase (Cat. no. 07920)

[0321] 6. 0.5M EDTA stock (Lonza 51201).

[0322] 7. Quick-RNA Miniprep Kit (Zymo Research, Cat No. R1055).

[0323] 8. Anti -Adherence Rinsing Solution (Stem cell technologies, Cat No.07010).

[0324] 9. Solution 1 for Primary Antibodies (EMD Millipore, Cat No.KP31812-200ml)

[0325] 10. Solution 2 for Secondary Antibodies (EMD Millipore, Cat No.KP31855-200ml)

[0326] 11. 0.5M EDTA stock (Lonza 51201)

[0327] 12. Rinsing Solution

[0328] 13. CHIR99021 (R&D, cat. no. 9902): Stock Solution (10 mM) — Centrifuge the tube briefly before opening. Reconstitute 10 mg of CHIR99021 into 2.149 ml of DMSO to make 10 mM stock. Prepare 20 pl aliquots and label tubes as “Ch” store at -20°C. The thawed tube can be kept at 4°C for 24hr.

[0329] 14. Y27632 ROCK inhibitor (Enzo, Cat. no. ALX-270-333): Stock Solution (5mM) — Dissolve Img in 625 pl sterile TC water Prepare 20 pl aliquots and label tubes as “Y”. Store at -20°C at stem cell core. Dilute in lOmL culture medium for final lOpM. Discard the tube after use. 15. Heparin (Sigma Aldrich, cat. no. H4784-250 mg): Stock solution (1 mg ml 1) — Reconstitute to 1 mg ml- ' in ultrapure water and filter through polyether sulfone (PES) 0.22 pm filter (syringe-driven filter unit). Prepare 40, u I aliquots and store at 4°C for up to 12 months.

[0330] 16. FGF9 (R&D, cat. no. 273-F9-025): Stock Solution FGF9 (100 pg / ml) — Centrifuge the tube briefly before opening. Reconstitute to 100 pg / ml in filtered DPBS containing 0.1% (wt / vol) Bovine serum albumin. Prepare 20 pl aliquots and store them at -80°C for up to 6 months. Thawed FGF9 aliquot can be stored at 4°C for up to 2 weeks.

[0331] 17. RA (Sigma, cat. no. R2625-5 mg): Stock Solution (0.5Mm) — Dissolve Img of RA dissolve in 3 ml of the DMSO to make 1.11 mM Stock Solution. Dilute the stock solution to 0.5 mM, prepare 50pl or lOOpl aliquots and this working solution can be stored at -20°C. Use 0.5 mM of stock solution to the cell culture media according to the experimental desired concentrations. Discard the tube after use.

[0332] 18. Insulin (Sigma, cat. no. 19278): Stock Solution (1.7mM) from Vendor.

[0333] 19. IGF1-50 pg (Sigma, Cat. no. 13769): Stock solution (lOMm) — 50 pg of IGF1 is dissolved in 658 pl of 0.2% acetic acid to a stock concentration of 10 mM. Prepare lOpl aliquots and store at -80°C. Dilute the stock solution to a concentration of 100 pM in 0.2% acetic acid and add to the cell culture media according to the experimental desired concentrations. The thawed tube can be stored at 4°C for up to 1 month.

[0334] 20. PGD2 (Cayman, cat. no.12010): Stock Solution (2mg / ml) — Dissolve PGD2 in 200pl PBS pH 7.4 to a stock concentration of 2mg / ml, prepare 5pl aliquots and store at -80°C. Discard the tube after use.

[0335] 21. Matrigel (Coming, cat. no. 354234, Lot no. 9133008): Stock Solution (2mg / ml or Img / ml) — Dilute the Matrigel to desired concentration 2mg / ml or 1 mg / ml in DMEM / F12 media and prepare aliquots and store at -20°C until use.

[0336] 22. Testosterone (Sigma, cat no. T1500-5g): Stock Solution(5mM)-6g of Testosterone was dissolved in 1ml of DMSO then serial diluted to make concentration 5mM.

[0337] 23. Follicle stimulating hormone (Sigma: cat no. F4021-25ug)- Stock Solution (100 pg / ml)- Dissolve 25ug of FSH in 250ul of ultrapure water and prepare aliquots and store at -20°C until use.

[0338] 24. Luteinizing Hormone (Sigma: cat no. L6420-10ug): Stock Solution (100 pg / ml)- Dissolve 25ug of LH in lOOul of ultrapure water and prepare aliquots and store at -20°C until use. 25. Epidermal Growth Factor from murine submaxillary gland, EGF (Sigma, cat no. E4127-.1 mg): Stock Solution(100ug / ml)-0.1mg of EGF was dissolved in 10ml of 0.1%BSA / PBS in 10ml then aliquoted in 1 ml and store at -20°C until use.

[0339] 26. IWR1 (Sigma: cat no. 681669-lOmg)- Dissolve 10 mg / ml in DMSO and prepare aliquots and store at -20°C until use.

[0340] Materials

[0341] 1. Nunc™ 4-Well Dishes for IVF (Fisher, cat no. 144444)

[0342] 2. Nunc™ Cell-Culture Treated Multidishes - 24well (Fisher, cat. no. 142475)

[0343] 3. Ultra-Low Binding, U-Shaped-Bottom Microplate(Corning,cat.no.l2-456-721)

[0344] 4. Greiner Bio-One ThinCert® Tissue Culture Inserts(Greiner Bio-One, cat.no. 10443845)

[0345] 5. Costar 24 well plate (Corning, Cat No.3526)

[0346] 6. Coverslips 12mm (Hampton Research, cat no. HR3-277)

[0347] 7. Bench top centrifuge (Eppendorf, 5417c)

[0348] 8. Biological safety cabinet

[0349] 9. Co2 Incubator (Heraeus)

[0350] 10. Conical tubes 15ml (Falcon, cat no. 352096)

[0351] 11. Conical tubes 50ml (Falcon, cat no. 352070)

[0352] 12. Freezing container (Nalgene, Mr. Frosty)

[0353] 13. Inverted contrasting tissue culture microscope (KL1500CD, Leica)

[0354] 14. Laser confocal microscope (Nikon Eclipse Ti2)

[0355] 15. Pipette boy (Drummond)

[0356] 16. Pipettes (Gilson)

[0357] 17. Automated cell counter (BioRad, TC20)

[0358] 18. Serological pipettes (Fisher, Cat No, 13-678-1 ID, 13-676-10J, 13-676-10R)

[0359] 19. Stericup 0.22pm Filter unit (Millipore, 03290)

[0360] 20. Sterile filter pipette tips (Genesee Scientific, 24-815, 24-804,24-830, 26-401)

[0361] 21. Sterile Microcentrifuge tubes (Fisher, Cat No.05-408-120)

[0362] 22. Aggrewell® 800 24-well,5pk (Stem cell technologies, Cat No.34815)

[0363] Coverslip cleaning method:

[0364] • Shake coverslips in xylene in 50mL conical tube for 2-3 hours.

[0365] • Discard xylenes and rinse with acetone. Discard acetone and rinse in fresh acetone in 50 mL conical tube for 2-3 hours. • Discard acetone. Autoclave in water if desired on liquid cycle. Rinse coverslips with 100% ethanol two times. Store coverslips in 100% ethanol at 4°C.

[0366] Dav -1 Plating the ESC in Single cell suspension for Differentiation to improve reproducibility

[0367] Coating plates for Cell pellet (RNA seq analysis) or staining of coverslip.

[0368] • Dry the coverslips at room temperature until all the alcohol evaporates completely in the Biosafety cabinet hood under sterile conditions. After drying, transfer the coverslip with forceps to 24 or 4 well plate.

[0369] • Coat the 24 or 4 well plate with Matrigel for Ihour before plating the cells.

[0370] Single cell suspension for Monolayer

[0371] • Clean any differentiating colonies from cultures colonies on 60mm petri plates.

[0372] • Wash with 2ml DPBS buffer.

[0373] • Add 0.5 ml of lx accutase and place in incubator for 5-10 min.

[0374] • Add 2 - 5 ml of DMEM / F12 media to stop the dissociation.

[0375] • Pellet cells by centrifuging for 3min at 1000 rpm.

[0376] • Resuspended the pellet in 5 ml of mTeSR media with 10 p M (Y27632) Rock inhibitor and count the cells.

[0377] • Plate 10,000 cells per well into a Matrigel coated 24 well plate.

[0378] Dav 0 ESC cell collection as a control:

[0379] • ES cells were collected on day 0.

[0380] • Cells were washed with 0.5 ml of DPBS buffer for each well of 24 well plate.

[0381] • 0.5 ml of lx accutase was added and placed in incubator for 3 - 5 min.

[0382] • Add 1ml of DMEM / F12 media to stop the digestion reaction.

[0383] • Centrifuge at 10,000g for 2 min.

[0384] • Aspirate the media.

[0385] • Snap freeze in Liquid N2 and store at -80°C until later use.

[0386] • Fix the cells in 4% PFA for 20 min and wash 3 times with DPBS, store in DPBS at 4°C until use.

[0387] Cells used to start differentiation. • Remove mTeSR media with Rock inhibitor and add 0.5 ml of APEL2 media + CHIR 3pM.

[0388] Media changed every 2 days.

[0389] Day 2 Change media with APEL2 media + CHIR 3 pM.

[0390] Dav 4: Presomitic Mesoderm

[0391] • Collect cells from required number of wells.

[0392] • Add 0.5 ml of lx accutase and placed in incubator for 3 - 5 min and add 1ml of DMEM / F12 to stop reaction.

[0393] • Centrifuge at 10,000g for 2 min.

[0394] • Aspirate the media.

[0395] • Snap freeze in Liquid N2 and store at -80°C until later use (Presomitic Mesoderm).

[0396] • Fix the cells in 4%PFA for 20 min and wash 3times with DPBS, store in DPBS at 4°C until use.

[0397] Start FGF9 Phase

[0398] • Change media with 200 ng / ml FGF9 + 1 g / ml Heparin.

[0399] • Change the media every two days.

[0400] Day 6 Change Media with 200 ng / ml FGF9 + 1 pg / ml Heparin.

[0401] On Dav 7 (Intermediate Mesoderm)

[0402] • Collect cells from required number of wells on day 7.

[0403] • Wash cells with 0.5 ml of DPBS buffer for each well of 24 well plate.

[0404] • Add 0.5 ml of lx accutase and placed in incubator for 3 - 5 min and add 1ml of DMEM / F12 to stop reaction.

[0405] • Centrifuge at 10,000g for 2 min.

[0406] • Aspirate the media.

[0407] • Snap freeze in Liquid N2 and store at -80°C until later use (Intermediate Mesoderm).

[0408] • Fix the cells in 4% PFA for 20 min and wash 3times with DPBS, store in DPBS at 4°C until use. o However, the remaining wells will continue with the differentiation protocol: for these wells we will APEL ± 0.1 pM RA + 100 nM Insulin + 17 nM IGF1 + 500 ng / ml PGD2± 200 ng / ml FGF9. On Dav 8

[0409] Seed the dissociated gonadal cells into Aggrewell® plate or 96 well u-bottom plate.

[0410] Preparation of Aggrewell® plate to receive cells.

[0411] • Add surface rinsing solution of 0.5 ml for each well. Make sure there is no air bubble present in microwell. To remove air bubbles from each microwell centrifuge at 200g for 5 min.

[0412] • Observe under the Bright Field microscope to make sure there is no air bubbles present. If the air bubbles are still present, then recentrifuged for 200g for 5 min.

[0413] • Incubate plate at room temperature or at 37°C for 30min. After incubation remove the rinsing solution and wash with 0.5 ml DBPS buffer and make sure the wells do not dry out before plating the cells.

[0414] • Add 0.5 ml of APEL Media with corresponding growth factors and centrifuge for 200g for 5 min to remove air bubbles. Observe under the Bright field microscope to make sure there is no air bubbles.

[0415] • For 96 well plate add 0.1 ml of APEL Media with corresponding growth factors and centrifuge for 200g for 5 min to remove air bubbles. Observe under the Bright field microscope to make sure there is no air bubbles.

[0416] Preparation of cells for Plating

[0417] • Dissociate the remaining wells of differentiated cells into single cells.

[0418] • Wash cells with 0.5 ml of DPBS buffer for each well of 24 well plate.

[0419] • Add 0.5 ml of lx accutase and place in incubator for 3 - 5 min and add 1ml of DMEM / F12 to stop reaction.

[0420] • Centrifuge at 1000 rpm for 3 min.

[0421] • Aspirate the media.

[0422] • Resuspended the cells with APEL media with corresponding growth factors with 10pm (Y27632) Rock inhibitor and count the cells.

[0423] • Add 0.5 ml of (1 x 106- 2 x 106) cells per well with APEL media with corresponding growth factors and mix up and down slowly and centrifuge 500g for 5 min. Observe under the microscope to see all the cells are clustered together. If using 96 well seed 30K cells in lOOul. • Change the half media every other day by slowing touching the side of the wells by removing 0.5 ml of old media and add 0.5 ml of the fresh media.

[0424] Day 9

[0425] • Change half the media every other day by slowly touching the side of the wells and removing 1 ml of old media and adding 1 ml of the fresh media with APEL ± 0.1 p M RA + 100 nM Insulin + 17 nM IGF1 + 500 ng / ml PGD2± 200 ng / ml FGF9+ IpM Testosterone ± lOOng / ml Follicle stimulating hormone ± 200ng / ml Luteinizing Hormone or 2U HCG+ IpM IWRl+50ng / ml EGF.

[0426] Dav 10 (Genital Ridge)

[0427] • Collect organoids from required number of wells on day 10. To collect the cells, use uncut the tip and pipette up and down to loosen and lift the organoids and collect the floating organoids in to 0.5 ml tube. Snap freeze in Liquid N2 and store at -80°C until later use.

[0428] • Fix the some of the wells of organoids 4% PFA for 10 min and wash 3X with DPBS.

[0429] • Incubate in the 30% sucrose at 4°C for 72hours. After incubation remove sucrose, wash with DPBS buffer for 3 times for 5min and store in DBFS buffer at 4°C until staining.

[0430] • Remaining wells will get various combination of growth factors: media will be changed every 2 days.

[0431] • APEL ± 0.1 pM RA + 100 nM Insulin + 17 nM IGF1 + 500 ng / ml PGD2± 200 ng / ml FGF91pM Testosterone ± lOOng / ml Follicle stimulating hormone ± 200ng / ml Luteinizing Hormone± I M IWRl+50ng / ml EGF.

[0432] Day 12

[0433] • Transfer organoids to ThinCert® cell culture inserts and add 700ul of media (APEL ± 0.1 (iM RA + 100 nM Insulin + 17 nM IGF1 + 500 ng / ml PGD2± 200 ng / ml FGF91pM Testosterone ± lOOng / ml Follicle stimulating hormone ± 200ng / ml Luteinizing Hormone± I pM lWRl+50ng / ml EGF) to the lower chamber.

[0434] Day 14

[0435] • Change the media. • APEL ± 0.1 |iM RA + 100 nM Insulin + 17 nM IGF1 + 500 ng / ml PGD2± 200 ng / ml FGF9l pM Testosterone ± lOOng / ml Follicle stimulating hormone ± 200ng / ml Luteinizing Hormonei I ,u M lWRl+50ng / ml EGF.

[0436] Day 16

[0437] • Collect organoids from required number of wells on day 16. To collect the cells, use uncut the tip and pipette up and down to loosen and lift the organoids and collect the floating organoids in to 0.5 ml tube. Snap freeze in Liquid N2 and store at -80°C until later use.

[0438] • Fix the some of the wells of organoids 4% PFA for 10 min and wash 3times with DPBS.

[0439] • Incubate in the 30% sucrose at 4°C for 72hours. After incubation remove sucrose, wash with DPBS buffer for 3 times for 5min and store in DBFS buffer at 4°C until staining.

[0440] • Remaining wells will get various combination of growth factors: media will be changed every 2 days.

[0441] • APEL ± O.lpM RA + WOnM Insulin + 17nM IGF1 + 500 ng / ml PGD2± 200ng / ml FGF91pM Testosterone ± lOOng / ml Follicle stimulating hormone ± 200ng / ml Luteinizing Hormone+± lpM IWRl+50ng / ml EGF.

[0442] Dav 18

[0443] • Change the media for every two days with growth factors.

[0444] • APEL ± 0.1 pM RA + 100 nM Insulin + 17 nM IGF1 + 500 ng / ml PGD2± 200 ng / ml FGF91pM Testosterone + lOOng / ml Follicle stimulating hormone ± 200ng / ml Luteinizing Hormonei IpM IWRl+50ng / ml EGF.

[0445] Dav 20

[0446] • Change the media for every two days with growth factors.

[0447] • APEL ± 0.1 pM RA + 100 nM Insulin + 17 nM IGF1 + 500 ng / ml PGD2± 200 ng / ml FGF91pM Testosterone + lOOng / ml Follicle stimulating hormone ± 200ng / ml Luteinizing Hormonei l uM IWRl+50ng / ml EGF.

[0448] Dav 22

[0449] • Collect organoids from required number of wells on day 22(Testis Organoids). To collect the cells, use uncut the tip and pipette up and down to loosen and lift the organoids and collect the floating organoids in to 0.5 ml tube. Snap freeze in Liquid N2 and store at -80 °C until later use.

[0450] • Fix the some of the wells of organoids 4% PFA for 10 min and wash 3times with DPBS.

[0451] • Incubate in the 30% sucrose at 4°C for 72hours. After incubation remove sucrose, wash with DPBS buffer for 3 times for 5min and store in DBPS buffer at 4°C until staining. These samples are ready for immunostaining.

[0452] Coverslip Staining Protocol d4 / d7 Monolayer cells.

[0453] • Fix the cells in 4% PFA for 20 min.

[0454] • Wash with PBS- / - for 5 min for 3 times and store in PBS- / - at 4°C until staining.

[0455] • Quick wash with PBS- / - before staining for 3times.

[0456] • Blocking buffer PBS+ / +, 0.1% Triton -x 100, 10% Donkey serum or goat serum and 10% Na-Azide

[0457] • (8.85ml PBS+ / +, 50 pl Triton-X 100 + 1 ml Donkey serum or Goat serum-i- 100 pl Na- Azide).

[0458] • Add 200 pl of blocking buffer in 24 well plate and incubate for Ihour in a covered humidity chamber at room temperature.

[0459] • Add 200 pl of Primary antibody in 24 well plate and incubate in a covered humidity chamber at 4 °C for overnight.

[0460] • Dilute the blocking buffer to 50% for primary antibody buffer (PBS+ / +, 0.25% Triton-X, 2.5% Donkey serum or Goat serum + 50 pl Na-Azide).

[0461] • Next day wash the slides with PBS- / - & 0.1% Triton-X for 10 min for 3times each.

[0462] • Add 200 pl of Secondary Ab along with DAPI for 1.5 hour at room temperature in a covered humidity chamber.

[0463] • (8.85ml PBS+ / +, 1 ml Donkey serum or Goat serum + 100 pl Na-Azide)

[0464] • Wash with PBS- / - for 10 min for 3times each.

[0465] • Mount on coverslip with Vectashield or prolong gold and dry it out and store at 4°Cuntil Imaging.

[0466] Staining Protocol for Whole Mount Organoid

[0467] • Fix the cells in 4% PFA for 10 min.

[0468] • Wash with PBS- / - for 5 min. for 3 times. • Incubate in the 30% sucrose at 4°C for 72 hours. After incubation remove sucrose, wash with DPBS buffer for 3 times for 5 min and store in DBPS buffer at 4°C until staining.

[0469] • Quick wash with PBS- / - before staining for 3 times.

[0470] • Blocking buffer PBS+ / +,0.1% Triton -x 100, 10% Donkey serum or goat serum and 10% Na-Azide

[0471] • (8.85 ml PBS+ / +, 50 pl Triton-X 100 + 1 ml Donkey serum or Goat serum-i- 100 pl Na-Azide).

[0472] • Add 50 - 100 pl of blocking buffer in 96 well plate or 0.5 ml tube and incubate for 2 - 3hour at room temperature on shaker.

[0473] • Add 50 - 100 pl of Primary Antibody in 96 well plate or 0.5 ml tube and incubate at 4°C for overnight on shaker.

[0474] • Dilute the blocking buffer to 50% for primary antibody buffer (PBS+ / +, 0.25% Triton-X, 2.5% Donkey serum or Goat serum + 50 pl Na-Azide) or Solution 1.

[0475] • Next day wash the slides with PBS- / - & 0. 1% Triton-X for 10 min for 6 times each.

[0476] • Add 50 - 100 pl of Secondary Antibody in 96 well plate or 0.5 ml tube along with DAPI for 4 °C for overnight on shaker.

[0477] • (8.85 ml PBS+ / +, 1 ml Donkey serum or Goat serum + 100 pl Na-Azide) or Solution 2.

[0478] • Wash with PBS- / - for 10 min for 3 times each.

[0479] • Mount on coverslip with Vectashield or prolong gold and let it dry and store at 4°C until imaging.

[0480] Directed differentiation of human iPSCs into multiple testis-like somatic cells.

[0481] A schematic of this protocol is provided as FIG. 6.

[0482] Reagents

[0483] Media and supplements

[0484] 1. STEMdiff APEL Medium (Stem Cell Technologies, cat. no. 05270 or 05275)

[0485] 2. DMEM / F-12 (Thermo Fisher Scientific, cat. no. 11320-082)

[0486] 3. DMSO (Sigma Aldrich, cat. no. D5879)

[0487] 4. Dulbecco’s phosphate -buffered saline (DPBS) (Thermo Fisher Scientific, Cat. no.14190- 144) 5. Accutase (Cat. no. 07920)

[0488] 6. 0.5M EDTA stock (Lonza 51201).

[0489] 7. Quick-RNA Miniprep Kit (Zymo Research, Cat No. R1055).

[0490] 8. Anti -Adherence Rinsing Solution (Stem cell technologies, Cat No.07010).

[0491] 9. Solution 1 for Primary Antibodies (EMD Millipore, Cat No.KP31812-200ml)

[0492] 10. Solution 2 for Secondary Antibodies (EMD Millipore, Cat No.KP31855-200ml)

[0493] 11. 0.5M EDTA stock (Lonza 51201)

[0494] 12. Rinsing Solution

[0495] 13. CHIR99021 (R&D, cat. no. 9902): Stock Solution (10 mM) — Centrifuge the tube briefly before opening. Reconstitute 10 mg of CHIR99021 into 2.149 ml of DMSO to make 10 mM stock. Prepare 20 pl aliquots and label tubes as “Ch” store at -20°C. The thawed tube can be kept at 4°C for 24hr.

[0496] 14. Y27632 ROCK inhibitor (Enzo, Cat. no. ALX-270-333): Stock Solution (5mM) — Dissolve Img in 625 pl sterile TC water Prepare 20 pl aliquots and label tubes as “Y”. Store at -20°C at stem cell core. Dilute in lOmL culture medium for final lOpM. Discard the tube after use.

[0497] 15. Heparin (Sigma Aldrich, cat. no. H4784-250 mg): Stock solution (1 mg ml“l) — Reconstitute to 1 mg ml- ' in ultrapure water and filter through polyether sulfone (PES) 0.22 pm filter (syringe-driven filter unit). Prepare 40pl aliquots and store at 4°C for up to 12 months.

[0498] 16. FGF9 (R&D, cat. no. 273-F9-025): Stock Solution FGF9 (100 pg / ml) — Centrifuge the tube briefly before opening. Reconstitute to 100 pg / ml in filtered DPBS containing 0.1% (wt / vol) Bovine serum albumin. Prepare 20 pl aliquots and store them at -80°C for up to 6 months. Thawed FGF9 aliquot can be stored at 4°C for up to 2 weeks.

[0499] 17. RA (Sigma, cat. no. R2625-5 mg): Stock Solution (0.5Mm) — Dissolve Img of RA dissolve in 3 ml of the DMSO to make 1.11 mM Stock Solution. Dilute the stock solution to 0.5 mM, prepare 50pl or lOOpl aliquots and this working solution can be stored at -20°C. Use 0.5 mM of stock solution to the cell culture media according to the experimental desired concentrations. Discard the tube after use.

[0500] 18. Insulin (Sigma, cat. no. 19278): Stock Solution (1.7mM) from Vendor.

[0501] 19. IGF1-50 pg (Sigma, Cat. no. 13769): Stock solution (lOMm) — 50 pg of IGF1 is dissolved in 658 pl of 0.2% acetic acid to a stock concentration of 10 mM. Prepare lOpl aliquots and store at -80°C. Dilute the stock solution to a concentration of 100 pM in 0.2% acetic acid and add to the cell culture media according to the experimental desired concentrations. The thawed tube can be stored at 4°C for up to 1 month.

[0502] 20. PGD2 (Cayman, cat. no. 12010): Stock Solution (2mg / ml) — Dissolve PGD2 in 200pl PBS pH 7.4 to a stock concentration of 2mg / ml, prepare 5p 1 aliquots and store at -80°C. Discard the tube after use.

[0503] 21. Matrigel (Coming, cat. no. 354234, Lot no. 9133008): Stock Solution (2mg / ml or Img / ml) — Dilute the Matrigel to desired concentration 2mg / ml or 1 mg / ml in DMEM / F12 media and prepare aliquots and store at -20°C until use.

[0504] 22. Testosterone (Sigma, cat no. T1500-5g): Stock Solution(5mM)-6g of Testosterone was dissolved in 1ml of DMSO then serial diluted to make concentration 5mM.

[0505] 23. Follicle stimulating hormone (Sigma: cat no. F4021-25ug)- Stock Solution (100 pg / ml)- Dissolve 25ug of FSH in 250ul of ultrapure water and prepare aliquots and store at -20°C until use.

[0506] 24. Luteinizing Hormone (Sigma: cat no. L6420-10ug): Stock Solution (100 pg / ml)- Dissolve 25ug of LH in lOOul of ultrapure water and prepare aliquots and store at -20°C until use.

[0507] 25. Epidermal Growth Factor from murine submaxillary gland, EGF (Sigma, cat no. E4127-.1 mg): Stock Solution(100ug / ml)-0.1mg of EGF was dissolved in 10ml of 0.1%BSA / PBS in 10ml then aliquoted in 1 ml and store at -20°C until use.

[0508] 26. IWR1 (Sigma: cat no. 681669-lOmg)- Stock Solution ()- Dissolve 10 mg / ml in DMSO and prepare aliquots and store at -20°C until use.

[0509] Materials

[0510] 1. Nunc™ 4-Well Dishes for IVF (Fisher, cat no. 144444)

[0511] 2. Nunc™ Cell-Culture Treated Multidishes - 24well (Fisher, cat. no. 142475)

[0512] 3. Uitra-Low Binding, U-Shaped-Bottom Microplate(Corning, cat.no.12-456-721)

[0513] 4. Greiner Bio-One ThinCert® Tissue Culture Inserts(Greiner Bio-One, cat.no. 10443845)

[0514] 5. Costar 24 well plate (Corning, Cat No.3526)

[0515] 6. Coverslips 12mm (Hampton Research, cat no. HR3-277)

[0516] 7. Bench top centrifuge (Eppendorf, 5417c)

[0517] 8. Biological safety cabinet

[0518] 9. Co2 Incubator (Heraeus)

[0519] 10. Conical tubes 15ml (Falcon, cat no. 352096)

[0520] 11. Conical tubes 50ml (Falcon, cat no. 352070)

[0521] 12. Freezing container (Nalgene, Mr. Frosty) 13. Inverted contrasting tissue culture microscope (KL1500CD, Leica)

[0522] 14. Laser confocal microscope (Nikon Eclipse Ti2)

[0523] 15. Pipette boy (Drummond)

[0524] 16. Pipettes (Gilson)

[0525] 17. Automated cell counter (BioRad, TC20)

[0526] 18. Serological pipettes (Fisher, Cat No, 13-678-1 ID, 13-676-10J, 13-676-10R)

[0527] 19. Stericup 0.22pm Filter unit (Millipore, 03290)

[0528] 20. Sterile filter pipette tips (Genesee Scientific, 24-815, 24-804,24-830, 26-401)

[0529] 21. Sterile Microcentrifuge tubes (Fisher, Cat No.05-408-120)

[0530] 22. Aggrewell® 800 24-well,5pk (Stem cell technologies, Cat No.34815)

[0531] Coverslip cleaning method:

[0532] • Shake coverslips in xylenes in 50mL conical tube for 2-3 hours.

[0533] • Discard xylenes and rinse with acetone. Discard acetone and rinse in fresh acetone in 50 mL conical tube for 2-3 hours.

[0534] • Discard acetone. Autoclave in water if desired on liquid cycle. Rinse coverslips with 100% ethanol 2times. Store coverslips in 100% ethanol at 4°C.

[0535] Dav -1 Plating the IPSC into Single cell suspension for Differentiation to improve reproducibility.

[0536] Coating plates for Cell pellet (RNA seq analysis) or staining of coverslip.

[0537] • Dry the coverslips at room temperature until all the alcohol evaporates completely in the Biosafety cabinet hood under sterile conditions. After drying, transfer the coverslip with forceps to 24 or 4 well plate.

[0538] • Coat the 24 or 4 well plate with Matrigel for Ihour before plating the cells.

[0539] Single cell suspension for Monolayer

[0540] • Clean any differentiating colonies from cultures colonies on 60mm petri plates.

[0541] • Wash with 2ml DPBS buffer.

[0542] • Add 0.5 ml of lx accutase and place in incubator for 5-10 min.

[0543] • Add 2 - 5 ml of DMEM / F12 media to stop the dissociation.

[0544] • Pellet cells by centrifuging for 3min at 1000 rpm. • Resuspended the pellet in 5 ml of mTeSR media with 10 pM (Y27632) Rock inhibitor and count the cells.

[0545] • Plate 10,000 cells per well onto Matrigel coated 24 well plate.

[0546] Dav 0 IPSC cell collection as a control:

[0547] • IPSC cells were collected on day 0.

[0548] • Cells were washed with 0.5 ml of DPBS buffer for each well of 24 well plate.

[0549] • 0.5 ml of lx accutase was added and placed in incubator for 3 - 5 min.

[0550] • Add 1ml of DMEM / F12 media to stop the digestion reaction.

[0551] • Centrifuge at 10,000g for 2 min.

[0552] • Aspirate the media.

[0553] • Snap freeze in Liquid N2 and store at -80°C until later use.

[0554] • Fix the cells in 4% PFA for 20 min and wash 3times with DPBS, store in DPBS at 4°C until use.

[0555] • Cells used to start differentiation.

[0556] • Remove mTeSR media with Rock inhibitor and add 0.5 ml of APEL2 media + CHIR 8pM.

[0557] Media changed every 2 days.

[0558] Day 2 Remove media and add APEL2 media + CHIR 8 pM.

[0559] Dav 4; Presomitic Mesoderm

[0560] • Collect cells from required number of wells.

[0561] • Add 0.5 ml of lx accutase and placed in incubator for 3 - 5 min and add 1ml of DMEM / F12 to stop reaction.

[0562] • Centrifuge at 10,000g for 2 min.

[0563] • Aspirate the media.

[0564] • Snap freeze in Liquid N2 and store at -80°C until later use (Presomitic Mesoderm).

[0565] • Fix the cells in 4%PFA for 20 min and wash 3times with DPBS, store in DPBS at 4°C until use.

[0566] Start FGF9 Phase

[0567] • Change media with 200 ng / ml FGF9 + 1 pg / ml Heparin.

[0568] • Change the media every two days. Day 6 Change Media with 200 ng / ml FGF9 + 1 ,u g / ml Heparin.

[0569] On Dav 7 (Intermediate Mesoderm)

[0570] • Collect cells from required number of wells on day 7.

[0571] • Wash cells with 0.5 ml of DPBS buffer for each well of 24 well plate.

[0572] • Add 0.5 ml of lx accutase and placed in incubator for 3 - 5 min and add 1ml of DMEM / F12 to stop reaction.

[0573] • Centrifuge at 10,000g for 2 min.

[0574] • Aspirate the media.

[0575] • Snap freeze in Liquid N2 and store at -80°C until later use (Intermediate Mesoderm).

[0576] • Fix the cells in 4% PFA for 20 min and wash 3times with DPBS, store in DPBS at 4°C until use.

[0577] • The remaining wells will continue with the differentiation protocol: for these wells the following media is used: APEL ± 0.1 pM RA + 100 nM Insulin + 17 nM IGF1 + 500 ng / ml PGD2± 200 ng / ml FGF9, 50ng / ml EGF1.

[0578] On Dav 8

[0579] Generation of Gonadal Organoids by Aggrewell® plate or 96 well plates

[0580] Preparation of Aggrewell® plate to receive cells.

[0581] • Add surface rinsing solution of 0.5 ml for each well. Make sure there is no air bubble present in microwell. To remove air bubbles from each microwell centrifuge at 200g for 5 min.

[0582] • Observe under the Bright Field microscope to make sure there is no air bubbles present. If the air bubbles are still present, then recentrifuged for 200g for 5 min.

[0583] • Incubate plate at room temperature or at 37°C for 30min. After incubation remove the rinsing solution and wash with 0.5 ml DBPS buffer and make sure the wells do not dry out before plating the cells.

[0584] • Add 0.5 ml of APEL Media with corresponding growth factors and centrifuge for 200g for 5 min to remove air bubbles. Observe under the Bright field microscope to make sure there is no air bubbles.

[0585] • If using a 96 well Add 0.1 ml of APEL Media with corresponding growth factors and centrifuge for 200g for 5 min to remove air bubbles. Observe under the Bright field microscope to make sure there is no air bubbles. Preparation of cells for Plating

[0586] • Dissociate the remaining wells of differentiated cells into single cells.

[0587] • Wash cells with 0.5 ml of DPBS buffer for each well of 24 well plate.

[0588] • Add 0.5 ml of lx accutase and placed in incubator for 3 - 5 min and add 1ml of DMEM / F12 to stop reaction.

[0589] • Centrifuge at 1000 rpm for 3 min.

[0590] • Aspirate the media.

[0591] • Resuspended the cells with APEL media with corresponding growth factors with 10pm (Y27632) Rock inhibitor and count the cells.

[0592] • Add 0.5 ml of (1 x 106- 2 x 106) cells per well with APEL media with corresponding growth factors and mix up and down slowly and centrifuge 500g for 5 min. Observe under the microscope to see all the cells are clustered together.

[0593] • If 96-well plate is used add 30K cells in lOOul of differentiation media. Mix cells and down slowly and centrifuge 500g for 5 min. Observe under the microscope to see all the cells are clustered together.

[0594] • Change the half media every other day by slowing touching the side of the wells by removing 0.5 ml of old media and add 0.5 ml of the fresh media.

[0595] Day 9

[0596] • Change half the media every other day by slowly touching the side of the wells and removing 1 ml of old media and adding 1 ml of the fresh media with APEL ± 0.1 pM RA + 100 nM Insulin + 17 nM IGF1 + 500 ng / ml PGD2+ 200 ng / ml FGF9+ IpM Testosterone ± lOOng / ml Follicle stimulating hormone ± 200ng / ml Luteinizing Hormone± IpM 1WR1.

[0597] Dav 10 (Genital Ridge)

[0598] • Collect organoids from required number of wells on day 10. To collect the cells, use uncut the tip and pipette up and down to loosen and lift the organoids and collect the floating organoids in to 0.5 ml tube. Snap freeze in Liquid N2 and store at -80°C until later use.

[0599] • Fix the some of the wells of organoids 4% PFA for 10 min and wash 3times with DPBS. • Incubate in the 30% sucrose at 4°C for 72hours. After incubation remove sucrose, wash with DPBS buffer for 3 times for 5min and store in DBFS buffer at 4°C until staining.

[0600] • Remaining wells will get various combination of growth factors: media will be changed every 2 days.

[0601] • APEL ± 0.1 |iM RA + 100 nM Insulin + 17 nM IGF1 + 500 ng / ml PGD2± 200 ng / ml FGF91|iM Testosterone ± lOOng / ml Follicle stimulating hormone ± 200ng / ml Euteinizing Hormonei IpM IWR1.

[0602] Day 12

[0603] • Transfer organoids to ThinCert® cell culture inserts and add 700ul of media ( APEE ± 0.1 |iM RA + 100 nM Insulin + 17 nM IGF1 + 500 ng / ml PGD2± 200 ng / ml FGF9 l pM Testosterone ± lOOng / ml Follicle stimulating hormone ± 200ng / ml Luteinizing Hormonei IpM IWR1) in the bottom well.

[0604] Day 14

[0605] • Change the media for every two days with growth factors.

[0606] • APEL ± 0.1 pM RA + 100 nM Insulin + 17 nM IGF1 + 500 ng / ml PGD2± 200 ng / ml FGF91|iM Testosterone ± lOOng / ml Follicle stimulating hormone ± 200ng / ml Luteinizing Hormonei I pM IWR1.

[0607] Day 16

[0608] • Collect organoids from required number of wells on day 16. To collect the cells, use uncut the tip and pipette up and down to loosen and lift the organoids and collect the floating organoids in to 0.5 ml tube. Snap freeze in Liquid N2 and store at -80°C until later use.

[0609] • Fix the some of the wells of organoids 4% PFA for 10 min and wash 3 times with DPBS .

[0610] • Incubate in the 30% sucrose at 4°C for 72hours. After incubation remove sucrose, wash with DPBS buffer for 3 times for 5min and store in DBFS buffer at 4°C until staining.

[0611] • Remaining wells will get various combination of growth factors: media will be changed every 2 days.

[0612] • APEL ± O. lpM RA + WOnM Insulin + 17nM IGF1 + 500 ng / ml PGD2± 200ng / ml FGF91pM Testosterone + lOOng / ml Follicle stimulating hormone ± 200ng / ml Luteinizing Hormone+± I pM IWR1.

[0613] Dav 18 • Change the media for every two days with growth factors.

[0614] • APEL ± 0. 1 pM RA + 100 nM Insulin + 17 nM IGF1 + 500 ng / ml PGD2± 200 ng / ml FGF91|iM Testosterone + lOOng / ml Follicle stimulating hormone ± 200ng / ml Luteinizing Hormonei IpM IWR1.

[0615] Dav 20

[0616] • Change the media for every two days with growth factors.

[0617] • APEL ± 0. 1 pM RA + 100 nM Insulin + 17 nM IGF1 + 500 ng / ml PGD2± 200 ng / ml FGF91|iM Testosterone ± lOOng / ml Follicle stimulating hormone ± 200ng / ml Luteinizing Hormonei I p M IWR1.

[0618] Dav 22

[0619] • Collect organoids from required number of wells on day 22(Testis Organoids). To collect the cells, use uncut the tip and pipette up and down to loosen and lift the organoids and collect the floating organoids in to 0.5 ml tube. Snap freeze in Liquid N2 and store at -80 °C until later use.

[0620] • Fix the some of the wells of organoids 4% PFA for 10 min and wash 3times with DPBS.

[0621] • Incubate in the 30% sucrose at 4°C for 72 hours. After incubation remove sucrose, wash with DPBS buffer for 3 times for 5min and store in DBFS buffer at 4°C until staining.

[0622] Coverslip Staining Protocol for Monolayer

[0623] • Fix the cells in 4% PFA for 20 min.

[0624] • Wash with PBS- / - for 5 min for 3 times and store in PBS- / - at 4°C until staining.

[0625] • Quick wash with PBS- / - before staining for 3 times.

[0626] • Blocking buffer PBS+ / +, 0.1% Triton -x 100, 10% Donkey serum or goat serum and 10% Na-Azide

[0627] • (8.85ml PBS+ / +, 50 pl Triton-X 100 + 1 ml Donkey serum or Goat serum-i- 100 pl Na-Azide).

[0628] • Add 200 pl of blocking buffer in 24 well plate and incubate for Ihour in a covered humidity chamber at room temperature.

[0629] • Add 200 pl of Primary antibody in 24 well plate and incubate in a covered humidity chamber at 4 °C for overnight.

[0630] • Dilute the blocking buffer to 50% for primary antibody buffer (PBS+ / +, 0.25% Triton-X, 2.5% Donkey serum or Goat serum + 50 pl Na-Azide). • Next day wash the slides with PBS- / - & 0.1% Triton-X for 10 min for 3times each.

[0631] • Add 200 pl of Secondary Ab along with DAPI for 1.5 hour at room temperature in a covered humidity chamber.

[0632] • (8.85ml PBS+ / +, 1 ml Donkey serum or Goat serum + 100 pl Na- Azide)

[0633] • Wash with PBS- / - for 10 min for 3times each.

[0634] • Mount on coverslip with Vectashield or prolong gold and dry it out and store at 4°Cuntil Imaging.

[0635] Staining Protocol for Whole Mount Organoid

[0636] • Fix the cells in 4% PFA for 10 min.

[0637] • Wash with PBS- / - for 5 min for 3 times.

[0638] • Incubate in the 30% sucrose at 4°C for 72 hours. After incubation remove sucrose, wash with DPBS buffer for 3 times for 5 min and store in DBPS buffer at 4°C until staining.

[0639] • Quick wash with PBS- / - before staining for 3 times.

[0640] • Blocking buffer PBS+ / +,0.1% Triton -x 100, 10% Donkey serum or goat serum and 10% Na-Azide

[0641] • (8.85 ml PBS+ / +, 50 pl Triton-X 100 + 1 ml Donkey serum or Goat serum-i- 100 pl Na-Azide).

[0642] • Add 50 - 100 pl of blocking buffer in 96 well plate or 0.5 ml tube and incubate for 2 - 3hour at room temperature on shaker.

[0643] • Add 50 - 100 pl of Primary Antibody in 96 well plate or 0.5 ml tube and incubate at 4°C for overnight on shaker.

[0644] • Dilute the blocking buffer to 50% for primary antibody buffer (PBS+ / +, 0.25% Triton-X, 2.5% Donkey serum or Goat serum + 50 pl Na-Azide) or Solution 1.

[0645] • Next day wash the slides with PBS- / - & 0.1% Triton-X for 10 min for 6 times each.

[0646] • Add 50 - 100 pl of Secondary Antibody in 96 well plate or 0.5 ml tube along with DAPI for 4 °C for overnight on shaker.

[0647] • (8.85 ml PBS+ / +, 1 ml Donkey serum or Goat serum + 100 pl Na-Azide) or Solution 2.

[0648] • Wash with PBS- / - for 10 min for 3 times each.

[0649] • Mount on coverslip with Vectashield or prolong gold and let it dry and store at 4°C until imaging. Combining testis somatic like cells derived from ESC with primordial germ cell like cells to promote PGCLC maturation.

[0650] A schematic of this protocol is provided as FIG. 7.

[0651] Reagents

[0652] Media and supplements

[0653] 1. STEMdiff APEL Medium (Stem Cell Technologies, cat. no. 05270 or 05275)

[0654] 2. DMEM / F-12 (Thermo Fisher Scientific, cat. no. 11320-082)

[0655] 3. DMSO (Sigma Aldrich, cat. no. D5879)

[0656] 4. Dulbecco's phosphate-buffered saline (DPBS) (Thermo Fisher Scientific, Cat. no.14190- 144)

[0657] 5. Accutase (Cat. no. 07920)

[0658] 6. 0.5M EDTA stock (Lonza 51201).

[0659] 7. Quick-RNA Miniprep Kit (Zymo Research, Cat No. R1055).

[0660] 8. Anti -Adherence Rinsing Solution (Stem cell technologies, Cat No.07010).

[0661] 9. Solution 1 for Primary Antibodies (EMD Millipore, Cat No.KP31812-200ml)

[0662] 10. Solution 2 for Secondary Antibodies (EMD Millipore, Cat No.KP31855-200ml)

[0663] 11. 0.5M EDTA stock (Lonza 51201)

[0664] 12. Rinsing Solution

[0665] 13. CHIR99021 (R&D, cat. no. 9902): Stock Solution (10 mM) — Centrifuge the tube briefly before opening. Reconstitute 10 mg of CHIR99021 into 2.149 ml of DMSO to make 10 mM stock. Prepare 20 pl aliquots and label tubes as “Ch” store at -20°C. The thawed tube can be kept at 4°C for 24hr.

[0666] 14. Y27632 ROCK inhibitor (Enzo, Cat. no. ALX-270-333): Stock Solution (5mM) — Dissolve Img in 625 pl sterile TC water Prepare 20 pl aliquots and label tubes as “Y”. Store at -20°C at stem cell core. Dilute in lOmL culture medium for final lOpM. Discard the tube after use.

[0667] 15. Heparin (Sigma Aldrich, cat. no. H4784-250 mg): Stock solution (1 mg ml“l) — Reconstitute to 1 mg ml- ' in ultrapure water and filter through polyether sulfone (PES) 0.22 pm filter (syringe-driven filter unit). Prepare 40pl aliquots and store at 4°C for up to 12 months.

[0668] 16. FGF9 (R&D, cat. no. 273-F9-025): Stock Solution FGF9 (100 pg / ml) — Centrifuge the tube briefly before opening. Reconstitute to 100 pg / ml in filtered DPBS containing 0.1% (wt / vol) Bovine serum albumin. Prepare 20 pl aliquots and store them at -80°C for up to 6 months. Thawed FGF9 aliquot can be stored at 4°C for up to 2 weeks.

[0669] 17. RA (Sigma, cat. no. R2625-5 mg): Stock Solution (0.5Mm) — Dissolve Img of RA dissolve in 3 ml of the DMSO to make 1.1 1 mM Stock Solution. Dilute the stock solution to 0.5 mM, prepare 50pl or lOOpl aliquots and this working solution can be stored at -20°C. Use 0.5 mM of stock solution to the cell culture media according to the experimental desired concentrations. Discard the tube after use.

[0670] 18. Insulin (Sigma, cat. no. 19278): Stock Solution (1.7mM) from Vendor.

[0671] 19. IGF1-50 pg (Sigma, Cat. no. 13769): Stock solution (lOMm) — 50 pg of IGF1 is dissolved in 658 pl of 0.2% acetic acid to a stock concentration of 10 mM. Prepare lOpl aliquots and store at -80°C. Dilute the stock solution to a concentration of 100 pM in 0.2% acetic acid and add to the cell culture media according to the experimental desired concentrations. The thawed tube can be stored at 4°C for up to 1 month.

[0672] 20. PGD2 (Cayman, cat. no.12010): Stock Solution (2mg / ml) — Dissolve PGD2 in 200pl PBS pH 7.4 to a stock concentration of 2mg / ml, prepare 5pl aliquots and store at -80°C. Discard the tube after use.

[0673] 21. Matrigel (Coming, cat. no. 354234, Lot no. 9133008): Stock Solution (2mg / ml or Img / ml) — Dilute the Matrigel to desired concentration 2mg / ml or 1 mg / ml in DMEM / F12 media and prepare aliquots and store at -20°C until use.

[0674] 22. Testosterone (Sigma, cat no. T1500-5g): Stock Solution(5mM)-6g of Testosterone was dissolved in 1ml of DMSO then serial diluted to make concentration 5mM.

[0675] 23. Follicle stimulating hormone (Sigma: cat no. F4021-25ug)- Stock Solution (100 pg / ml)- Dissolve 25ug of FSH in 250ul of ultrapure water and prepare aliquots and store at -20°C until use.

[0676] 24. Luteinizing Hormone (Sigma: cat no. L6420-10ug): Stock Solution (100 pg / ml)- Dissolve 25ug of LH in lOOul of ultrapure water and prepare aliquots and store at -20°C until use.

[0677] 25. Epidermal Growth Factor from murine submaxillary gland, EGF (Sigma, cat no. E4127-.1 mg): Stock Solution(100ug / ml)-0.1mg of EGF was dissolved in 10ml of 0.1%BSA / PBS in 10ml then aliquoted in 1 ml and store at -20°C until use.

[0678] 26. IWR1 (Sigma: cat no. 681669-lOmg)- Stock Solution ()- Dissolve 10 mg / ml in DMSO and prepare aliquots and store at -20°C until use.

[0679] Materials

[0680] 1. Nunc™ 4-Well Dishes for IVF (Fisher, cat no. 144444) 2. Nunc™ Cell-Culture Treated Multidishes - 24well (Fisher, cat. no. 142475)

[0681] 3. Uitra-Low Binding, U-Shaped-Bottom Microplate(Corning, cat.no.12-456-721)

[0682] 4. Greiner Bio-One ThinCert® Tissue Culture Inserts(Greiner Bio-One, cat.no. 10443845)

[0683] 5. Costar 24 well plate (Corning, Cat No.3526)

[0684] 6. Coverslips 12mm (Hampton Research, cat no. HR3-277)

[0685] 7. Bench top centrifuge (Eppendorf, 5417c)

[0686] 8. Biological safety cabinet

[0687] 9. Co2 Incubator (Heraeus)

[0688] 10. Conical tubes 15ml (Falcon, cat no. 352096)

[0689] 11. Conical tubes 50ml (Falcon, cat no. 352070)

[0690] 12. Freezing container (Nalgene, Mr. Frosty)

[0691] 13. Inverted contrasting tissue culture microscope (KL1500CD, Leica)

[0692] 14. Laser confocal microscope (Nikon Eclipse 772)

[0693] 15. Pipette boy (Drummond)

[0694] 16. Pipettes (Gilson)

[0695] 17. Automated cell counter (BioRad, TC20)

[0696] 18. Serological pipettes (Fisher, Cat No, 13-678-1 ID, 13-676-10J, 13-676-10R)

[0697] 19. Stericup 0.22 m Filter unit (Millipore, 03290)

[0698] 20. Sterile filter pipette tips (Genesee Scientific, 24-815, 24-804,24-830, 26-401)

[0699] 21. Sterile Microcentrifuge tubes (Fisher, Cat No.05-408-120)

[0700] 22. Aggrewell® 800 24-well,5pk (Stem cell technologies, Cat No.34815)

[0701] Coverslip cleaning method:

[0702] • Shake coverslips in xylenes in 50mL conical tube for 2-3 hours.

[0703] • Discard xylenes and rinse with acetone. Discard acetone and rinse in fresh acetone in 50 mL conical tube for 2-3 hours.

[0704] • Discard acetone. Autoclave in water if desired on liquid cycle. Rinse coverslips with 100% ethanol 2 times. Store coverslips in 100% ethanol at 4°C.

[0705] Dav -1 Plating the ESC in Single cell suspension for Differentiation to improve reproducibility.

[0706] Coating plates for Cell pellet (RNA seq analysis) or staining of coverslip. • Dry the coverslips at room temperature until all the alcohol evaporates completely in the Biosafety cabinet hood under sterile conditions. After drying, transfer the coverslip with forceps to 24 or 4 well plate.

[0707] • Coat the 24 or 4 well plate with Matrigel for Ihour before plating the cells.

[0708] Single cell suspension for Monolayer

[0709] • Clean any differentiating colonies from cultures colonies on 60mm petri plates.

[0710] • Wash with 2ml DPBS buffer.

[0711] • Add 0.5 ml of lx accutase and place in incubator for 5-10 min.

[0712] • Add 2 - 5 ml of DMEM / F12 media to stop the dissociation.

[0713] • Pellet cells by centrifuging for 3min at 1000 rpm.

[0714] • Resuspended the pellet in 5 ml of mTeSR media with 10 pM (Y27632) Rock inhibitor and count the cells.

[0715] Plate 10,000 cells per well onto Matrigel coated 24 well plate.

[0716] Next morning cells can be used to start differentiation.

[0717] • Remove mTeSR media with Rock inhibitor and add 0.5 ml of APEL2 media + CHIR 3pM.

[0718] Day 2 Change media with APEL2 media + CHIR 3 pM.

[0719] Dav 4: Presomitic Mesoderm

[0720] • Collect cells from required number of wells.

[0721] • Add 0.5 ml of lx accutase and placed in incubator for 3 - 5 min and add 1ml of DMEM / F12 to stop reaction.

[0722] • Centrifuge at 10,000g for 2 min.

[0723] • Aspirate the media.

[0724] • Snap freeze in Liquid N2 and store at -80°C until later use (Presomitic Mesoderm).

[0725] • Fix the cells in 4%PFA for 20 min and wash 3times with DPBS, store in DPBS at 4°C until use.

[0726] Start FGF9 Phase

[0727] • Change media with 200 ng / ml FGF9 + 1 pg / ml Heparin.

[0728] • Change the media every two days. Day 6 Change Media with 200 ng / ml FGF9 + 1 p g / ml Heparin.

[0729] On Dav 7 (Intermediate Mesoderm)

[0730] • Change the media to (APEL ± 0.1 pM RA + 100 nM Insulin + 17 nM IGF1 + 500 ng / ml PGD2± 200 ng / ml FGF9).

[0731] On Dav 8: Germ cell + Somatic cell mixing.

[0732] Differentiated Cells to Gonadal Organoids by U-Bottom Microplates

[0733] Preparation of cells for U-Bottom Microplate Plating

[0734] • Dissociate the in vitro derived testis like somatic cells into single cells.

[0735] • Wash cells with 0.5 ml of DPBS buffer for each well of 24 well plate.

[0736] • Add 0.5 ml of lx accutase and placed in incubator for 3 - 5 min and add 1ml of DMEM / F12 to stop reaction.

[0737] • Centrifuge at 1000 rpm for 3 min.

[0738] • Aspirate the media.

[0739] • Resuspended the cells with APEL media with corresponding growth factors with 10 m (Y27632) Rock inhibitor and count the cells.

[0740] • Add lOOul of (90% somatic cells and 10% germ cells with total of 30,000) cells per well with APEL media with corresponding growth factors and mix up and down slowly and transfer the cells into U-Bottom Microplates

[0741] • Centrifuge U-bottom Microplates at 200g for 3min.

[0742] • Observe under the microscope to see all the cells are clustered together.

[0743] Day 9

[0744] • Change the half the media every other day by slowly touching the side of the wells by removing 1 ml of old media and add 1 ml of the fresh media with APEL + 0.1 pM RA + 100 nM Insulin + 17 nM IGF1 + 500 ng / ml PGD2± 200 ng / ml FGF9+ IpM Testosterone + lOOng / ml Follicle stimulating hormone + 200ng / ml Luteinizing Hormone or 2U HCG+ IpM IWRl+50ng / ml EGF.

[0745] • Day 10 - change media and remove IWR1 but keep all media composition the same.

[0746] Dav 11 • Collect organoids from required number of wells on day 11 and transfer the Organoids into ThinCert® plates.

[0747] Combine the at least 5 organoids and transfer them into ThinCert® plates with 200 til wide pipette.

[0748] • Lower chamber receives at least 700ul of APEL ± 0.1 pM RA + 100 nM Insulin + 17 nM IGF1 + 500 ng / ml PGD2 + 200 ng / ml FGF9 I pM Testosterone ± lOOng / ml Follicle stimulating hormone ± 200ng / ml Luteinizing Hormonei +50ng / ml EGF.

[0749] Day 13

[0750] • Upper chamber receives at least 50ul of will get various combination of growth factors: media will be changed every 2 days.

[0751] • Lower chamber receives at least 700ul of will get various combination of growth factors: media will be changed every 2 days.

[0752] • APEL ± 0.1 pM RA + 100 nM Insulin + 17 nM IGF1 + 500 ng / ml PGD2 ± 200 ng / ml FGF91pM Testosterone ± lOOng / ml Follicle stimulating hormone ± 200ng / ml Luteinizing Hormonei 50ng / ml EGF.

[0753] Day 16

[0754] • Collect organoids from required number of wells on day 16 for RNA. Snap freeze in Liquid N2 and store at -80°C until later use.

[0755] • Fix the some of the wells of organoids 4% PFA for 20 min and wash 3times with DPBS.

[0756] • Incubate in the 30% sucrose at 4°C for 72hours. After incubation remove sucrose, wash with DPBS buffer for 3 times for 5min and store in DBFS buffer at 4°C until staining.

[0757] Staining Protocol for Whole Mount Organoid

[0758] • Fix the cells in 4% PFA for 10 min.

[0759] • Wash with PBS- / - for 5 min for 3 times.

[0760] • Incubate in the 30% sucrose at 4°C for 72 hours. After incubation remove sucrose, wash with DPBS buffer for 3 times for 5 min and store in DBPS buffer at 4°C until staining.

[0761] • Quick wash with PBS- / - before staining for 3 times.

[0762] • Blocking buffer PBS+ / +,0.1% Triton -x 100, 10% Donkey serum or goat serum and 10% Na-Azide • (8.85 ml PBS+ / +, 50 pl Triton-X 100 + 1 ml Donkey serum or Goat serum+ 100 l Na- Azide).

[0763] • Add 50 - 100 pl of blocking buffer in 96 well plate or 0.5 ml tube and incubate for 2 - 3 hours at room temperature on shaker.

[0764] • Add 50 - 100 pl of Primary Antibody in 96 well plate or 0.5 ml tube and incubate at 4°C for overnight on shaker.

[0765] • Dilute the blocking buffer to 50% for primary antibody buffer (PBS+ / +, 0.25% Triton-X, 2.5% Donkey serum or Goat serum + 50 pl Na- Azide) or Solution 1.

[0766] • Next day wash the slides with PBS- / - & 0.1% Triton-X for 10 min for 6 times each.

[0767] • Add 50 - 100 pl of Secondary Antibody in 96 well plate or 0.5 ml tube along with DAPI for 4 °C for overnight on shaker.

[0768] • (8.85 ml PBS+ / +, 1 ml Donkey serum or Goat serum + 100 pl Na-Azide) or Solution 2.

[0769] • Wash with PBS- / - for 10 min for 3 times each.

[0770] • Mount on coverslip with Vectashield or prolong gold and let it dry and store at 4°C until imaging.

[0771] ICC protocol for OCT sections for Tissue and Organoid

[0772] • Section the Organoid 8- 12pm and frozen in -80°C.

[0773] • Take the slides out of the freezer. Let them sit for 10 min.

[0774] • Post- fix the slides with 4% PFA (on the slides) for 10 min.

[0775] • Wash slides with PBS 3x 5 min.

[0776] • Draw circles on the slides.

[0777] • Penetrate the tissues with 0.1% Triton X-100 / PBS for 15 min.

[0778] • Block the slides in blocking solution (200 pl 2.5M glycine / 900 pl PBS / 3% BSA) for Ihr.

[0779] • Dilute primary antibodies in solution I and incubate the slides with the antibodies at 4°C overnight.

[0780] • Wash the slides in PBST 4x 15 min.

[0781] • Incubate the slides with Solution II secondary antibodies for 2 hours at room temp.

[0782] • Wash with PBS 4x 15 min.

[0783] • Mount with Vectashield or prolong gold. Combining testis somatic like cells derived from iPSC with primordial germ cell like cells to promote PGCLC maturation.

[0784] Reagents

[0785] Media and supplements

[0786] 1. STEMdiff APEL Medium (Stem Cell Technologies, cat. no. 05270 or 05275)

[0787] 2. DMEM / F-12 (Thermo Fisher Scientific, cat. no. 11320-082)

[0788] 3. DMSO (Sigma Aldrich, cat. no. D5879)

[0789] 4. Dulbecco's phosphate-buffered saline (DPBS) (Thermo Fisher Scientific, Cat. no.14190- 144)

[0790] 5. Accutase (Cat. no. 07920)

[0791] 6. 0.5M EDTA stock (Lonza 51201).

[0792] 7. Quick-RNA Miniprep Kit (Zymo Research, Cat No. R1055).

[0793] 8. Anti -Adherence Rinsing Solution (Stem cell technologies, Cat No.07010).

[0794] 9. Solution 1 for Primary Antibodies (EMD Millipore, Cat No.KP31812-200ml)

[0795] 10. Solution 2 for Secondary Antibodies (EMD Millipore, Cat No.KP31855-200ml)

[0796] 11. 0.5M EDTA stock (Lonza 51201)

[0797] 12. Rinsing Solution

[0798] 13. CHIR99021 (R&D, cat. no. 9902): Stock Solution (10 mM) — Centrifuge the tube briefly before opening. Reconstitute 10 mg of CHIR99021 into 2.149 ml of DMSO to make 10 mM stock. Prepare 20 pl aliquots and label tubes as “Ch” store at -20°C. The thawed tube can be kept at 4°C for 24hr.

[0799] 14. Y27632 ROCK inhibitor (Enzo, Cat. no. ALX-270-333): Stock Solution (5mM) — Dissolve Img in 625 pl sterile TC water Prepare 20 pl aliquots and label tubes as “Y”. Store at -20°C at stem cell core. Dilute in lOmL culture medium for final lOpM. Discard the tube after use.

[0800] 15. Heparin (Sigma Aldrich, cat. no. H4784-250 mg): Stock solution (1 mg ml_l) — Reconstitute to 1 mg ml- ' in ultrapure water and filter through polyether sulfone (PES) 0.22 pm filter (syringe-driven filter unit). Prepare 40pl aliquots and store at 4°C for up to 12 months.

[0801] 16. FGF9 (R&D, cat. no. 273-F9-025): Stock Solution FGF9 (100 pg / ml) — Centrifuge the tube briefly before opening. Reconstitute to 100 pg / ml in filtered DPBS containing 0.1% (wt / vol) Bovine serum albumin. Prepare 20 pl aliquots and store them at -80°C for up to 6 months. Thawed FGF9 aliquot can be stored at 4°C for up to 2 weeks.

[0802] 17. RA (Sigma, cat. no. R2625-5 mg): Stock Solution (0.5Mm) — Dissolve Img of RA dissolve in 3 ml of the DMSO to make 1.1 1 mM Stock Solution. Dilute the stock solution to 0.5 mM, prepare 50pl or lOOpl aliquots and this working solution can be stored at -20°C. Use 0.5 mM of stock solution to the cell culture media according to the experimental desired concentrations. Discard the tube after use.

[0803] 18. Insulin (Sigma, cat. no. 19278): Stock Solution (1.7mM) from Vendor.

[0804] 19. IGF1-50 pg (Sigma, Cat. no. 13769): Stock solution (lOMm) — 50 pg of IGF1 is dissolved in 658 pl of 0.2% acetic acid to a stock concentration of 10 mM. Prepare lOpl aliquots and store at -80°C. Dilute the stock solution to a concentration of 100 pM in 0.2% acetic acid and add to the cell culture media according to the experimental desired concentrations. The thawed tube can be stored at 4°C for up to 1 month.

[0805] 20. PGD2 (Cayman, cat. no.12010): Stock Solution (2mg / ml) — Dissolve PGD2 in 200pl PBS pH 7.4 to a stock concentration of 2mg / ml, prepare 5pl aliquots and store at -80°C. Discard the tube after use.

[0806] 21. Matrigel (Coming, cat. no. 354234, Lot no. 9133008): Stock Solution (2mg / ml or Img / ml) — Dilute the Matrigel to desired concentration 2mg / ml or 1 mg / ml in DMEM / F12 media and prepare aliquots and store at -20°C until use.

[0807] 22. Testosterone (Sigma, cat no. T1500-5g): Stock Solution(5mM)-6g of Testosterone was dissolved in 1ml of DMSO then serial diluted to make concentration 5mM.

[0808] 23. Follicle stimulating hormone (Sigma: cat no. F4021-25ug)- Stock Solution (100 pg / ml)- Dissolve 25ug of FSH in 250ul of ultrapure water and prepare aliquots and store at -20°C until use.

[0809] 24. Luteinizing Hormone (Sigma: cat no. L6420-10ug): Stock Solution (100 pg / ml)- Dissolve 25ug of LH in lOOul of ultrapure water and prepare aliquots and store at -20°C until use.

[0810] 25. Epidermal Growth Factor from murine submaxillary gland, EGF (Sigma, cat no. E4127-.1 mg): Stock Solution(100ug / ml)-0.1mg of EGF was dissolved in 10ml of 0.1%BSA / PBS in 10ml then aliquoted in 1 ml and store at -20°C until use.

[0811] 26. IWR1 (Sigma: cat no. 681669-lOmg)- Stock Solution ()- Dissolve 10 mg / ml in DMSO and prepare aliquots and store at -20°C until use.

[0812] Materials

[0813] 1. Nunc™ 4-Well Dishes for IVF (Fisher, cat no. 144444) 2. Nunc™ Cell-Culture Treated Multidishes - 24well (Fisher, cat. no. 142475)

[0814] 3. Uitra-Low Binding, U-Shaped-Bottom Microplate(Corning, cat.no.12-456-721)

[0815] 4. Greiner Bio-One ThinCert® Tissue Culture Inserts(Greiner Bio-One, cat.no. 10443845)

[0816] 5. Costar 24 well plate (Corning, Cat No.3526)

[0817] 6. Coverslips 12mm (Hampton Research, cat no. HR3-277)

[0818] 7. Bench top centrifuge (Eppendorf, 5417c)

[0819] 8. Biological safety cabinet

[0820] 9. Co2 Incubator (Heraeus)

[0821] 10. Conical tubes 15ml (Falcon, cat no. 352096)

[0822] 11. Conical tubes 50ml (Falcon, cat no. 352070)

[0823] 12. Freezing container (Nalgene, Mr. Frosty)

[0824] 13. Inverted contrasting tissue culture microscope (KL1500CD, Leica)

[0825] 14. Laser confocal microscope (Nikon Eclipse 772)

[0826] 15. Pipette boy (Drummond)

[0827] 16. Pipettes (Gilson)

[0828] 17. Automated cell counter (BioRad, TC20)

[0829] 18. Serological pipettes (Fisher, Cat No, 13-678-1 ID, 13-676-10J, 13-676-10R)

[0830] 19. Stericup 0.22 m Filter unit (Millipore, 03290)

[0831] 20. Sterile filter pipette tips (Genesee Scientific, 24-815, 24-804,24-830, 26-401)

[0832] 21. Sterile Microcentrifuge tubes (Fisher, Cat No.05-408-120)

[0833] 22. Aggrewell® 800 24-well,5pk (Stem cell technologies, Cat No.34815)

[0834] Coverslip cleaning method:

[0835] • Shake coverslips in xylenes in 50mL conical tube for 2-3 hours.

[0836] • Discard xylenes and rinse with acetone. Discard acetone and rinse in fresh acetone in 50 mL conical tube for 2-3 hours.

[0837] • Discard acetone. Autoclave in water if desired on liquid cycle. Rinse coverslips with 100% ethanol 2times. Store coverslips in 100% ethanol at 4°C.

[0838] Dav -1 Plating the ESC in Single cell suspension for Differentiation to improve reproducibility.

[0839] Coating plates for Cell pellet (RNA seq analysis) or staining of coverslip. • Dry the coverslips at room temperature until all the alcohol evaporates completely in the Biosafety cabinet hood under sterile conditions. After drying, transfer the coverslip with forceps to 24 or 4 well plate.

[0840] • Coat the 24 or 4 well plate with Matrigel for 1 hour before plating the cells.

[0841] Single cell suspension for Monolayer

[0842] • Clean any differentiating colonies from cultures colonies on 60mm petri plates.

[0843] • Wash with 2ml DPBS buffer.

[0844] • Add 0.5 ml of lx accutase and place in incubator for 5-10 min.

[0845] • Add 2 - 5 ml of DMEM / F12 media to stop the dissociation.

[0846] • Pellet cells by centrifuging for 3min at 1000 rpm.

[0847] • Resuspended the pellet in 5 ml of mTeSR media with 10 pM (Y27632) Rock inhibitor and count the cells.

[0848] • Plate 10,000 cells per well onto Matrigel coated 24 well plate.

[0849] Dav 0 ESC cell collection as a control:

[0850] • Cells used to start differentiation.

[0851] • Remove mTeSR media with Rock inhibitor and add 0.5 ml of APEL2 media + CHIR 8pM.

[0852] Media changed every 2 days.

[0853] Day 2 Change media with APEL2 media + CHIR 8 p M.

[0854] Dav 4; Presomitic Mesoderm

[0855] Start FGF9 Phase

[0856] • Change media with 200 ng / ml FGF9 + 1 pg / ml Heparin.

[0857] • Change the media every two days.

[0858] Day 6 Change Media with 200 ng / ml FGF9 + 1 pg / ml Heparin.

[0859] On Dav 7 (Intermediate Mesoderm)

[0860] • Change media to (APEL ± 0.1 pM RA + 100 nM Insulin + 17 nM IGF1 + 500 ng / ml PGD2 ± 200 ng / ml FGF9). On Dav 8

[0861] • Dissociate the in vitro differentiated cells into single cells.

[0862] • Wash cells with 0.5 ml of DPBS buffer for each well of 24 well plate.

[0863] • Add 0.5 ml of lx accutase and place in incubator for 3 - 5 min and add 1ml of DMEM / F12 to stop reaction.

[0864] • Centrifuge at 1000 rpm for 3 min.

[0865] • Aspirate the media.

[0866] • Resuspended the cells with APEL media with corresponding growth factors with 10pm (Y27632) Rock inhibitor and count the cells.

[0867] • Add lOOul of (90% somatic cells and 10% germ cells with total of 30,000) cells per well with APEL media with corresponding growth factors and mix up and down slowly and plate the cells into U-Bottom Microplates

[0868] • Centrifuge U-bottom Microplates at 200g for 3min.

[0869] • Observe under the microscope to see all the cells are clustered together.

[0870] Day 9

[0871] • Change media to APEL ± 0.1 pM RA + 100 nM Insulin + 17 nM IGF1 + 500 ng / ml PGD2 + 200 ng / ml FGF9+ IpM Testosterone ± lOOng / ml Follicle stimulating hormone ± 200ng / ml Luteinizing Hormonei 50ng / ml EGF. Change media every other day.

[0872] Dav 12: Transfer the Organoids into ThinCert® plates

[0873] Combine the at least 5 organoids and transfer them into ThinCert® plates with 200ul wide pipette.

[0874] • Upper chamber receives at least 50pl of will get various combination of growth factors: media will be changed every 2 days.

[0875] • Lower chamber receives at least 700ul of will get various combination of growth factors: media will be changed every 2 days.

[0876] • APEL ± 0.1 pM RA + 100 nM Insulin + 17 nM IGF1 + 500 ng / ml PGD2± 200 ng / ml FGF91pM Testosterone ± lOOng / ml Follicle stimulating hormone ± 200ng / ml Luteinizing Hormonei lpM50ng / ml EGF.

[0877] Day 13 • Upper chamber receives at least 50ul of will get various combination of growth factors: media will be changed every 2 days.

[0878] • Lower chamber receives at least 700ul of will get various combination of growth factors: media will be changed every 2 days.

[0879] • APEL ± 0.1 pM RA + 100 nM Insulin + 17 nM IGF1 + 500 ng / ml PGD2± 200 ng / ml FGF91pM Testosterone ± lOOng / ml Follicle stimulating hormone ± 200ng / ml Luteinizing Hormonei 50ng / ml EGF.

[0880] Day 16

[0881] • Collect organoids from required number of wells on day 16 for RNA. Snap freeze in Liquid N2 and store at -80°C until later use.

[0882] • Fix the some of the wells of organoids 4% PFA for 20 min and wash 3times with DPBS.

[0883] • Incubate in the 30% sucrose at 4°C for 72hours. After incubation remove sucrose, wash with DPBS buffer for 3 times for 5min and store in DBFS buffer at 4°C until staining.

[0884] Staining Protocol for Whole Mount Organoid

[0885] • Fix the cells in 4% PFA for 10 min.

[0886] • Wash with PBS- / - for 5 min for 3 times.

[0887] • Incubate in the 30% sucrose at 4°C for 72 hours. After incubation remove sucrose, wash with DPBS buffer for 3 times for 5 min and store in DBPS buffer at 4°C until staining.

[0888] • Quick wash with PBS- / - before staining for 3 times.

[0889] • Blocking buffer PBS+ / +,0.1% Triton -x 100, 10% Donkey serum or goat serum and 10% Na-Azide

[0890] • (8.85 ml PBS+ / +, 50 pl Triton-X 100 + 1 ml Donkey serum or Goat serum-i- 100 pl Na-Azide).

[0891] • Add 50 - 100 pl of blocking buffer in 96 well plate or 0.5 ml tube and incubate for 2 - 3hour at room temperature on shaker.

[0892] • Add 50 - 100 pl of Primary Antibody in 96 well plate or 0.5 ml tube and incubate at 4°C for overnight on shaker.

[0893] • Dilute the blocking buffer to 50% for primary antibody buffer (PBS+ / +, 0.25% Triton-X, 2.5% Donkey serum or Goat serum + 50 pl Na-Azide) or Solution 1.

[0894] • Next day wash the slides with PBS- / - & 0. 1% Triton-X for 10 min for 6 times each. • Add 50 - 100 pl of Secondary Antibody in 96 well plate or 0.5 ml tube along with DAPI for 4 °C for overnight on shaker.

[0895] • (8.85 ml PBS+ / +, 1 ml Donkey serum or Goat serum + 100 pl Na-Azide) or Solution 2.

[0896] • Wash with PBS- / - for 10 min for 3 times each.

[0897] • Mount on coverslip with Vectashield or prolong gold and let it dry and store at 4°C until imaging.

[0898] ICC protocol for OCT sections for Tissue and Organoid

[0899] • Section the Organoid 8-12um and frozen in -80°C

[0900] • Take the slides out of the freezer. Let them sit for 10 min.

[0901] • Post- fix the slides with 4% PFA (on the slides) for 10 min.

[0902] • Wash slides with PBS 3x 5 min.

[0903] • Draw circles on the slides.

[0904] • Penetrate the tissues with 0.1 % Triton X- 100 / PBS for 15 min.

[0905] • Block the slides in blocking solution (200 pl 2.5M glycine / 900 pl PBS / 3% BSA) for Ihr.

[0906] • Dilute primary antibodies in solution I and incubate the slides with the antibodies at 4°C overnight.

[0907] • Wash the slides in PBST 4x 15 min.

[0908] • Incubate the slides with Solution II secondary antibodies for 2 hours at room temp.

[0909] • Wash with PBS 4x 15 min.

[0910] • Mount with Vectashield or prolong gold.

[0911] Differentiation into presomatic mesoderm. Intermediate Mesoderm and then into Peritubular Myoid Like cells.

[0912] A schematic of this protocol is provided as FIG. 8.

[0913] Reagents

[0914] Media and supplements

[0915] 1 . STEMdiff APEL Medium (Stem Cell Technologies, cat. no. 05270 or 05275)

[0916] 2. DMEM / F-12 (Thermo Fisher Scientific, cat. no. 11320-082)

[0917] 3. DMSO (Sigma Aldrich, cat. no. D5879) 4. Dulbecco's phosphate-buffered saline (DPBS) (Thermo Fisher Scientific, Cat. no.14190- 144)

[0918] 5. Accutase (Cat. no. 07920)

[0919] 6. 0.5M EDTA stock (Lonza 51201).

[0920] 7. Quick-RNA Miniprep Kit (Zymo Research, Cat No. R1055).

[0921] 8. Anti -Adherence Rinsing Solution (Stem cell technologies, Cat No.07010).

[0922] 9. Solution 1 for Primary Antibodies (EMD Millipore, Cat No.KP31812-200ml)

[0923] 10. Solution 2 for Secondary Antibodies (EMD Millipore, Cat No.KP31855-200ml)

[0924] 11. 0.5M EDTA stock (Lonza 51201)

[0925] 12. Rinsing Solution

[0926] 13. CHIR99021 (R&D, cat. no. 9902): Stock Solution (10 mM) — Centrifuge the tube briefly before opening. Reconstitute 10 mg of CHIR99021 into 2.149 ml of DMSO to make 10 mM stock. Prepare 20 pl aliquots and label tubes as “Ch” store at -20°C. The thawed tube can be kept at 4°C for 24hr.

[0927] 14. Y27632 ROCK inhibitor (Enzo, Cat. no. ALX-270-333): Stock Solution (5mM) — Dissolve Img in 625 pl sterile TC water Prepare 20 pl aliquots and label tubes as “Y”. Store at -20°C at stem cell core. Dilute in lOmL culture medium for final lOpM. Discard the tube after use.

[0928] 15. Heparin (Sigma Aldrich, cat. no. H4784-250 mg): Stock solution (1 mg ml_l) — Reconstitute to 1 mg ml-' in ultrapure water and filter through polyether sulfone (PES) 0.22 pm filter (syringe-driven filter unit). Prepare 40pl aliquots and store at 4°C for up to 12 months.

[0929] 16. FGF9 (R&D, cat. no. 273-F9-025): Stock Solution FGF9 (100 pg / ml) — Centrifuge the tube briefly before opening. Reconstitute to 100 pg / ml in filtered DPBS containing 0.1% (wt / vol) Bovine serum albumin. Prepare 20 pl aliquots and store them at -80°C for up to 6 months. Thawed FGF9 aliquot can be stored at 4°C for up to 2 weeks.

[0930] 17. RA (Sigma, cat. no. R2625-5 mg): Stock Solution (0.5Mm) — Dissolve Img of RA dissolve in 3 ml of the DMSO to make 1.11 mM Stock Solution. Dilute the stock solution to 0.5 mM, prepare 50pl or lOOpl aliquots and this working solution can be stored at -20°C. Use 0.5 mM of stock solution to the cell culture media according to the experimental desired concentrations. Discard the tube after use.

[0931] 18. Insulin (Sigma, cat. no. 19278): Stock Solution (1.7mM) from Vendor. 19. IGF1-50 pg (Sigma, Cat. no. 13769): Stock solution (lOMm) — 50 pg of IGF1 is dissolved in 658 pl of 0.2% acetic acid to a stock concentration of 10 mM. Prepare lOpl aliquots and store at -80°C. Dilute the stock solution to a concentration of 100 pM in 0.2% acetic acid and add to the cell culture media according to the experimental desired concentrations. The thawed tube can be stored at 4°C for up to 1 month.

[0932] 20. PGD2 (Cayman, cat. no.12010): Stock Solution (2mg / ml) — Dissolve PGD2 in 200pl PBS pH 7.4 to a stock concentration of 2mg / ml, prepare 5 pl aliquots and store at -80°C. Discard the tube after use.

[0933] 21. Matrigel (Corning, cat. no. 354234, Lot no. 9133008): Stock Solution (2mg / ml or Img / ml) — Dilute the Matrigel to desired concentration 2mg / ml or 1 mg / ml in DMEM / F12 media and prepare aliquots and store at -20°C until use.

[0934] 22. SAG (Millipore, cat.no.566661): Stock Solution(0.5mM). Prepare 5ul of aliquots and store at -20 °C.

[0935] 23. PDGFAA (Sigma, cat.no. SRP3228-10ug): Stock Solution(10ug / ml)- Dissolve lOug in 1ml of 0.1%BSA. Prepare 50ul of aliquots and store at -20°C.

[0936] 24. PDGFBB (Sigma, cat.no. SRP3229-10ug): Stock Solution(10ug / ml)-Dissolve lOug in 1ml of 0.1%BSA. Prepare 50ul of aliquots and store at -20°C.

[0937] 25. Valproic acid (Sigma, cat.no. P4543-10G): Stock Solution (ImM)-Dissolve 1.6619mg in 1ml of Sterile H20.Prepare 50ul of aliquots and store at-80°C.

[0938] 26. BMP2 (R&D, cat.no. 355-BM-050): Stock Solution(50ug / ml)-Dissolve 50ug in 1ml Sterile 4mM HCL containing 0.1%BSA and Store 50ul aliquots at 80°C.

[0939] 27. BMP4 (R&D: cat no. 314-BP-500): Stock Solution (200 pg / ml)- Dissolve 200 pg / mL in sterile 4 mM HC1 containing 0.1% BSA and store 50ul aliquots at -80°C until use.

[0940] 28. Activin A (R&D, cat.no. 338-AC-050 / CF). Stock Solution(50ug / ml)- Dissolve 50ug in 1ml Sterile 4mM HCL and Store 50ul aliquots at -80°C.

[0941] Materials

[0942] 1. Nunc™ 4-Well Dishes for IVF (Fisher, cat no. 144444)

[0943] 2. Nunc™ Cell-Culture Treated Multidishes - 24well (Fisher, cat. no. 142475)

[0944] 3. Ultra-Low Binding, U-Shaped-Bottom Microplate (Coming, cat.no.12-456-721)

[0945] 4. Greiner Bio-One ThinCert® Tissue Culture Inserts (Greiner Bio-One, cat.no. 10443845)

[0946] 5. Costar 24 well plate (Corning, Cat No.3526)

[0947] 6. Costar 6 well plate (Corning, Cat No.3506)

[0948] 7. Coverslips 12mm (Hampton Research, cat no. HR3-277) 8. Bench top centrifuge (Eppendorf, 5417c)

[0949] 9. Biological safety cabinet

[0950] 10. Co2 Incubator (Heraeus)

[0951] 11. Conical tubes 15ml (Falcon, cat no. 352096)

[0952] 12. Conical tubes 50ml (Falcon, cat no. 352070)

[0953] 13. Freezing container (Nalgene, Mr. Frosty)

[0954] 14. Inverted contrasting tissue culture microscope (KL1500CD, Leica)

[0955] 15. Laser confocal microscope (Nikon Eclipse 772)

[0956] 16. Pipette boy (Drummond)

[0957] 17. Pipettes (Gilson)

[0958] 18. Automated cell counter (BioRad, TC20)

[0959] 19. Serological pipettes (Fisher, Cat No, 13-678-1 ID, 13-676-10J, 13-676-10R)

[0960] 20. Stericup 0.22pm Filter unit (Millipore, 03290)

[0961] 21. Sterile filter pipette tips (Genesee Scientific, 24-815, 24-804,24-830, 26-401)

[0962] 22. Sterile Microcentrifuge tubes (Fisher, Cat No.05-408-120)

[0963] 23. Aggrewell® 800 24-well,5pk (Stem cell technologies, Cat No.34815)

[0964] Prepare cells as described in ESC or IPSCs

[0965] On Dav 7 (Intermediate Mesoderm)

[0966] • Remove fgf9 and heparin and add:

[0967] • APEL + 0.1 pM RA ± 100 nM Insulin ± 17 nM IGF1 + 500 ng / ml PGD2± 200 ng / ml FGF9.

[0968] On Dav 8

[0969] • Dissociate the remaining wells of differentiated cells into single cells.

[0970] • Wash cells with 0.5 ml of DPBS buffer for each well of 24 well plate

[0971] • Add 0.5 ml of lx accutase and placed in incubator for 3 - 5 min and add 1ml of DMEM / F12 to stop reaction.

[0972] • Centrifuge at 1000 rpm for 3 min.

[0973] • Aspirate the media.

[0974] • Resuspended the cells with APEL media with corresponding growth factors with 10pm (Y27632) Rock inhibitor and count the cells. • Add 3 ml of (5x 105) cells per well with APEL media with corresponding growth factors. o Change media every other day with APEL ± 0.5 pM SAG ±10 nM Valproic acid ± lOng / ml Activin A ± 10 ng / ml BMP2 ± 10 ng / ml BMP4 ± 10 ng / ml PDGFAA ± 10 ng / ml PDGFBB ±0.1%BSA.

[0975] Day 12

[0976] • Change the media once every two days with growth factors. o APEL ± 0.5 pM SAG ±10 nM Valproic acid ± lOng / ml Activin A ± 10 ng / ml BMP2 ± 10 ng / ml BMP4 ± 10 ng / ml PDGFAA ± 10 ng / ml PDGFBB ±0.1%BSA.

[0977] Day 14

[0978] • Change the media for every two days with growth factors. o APEL ± 0.5 pM SAG ±10 nM Valproic acid ± lOng / ml Activin A ± 10 ng / ml BMP2 ± 10 ng / ml BMP4 ± 10 ng / ml PDGFAA ± 10 ng / ml PDGFBB ±0.1%BSA.

[0979] Day 16

[0980] • Collect Cells for RNA.

[0981] • Wash the cells with 0.5ml DBPS.

[0982] • Add 0.5 ml of lx accutase and placed in incubator for 3 - 5 min and add 1ml of DMEM / F12 to stop reaction.

[0983] • Centrifuge at 10,000g for 2 min.

[0984] • Aspirate the media.

[0985] • Snap freeze in Liquid N2 and store at -80°C until later use (Leydig like cells).

[0986] • Fix the some of the wells with coverslips covered cells 4% PFA for 10 min and wash 3times with DPBS and store in DBPS buffer at 4°C until staining.

[0987] Coverslip Staining Protocol for Monolayer

[0988] • Fix the cells in 4% PFA for 20 min.

[0989] • Wash with PBS- / - for 5 min for 3 times and store in PBS- / - at 4°C until staining.

[0990] • Quick wash with PBS- / - before staining for 3 times.

[0991] • Blocking buffer PBS+ / +, 0.1% Triton -x 100, 10% Donkey serum or goat serum and 10% Na-Azide • (8.85ml PBS+ / +, 50 l Triton-X 100 + 1 ml Donkey serum or Goat serum+ 100 pl Na- Azide).

[0992] • Add 200 pl of blocking buffer in 24 well plate and incubate for 1 hour in a covered humidity chamber at room temperature.

[0993] • Add 200 pl of Primary antibody in 24 well plate and incubate in a covered humidity chamber at 4 °C for overnight.

[0994] • Dilute the blocking buffer to 50% for primary antibody buffer (PBS+ / +, 0.25% Triton-X, 2.5% Donkey serum or Goat serum + 50 pl Na-Azide).

[0995] • Next day wash the slides with PBS- / - & 0.1% Triton-X for 10 min for 3 times each.

[0996] • Add 200 pl of Secondary Ab along with DAPI for 1.5 hour at room temperature in a covered humidity chamber.

[0997] • (8.85ml PBS+ / +, 1 ml Donkey serum or Goat serum + 100 pl Na-Azide)

[0998] • Wash with PBS- / - for 10 min for 3 times each.

[0999] • Mount on coverslip with Vectashield or prolong gold and dry it out and store at 4°C until Imaging.

[1000] Differentiation into presomatic mesoderm. Intermediate Mesoderm and then into Levdiglike cells.

[1001] A schematic of this protocol is provided as FIG. 9.

[1002] Reagents

[1003] Media and supplements

[1004] 1 . STEMdiff APEL Medium (Stem Cell Technologies, cat. no. 05270 or 05275)

[1005] 2. DMEM / F-12 (Thermo Fisher Scientific, cat. no. 11320-082)

[1006] 3. DMSO (Sigma Aldrich, cat. no. D5879)

[1007] 4. Dulbecco's phosphate-buff ered saline (DPBS) (Thermo Fisher Scientific, Cat. no. 14190- 144)

[1008] 5. Accutase (Cat. no. 07920)

[1009] 6. 0.5M EDTA stock (Lonza 51201).

[1010] 7. Quick-RNA Miniprep Kit (Zymo Research, Cat No. R1055).

[1011] 8. Anti -Adherence Rinsing Solution (Stem cell technologies, Cat No.07010).

[1012] 9. Solution 1 for Primary Antibodies (EMD Millipore, Cat No.KP31812-200ml) 10. Solution 2 for Secondary Antibodies (EMD Millipore, Cat No.KP31855-200ml)

[1013] 11. 0.5M EDTA stock (Lonza 51201)

[1014] 12. Rinsing Solution

[1015] 13. CHIR99021 (R&D, cat. no. 9902): Stock Solution (10 mM) — Centrifuge the tube briefly before opening. Reconstitute 10 mg of CHIR99021 into 2.149 ml of DMSO to make 10 mM stock. Prepare 20 pl aliquots and label tubes as “Ch” store at -20°C. The thawed tube can be kept at 4°C for 24hr.

[1016] 14. Y27632 ROCK inhibitor (Enzo, Cat. no. ALX-270-333): Stock Solution (5mM) — Dissolve Img in 625 pl sterile TC water Prepare 20 pl aliquots and label tubes as “Y”. Store at -20°C at stem cell core. Dilute in lOmL culture medium for final lOpM. Discard the tube after use.

[1017] 15. Heparin (Sigma Aldrich, cat. no. H4784-250 mg): Stock solution (1 mg — Reconstitute to 1 mg ml- ' in ultrapure water and filter through polyether sulfone (PES) 0.22 pm filter (syringe-driven filter unit). Prepare 40pl aliquots and store at 4°C for up to 12 months.

[1018] 16. FGF9 (R&D, cat. no. 273-F9-025): Stock Solution FGF9 (100 pg / ml) — Centrifuge the tube briefly before opening. Reconstitute to 100 pg / ml in filtered DPBS containing 0.1% (wt / vol) Bovine serum albumin. Prepare 20 pl aliquots and store them at -80°C for up to 6 months. Thawed FGF9 aliquot can be stored at 4°C for up to 2 weeks.

[1019] 17. RA (Sigma, cat. no. R2625-5 mg): Stock Solution (0.5Mm) — Dissolve Img of RA dissolve in 3 ml of the DMSO to make 1.11 mM Stock Solution. Dilute the stock solution to 0.5 mM, prepare 50pl or lOOpl aliquots and this working solution can be stored at -20°C. Use 0.5 mM of stock solution to the cell culture media according to the experimental desired concentrations. Discard the tube after use.

[1020] 18. Insulin (Sigma, cat. no. 19278): Stock Solution (1.7mM) from Vendor.

[1021] 19. IGF1-50 pg (Sigma, Cat. no. 13769): Stock solution (lOMm) — 50 pg of IGF1 is dissolved in 658 pl of 0.2% acetic acid to a stock concentration of 10 mM. Prepare lOpl aliquots and store at -80°C. Dilute the stock solution to a concentration of 100 pM in 0.2% acetic acid and add to the cell culture media according to the experimental desired concentrations. The thawed tube can be stored at 4°C for up to 1 month.

[1022] 20. PGD2 (Cayman, cat. no.12010): Stock Solution (2mg / ml) — Dissolve PGD2 in 200pl PBS pH 7.4 to a stock concentration of 2mg / ml, prepare 5p 1 aliquots and store at -80°C. Discard the tube after use. 21. Matrigel (Coming, cat. no. 354234, Lot no. 9133008): Stock Solution (2mg / ml or Img / ml) — Dilute the Matrigel to desired concentration 2mg / ml or 1 mg / ml in DMEM / F12 media and prepare aliquots and store at -20°C until use.

[1023] 22. SAG (Millipore, cat.no.566661): Stock Solution(0.5mM). Prepare 5ul of aliquots and store at -20°C.

[1024] 23. PDGFAA (Sigma, cat.no. SRP3228-10ug): Stock Solution(10ug / ml)- Dissolve lOug in 1ml of 0.1%BSA. Prepare 50ul of aliquots and store at -20°C.

[1025] 24. PDGFBB (Sigma, cat.no. SRP3229-10ug): Stock Solution(10ug / ml)-Dissolve lOug in 1ml of 0.1%BSA. Prepare 50ul of aliquots and store at -20°C.

[1026] 25. LiCh(Sigma, cat.no. L7026-100ml): Stock Solution (8M) from Vendor. Store at room temp.

[1027] 26. Basic FGF2(Proteintech, cat.no. HZ-1285): Stock Solution(10ug / ml)-Dissolve lOug in 1 ml 0.2%BSA DPBS and Store 50ul aliquots at -20°C

[1028] 27. DAPT (Stem Cell Technologies, cat.no. 72082). Stock Solution(lOmM)- Dissolve 5mg of DAPT in 1.16ml of DMS0. Prepare 50ul of aliquots and store at -80°C.

[1029] Materials

[1030] 1. Nunc™ 4-Well Dishes for IVF (Fisher, cat no. 144444)

[1031] 2. Nunc™ Cell-Culture Treated Multidishes - 24well (Fisher, cat. no. 142475)

[1032] 3. Uitra-Low Binding, U-Shaped-Bottom Microplate(Corning,cat.no.l2-456-721)

[1033] 4. Greiner Bio-One ThinCert® Tissue Culture Inserts (Greiner Bio-One, cat.no. 10443845)

[1034] 5. Costar 24 well plate (Corning, Cat No.3526)

[1035] 6. Costar 6 well plate (Corning, Cat No.3506)

[1036] 7. Coverslips 12mm (Hampton Research, cat no. HR3-277)

[1037] 8. Bench top centrifuge (Eppendorf, 5417c)

[1038] 9. Biological safety cabinet

[1039] 10. Co2 Incubator (Heraeus)

[1040] 11. Conical tubes 15ml (Falcon, cat no. 352096)

[1041] 12. Conical tubes 50ml (Falcon, cat no. 352070)

[1042] 13. Freezing container (Nalgene, Mr. Frosty)

[1043] 14. Inverted contrasting tissue culture microscope (KL1500CD, Leica)

[1044] 15. Laser confocal microscope (Nikon Eclipse Ti2)

[1045] 16. Pipette boy (Drummond)

[1046] 17. Pipettes (Gilson)

[1047] 18. Automated cell counter (BioRad, TC20) 19. Serological pipettes (Fisher, Cat No, 13-678-1 ID, 13-676-10J, 13-676-10R)

[1048] 20. Stericup 0.22pm Filter unit (Millipore, 03290)

[1049] 21. Sterile filter pipette tips (Genesee Scientific, 24-815, 24-804,24-830, 26-401)

[1050] 22. Sterile Microcentrifuge tubes (Fisher, Cat No.05-408-120)

[1051] 23. Aggrewell® 800 24-well,5pk (Stem cell technologies, Cat No.34815)

[1052] Follow the same protocol as ESC or IPSC to day6.

[1053] On Dav 7 (Intermediate Mesoderm)

[1054] • Remove FGF9 media and add APEL ± 0.1 pM RA ± 100 nM Insulin ± 17 nM IGF1 ± 500 ng / ml PGD2± 200 ng / ml FGF9.

[1055] On Dav 8

[1056] Differentiated Cells to Leydig Cells

[1057] Preparation of cells for Plating

[1058] • Dissociate the in vitro derived gonadal progenitor cells.

[1059] • Wash cells with 0.5 ml of DPBS buffer for each well of 24 well plate.

[1060] • Add 0.5 ml of lx accutase and placed in incubator for 3 - 5 min and add 1ml of DMEM / F12 to stop reaction.

[1061] • Centrifuge at 1000 rpm for 3 min.

[1062] • Aspirate the media.

[1063] • Resuspended the cells with APEL media with corresponding growth factors with 10pm (Y27632) Rock inhibitor and count the cells.

[1064] • Add 3 ml of (5x 105) cells per well with APEL media with corresponding growth factors

[1065] • Change media every other day with APEL ± 0.5 pM SAG ± 5mM LiCh ± 5 pM DAPT± 10 ng / ml bFGf2± 10 ng / ml PDGFAA ± 10 ng / ml PDGFBB ± 0.1%BSA.

[1066] Day 12

[1067] • Change the media once every two days with growth factors.

[1068] • APEL ± 0.5 pM SAG ± 5mM LiCh ± 5 pM DAPT± 10 ng / ml bFGf2± 10 ng / ml PDGFAA ± 10 ng / ml PDGFBB ± 0.1%BSA.

[1069] Day 14

[1070] • Change the media for every two days with growth factors • APEL + 0.5 pM SAG ± 5mM LiCl2± 5 pM DAPT+ 10 ng / ml bFGf2± 10 ng / ml PDGFAA ± 10 ng / ml PDGFBB ± 0.1%BSAr

[1071] Day 16

[1072] • Collect Cells for RNA.

[1073] • Cells were washed with 0.5 ml of DPBS buffer for each well of 24 well plate.

[1074] • Add 0.5 ml of lx accutase and placed in incubator for 3 - 5 min and add 1ml of DMEM / F12 to stop reaction.

[1075] • Centrifuge at 10,000g for 2 min.

[1076] • Aspirate the media.

[1077] • Snap freeze in Liquid N2 and store at -80°C until later use (Leydig like cells).

[1078] • Fix the some of the wells Coverslips covered cells 4% PFA for 10 min and wash 3 times with DPBS and store in DBPS buffer at 4°C until staining.

[1079] Coverslip Staining Protocol for Monolayer

[1080] • Fix the cells in 4% PFA for 20 min.

[1081] • Wash with PBS- / - for 5 min for 3 times and store in PBS- / - at 4°C until staining.

[1082] • Quick wash with PBS- / - before staining for 3 times.

[1083] • Blocking buffer PBS+ / +, 0.1% Triton -x 100, 10% Donkey serum or goat serum and 10% Na- Azide

[1084] • (8.85ml PBS+ / +, 50 pl Triton-X 100 + 1 ml Donkey serum or Goat serum-i- 100 pl Na-Azide).

[1085] • Add 200 pl of blocking buffer in 24 well plate and incubate for Ihour in a covered humidity chamber at room temperature.

[1086] • Add 200 pl of Primary antibody in 24 well plate and incubate in a covered humidity chamber at 4 °C for overnight.

[1087] • Dilute the blocking buffer to 50% for primary antibody buffer (PBS+ / +, 0.25% Triton-X, 2.5% Donkey serum or Goat serum + 50 pl Na-Azide).

[1088] • Next day wash the slides with PBS- / - & 0.1% Triton-X for 10 min for 3times each.

[1089] • Add 200 pl of Secondary Ab along with DAPI for 1.5 hour at room temperature in a covered humidity chamber.

[1090] • (8.85ml PBS+ / +, 1 ml Donkey serum or Goat serum + 100 pl Na-Azide)

[1091] • Wash with PBS- / - for 10 min for 3times each. • Mount on coverslip with Vectashield or prolong gold and dry it out and store at 4°C until Imaging.

[1092] RESULTS AND DISCUSSION

[1093] A differentiation protocol that effectively enriched for Sertoli-like cells and testis interstitial cells has been previously described. (See PCT US2023 / 13608, the contents of which are incorporated herein by reference in their entirety). These previously-described protocols have been modified in these examples to improve organoid differentiation. The original protocol concluded at day 13, during which modest expression levels of key Sertoli cell markers such as Gata4, Nr5al, Sox9, and Fshr were observed. Notably, comprehensive single-cell RNA sequencing analysis and a comparative study between in vitro and in vivo derived cells indicated a correlation between the in vitro derived cells and early gonadal and pre-Sertoli cell progenitors, as outlined in PCT US2023 / 13608.

[1094] The differentiation process has been further optimized by introducing IWR1 and EGF1 into the previously established ALL GF cocktail on day 8 of the differentiation timeline. To sustain organoids' growth in the absence of primordial germ cell-like cells (PGCLC), the cells are maintained in ALLGF+ EGF+ TWR1+ Hormone media. It is noteworthy that, upon the introduction of germ cells, IWR1 supplementation is eliminated due to its observed impact on germ cell maturation and differentiation. An additional modification involves the transfer of the organoids to a Trans well® plate system on day 12. This alteration allows cultivation of the organoids at the air-liquid interphase, enabling prolonged culture periods that more accurately emulate the in vivo environment. By implementing this novel approach, a superior model for studying and understanding the intricacies of cell interactions and developmental processes within the testis is achieved.

[1095] The incorporation of IWR1 and EGF1, coupled with the transition to Transwell® plate culture, has yielded a marked increase in the expression of previously absent markers. Notably, these modifications have prompted the induction of key markers within the gonadal cell lineage, exemplified by the upregulation of LHX9. Similarly, Sertoli cell-specific markers such as DHH have been robustly induced, alongside Leydig cell markers STAR and 3BHSD (see FIG. 10: D13 compared to FIG. 11 : D16+ / -Hormone).

[1096] Furthermore, the introduction of primordial germ cell-like cells (PGCLC) into the testicular organoids has significantly bolstered the overall efficiency of gonadal progenitor differentiation, surpassing a remarkable 1,000-fold enrichment. Impressively, Sertoli cell markers have exhibited an induction surpassing 10-fold, encompassing the entire spectrum of identified markers. Additionally, the protocol's refinement has led to the successful induction of Leydig cells.

[1097] These adaptations to the previously described differentiation protocol, in tandem with the inclusion of in vitro derived germ cell-like cells, have collectively culminated in an enhanced determination of all gonadal cell fates. This refined protocol not only amplifies differentiation efficiency, thereby yielding a larger proportion of cells originating from the gonadal lineage, but also elicits the expression of hitherto unseen markers. Particularly noteworthy is the emergence of SRY, a pivotal gene implicated in testis sex determination (see Figure 11).

[1098] In sum, these modifications, and advancements in the differentiation protocol, paired with the integration of in vitro derived germ cell-like cells, have ushered in a new era of improved differentiation efficiency and marker induction. These findings underscore the substantial strides made in the ability to replicate and investigate the intricate processes governing gonadal cell fate determination.

[1099] In vitro derived somatic cells can support PGCLC transition to spermatogonia. Maintaining and differentiating the PGCLC into spermatogonia is an important step for clinical application of in vitro gametogenesis. The data presented herein demonstrates the capability of the in vitro derived cells to effectively nurture human PGCLC, thereby eliciting a reduction in the expression of PGCLC-associated markers such as Soxl7, TFAP2C, and PRDM1. This reduction in marker expression is accompanied by the induction of genes emblematic of spermatogonia, including DDX4, PLZF, DNMT3, and various other pertinent markers.

[1100] These findings underscore the significance of this transformational process, as it showcases the ability to steer the developmental trajectory of PGCLC towards the spermatogonial lineage. This accomplishment not only contributes to a deeper comprehension of the intricate mechanisms governing germ cell differentiation but also holds substantial promise for advancing the prospects of clinical application in the realm of in vitro gametogenesis.

[1101] Example 3

[1102] This example provides an alternative protocol for producing testis like organoids from mouse pluripotent stem cells. A schematic is provided in FIG. 12. In the protocol described in Example 1 above, intermediate mesoderm cells were dissociated and 300,000 cells were mixed with 500 pl of organoid differentiation media and added to a nonadherent Aggrewell® plate. In this protocol, intermediate mesoderm cells (Day4 cells) are dissociated and seeded at 25000 cells in 100 ul of organoid aggregation medium (DMEM / F12, 5% Knockout serum replacement, IX Pyruvate, IX Non-Essential Amino acids, IX Beta-Marketo Ethanol, 1% Matrigel, Y, PGD2, Insulin, IGF, 10 ng / ml FGF9, 20 ng / ml BMP4, 5 ng / ml EGF1, 10 ng / ml Luteinizing Hormone (or 2units / ml HCG), 20 ng / ml Follicle Stimulating Hormone) in a well from a U bottom nonadherent 96 well plate. After adding the lOOul suspension, the 96 well plate is centrifuged at 500 g and incubated in 5% CO2 incubator. The next day after aggregation (day 5), an additional 100 ul of the original organoid aggregation media is added into each well. On day 2 after aggregation, lOOul of old media is removed and lOOul of organoid aggregation medium is added. On day 7, the organoids are transplanted to ThinCert® Transwell® dishes. Organoids are placed in the upper compartment, while the lower compartment is filled with 500ul of organoid differentiation medium (DMEM / F12, 5% Knockout serum replacement, IX Pyruvate, IX Non-Essential Amino acids, IX Beta-Marketo Ethanol, 1% Matrigel, Y, PGD2, Insulin, IGF, 60 ng / ml FGF9, 20 ng / ml BMP4, 5 ng / ml EGF1, 10 ng / ml Luteinizing Hormone (or 2units / ml HCG), 20 ng / ml Follicle Stimulating Hormone). Half media changes are done every other day for the next 10 days. The organoids are imaged and collected at day 10 from ThinCert® cell culture inserts. The collected sample are used for qPCR, sequencing, immunostaining and the media is sent to a mass spectrometry facility to obtain testosterone measurements.

[1103] Using this differentiation schema, mature tubule structures are formed with Sertoli cells, spontaneously specified Leydig cells, peritubular myoid cells, and a few rare endothelial cells as demonstrated by the data in FIG. 13.

[1104] Next to confirm the outcomes of differentiation, the organoids from the two conditions (LH or HCG) were sectioned and protein expression and organization was assayed by immunostaining. See FIG. 14. FIG. 14 includes data for only the HCG treatment, but LH provided the same results.

[1105] To determine whether the Leydig cells present in the culture are functional, media was collected from either the HCG and LH treated organoids and analyzed by Mass Spectrometry to measure testosterone levels. As a control, basal media that has never been exposed to organoids was utilized background subtraction. The results are provided in FIG. 15. Although the HCG results in lower testosterone levels, these organoids self-organize more quickly in culture compared to LH-treated organoids and exhibit more extensive tubule networks. A preferred approach is to treat organoids with HCG for 10 days followed by another 10 days with LH.

[1106] Finally, whether the organoids could support germ cell proliferation was tested. A schematic depiction of the experiment is provided in FIG. 16. It was found that these organoid support germ cell proliferation and differentiation as shown by the data in FIG. 17. In the protocols in Example 1 , the in vivo-derived PND2 germ cells are undifferentiated and lack Stra8 expression. In this example, the organoids show a decrease in the percentage of OCT4+ cells, which are markers of primordial germ cells or prospermatogonia. Concurrently, a transition of these cells into undifferentiated spermatogonia (PLZF+) and / or differentiating spermatogonia (Stra8+) is observed.

[1107] Example 4

[1108] This example provides an alternative protocol for producing testis like organoids from human stem cells. A schematic is provided in FIG. 18.

[1109] 10,000 UCLA6 hESCs were plated on Geltrex-coated 24 well plates in mTESR+ media containing 10 uM Y27632. The following morning, media was changed to APEL2 (StemCell Technologies) containing 3 uM CHIR99021 (day 0) and cells were cultured in this media until day 4. From day 4 through day 7, cells were cultured in APEL2 + 200 ng / ml FGF9 and 1 ug / ml heparin. On day 7, media was changed to APEL2 + 0.1 uM retinoic acid + 17 nM IGF1 + 500 ng / ml PGD2 + 200 ng / ml FGF9 + 5 ng / ml EGF + 20 ng / ml FSH + 10 ng / ml LH + 1 uM testosterone. After 24 hours, cells were dissociated using accutase and 30,000 cells per well were re-aggregated into ultra-low attachment, U-bottom 96 well plates in the same media described above for day 7 + 10 uM Y27632. After 24 hours, fresh media was added (same media as above for day 8 without Y27632). After 3 days, 5-6 organoids were transferred to Thincert® cell culture inserts in a 24 well plate. Organoids were allowed to mature for an additional 11 days before being collected for analysis. This protocol includes the removal of the aggregation step in Aggrewell® plates. Instead, day 8 cells are seeded into a 96-well U-bottom plate. At Day 22, more robust expression levels of many Sertoli cell markers are detected that were previously missing (for example, AMH, SRY, and NR5A1), and improved tubule organization is observed. See FIGs. 19 and 20. REFERENCES:

[1110] 1 Jemal, A. et al. Cancer statistics, 2004. CA Cancer J Clin 54, 8-29, doi:10.3322 / canjclin.54.1.8 (2004).

[1111] 2 Gratwohl, A. et al. Hematopoietic stem cell transplantation: a global perspective. JAMA 303, 1617-1624, doi: 10.1001 / jama.2010.491 (2010).

[1112] 3 Wallace, W. H. Oncofertility and preservation of reproductive capacity in children and young adults. Cancer 117, 2301-2310, doi:10.1002 / cncr.26045 (2011).

[1113] 4 Lambertini, M. et al. Cancer and fertility preservation: international recommendations from an expert meeting. BMC Med 14, 1 , doi : 10.1186 / s 12916-015-0545-7 (2016).

[1114] 5 Nieman, C. L. et al. Fertility preservation and adolescent cancer patients: lessons from adult survivors of childhood cancer and their parents. Cancer Treat Res 138, 201-217, doi: 10. 1007 / 978-0-387-72293- 1_15 (2007).

[1115] 6 Hayashi, K., Ohta, H., Kurimoto, K., Aramaki, S. & Saitou, M. Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 146, 519-532, doi : 10.1016 / j .cell.2011.06.052 (2011 ).

[1116] 7 Hayashi, K. et al. Offspring from oocytes derived from in vitro primordial germ celllike cells in mice. Science 338, 971-975, doi:10.1126 / science.l226889 (2012).

[1117] 8 Ishikura, Y. et al. In vitro reconstitution of the whole male germ-cell development from mouse pluripotent stem cells. Cell Stem Cell, doi: 10. 1016 / j.stem.2021.08.005 (2021).

[1118] 9 Yamashiro, C. et al. Generation of human oogonia from induced pluripotent stem cells in vitro. Science 362, 356-360, doi:10.1126 / science.aatl674 (2018).

[1119] 10 Sosa, E. et al. Differentiation of primate primordial germ cell-like cells following transplantation into the adult gonadal niche. Nat Commun 9, 5339, doi:10.1038 / s41467-018- 07740-7 (2018).

[1120] 11 Hwang, Y. S. et al. Reconstitution of prospermatogonial specification in vitro from human induced pluripotent stem cells. Nat Commun 1 1, 5656, doi:10.1038 / s41467-020- 19350-3 (2020). 12 Stevant, I. et al. Dissecting Cell Lineage Specification and Sex Fate Determination in Gonadal Somatic Cells Using Single-Cell Transcriptomics. Cell reports 26, 3272-3283 e3273, doi:10.1016 / j.celrep.2019.02.069 (2019).

[1121] 13 Stevant, 1. et al. Deciphering Cell Lineage Specification during Male Sex Determination with Single-Cell RNA Sequencing. Cell reports 22, 1589-1599, doi:10.1016 / j.celrep.2018.01.043 (2018).

[1122] 14 Guo, J. et al. Single-cell analysis of the developing human testis reveals somatic niche cell specification and fetal germline stem cell establishment. Cell Stem Cell 28, 764-778 e764, doi: 10.1016 / j.stem.2020.12.004 (2021).

[1123] 15 Luz Garcia- Alonso et al. Single-cell roadmap of human gonadal development. Nature, doi:doi: 10.1038 / s41586-022-04918-4.(2022).

[1124] 16 Shen, Y.-c. et al. TCF21+ mesenchymal cells contribute to testis somatic cell development, homeostasis, and regeneration in mice. Nature Communications 12, 3876, doi: 10. 1038 / s41467-021 -24130- 8 (2021 ) .

[1125] 17 Yokonishi, T., McKey, J., Ide, S. & Capel, B. Sertoli cell ablation and replacement of the spermatogonial niche in mouse. Nat Commun 11, 40, doi: 10.1038 / s41467-019-13879-8 (2020).

[1126] All publications, patents, patent applications and accession numbers mentioned in the above specification are herein incorporated by reference in their entirety. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications and variations of the described compositions and methods of the invention will be apparent to those of ordinary skill in the art and are intended to be within the scope of the following claims.

Claims

Claims1. An in vitro method for production of artificial testis cells from vertebrate pluripotent stem cells comprising: deriving genital ridge cells from pluripotent stem cells; and treating the genital ridge cells with a base medium comprising fibroblast growth factor 9 (FGF9), insulin and / or Insulin-like Growth Factor 1 (IGFl),and Epidermal Growth Factor (EGF) so that the genital ridge cells differentiate into testis cells.

2. The method of claim 1 , wherein the base medium further comprises Prostaglandin D2 (PGD2) and / or retinoic acid (RA).

3. The method of any one of claim 1 to 2, wherein the base medium further comprises Follicle Stimulating Hormone (FSH) and / or luteinizing hormone (LH) or Human Chorionic Gonadotropin (HCG).

4. The method of any one of claims 1 to 3, wherein the base medium further comprises testosterone (T).

5. The method of any one of claims 1 to 4, wherein the base medium further comprises IWR1.

6. The method of claim 1 , wherein the step of deriving genital ridge cells further comprises: providing vertebrate pluripotent stem cells in a maintenance medium comprising a ROCK inhibitor; at day 0, removing the maintenance medium comprising a ROCK inhibitor and culturing the vertebrate pluripotent stem cells with the base medium comprising CHIR99021 so that the vertebrate pluripotent stem cells differentiate into presomitic mesoderm cells; on about day 4, removing the base medium comprising CHIR99021 and culturing the presomitic mesoderm cells in base medium comprising fibroblast growth factor 9 (FGF9) and heparin so that the presomitic mesoderm cells differentiate into intermediate mesoderm cells; andon about day 7 or 8, removing the medium comprising FGF9 and heparin and culturing the cells in a base medium comprising FGF9, insulin and / or IGF1, EGF, RA,PGD2, LH or HCG, FSH and / or T or a base medium comprising insulin and / r IGF1, FGF9, RA, and PDG2 so that the cells differentiate into genital ridge cells.

7. The method of any one of claim 6, wherein the genital ridge cells are treated with the base medium comprising FGF9, insulin and / or IGF1, EGF, LH or HCG, FSH and / or T on about day 8 or 9 to provide gonadogenesis -induced cells.

8. The method of claim 7, wherein the gonadogenesis-induced cells are cultured to induce the formation of organoids.

9. The method of any one of claims 1 to 8, wherein the genital ridge cells are dissociated into single cells on about day 8.

10. The method of any one of claims 1 to 9, further comprising on about day 12 removing organoids from the culture and culturing the organoids at an air- liquid interphase to allow maturation of testis organoids.

11. The method of claim 10, where the testis organoids comprise one or more of artificial Sertoli cells, artificial Leydig cells, artificial myoid cells and artificial stromal cells.

12. The method of claim 10, wherein the testis organoids comprise two or more of artificial Sertoli cells, artificial Leydig cells, artificial myoid cells and artificial stromal cells.

13. The method of claim 10, wherein the testis organoids comprise three or more of artificial Sertoli cells, artificial Leydig cells, artificial myoid cells and artificial stromal cells.

14. The method of claim 10, wherein the testis organoids comprise artificial Sertoli cells, artificial Leydig cells, artificial myoid cells and artificial stromal cells.

15. The method of any one of claims 10 to 14, wherein the organoids demonstrate upregulation or expression of one or more markers selected from the group consisting of LHX9, PDGRA, COUPTFII, TCF21, SOX9, GATA4, SF1, SMA, DHH, STAR and 3BHSD.

16. The method of any one of claims 1 to 12, wherein the vertebrate pluripotent stem cells are human stem cells.

17. The method of claim 16, wherein the human stem cells are human embryonic stem cells.

18. The method of claim 16, wherein the human stem cells are induced pluripotent stem cells.

19. The method of any one of claims 1 to 18, further comprising the step of isolating 1) the artificial testis cells from the organoids or 2) the testis organoid.

20. The method of claim 19, wherein the artificial testis cells are Sertoli-like cells.

21. The method of claim 19, wherein the artificial testis cells are Leydig-like cells.

22. The method of claim 19, wherein the artificial testis cells are Myoid-like cells.

23. The method of claim 19, wherein the artificial testis cells are stromal progenitor cells.

24. The method of any one of claims 19 to 23, further comprising transplanting the isolated artificial testis cells or artificial testis organoid into a mammal.

25. The method of any one of claims 1 to 23, further comprising contacting the artificial testis cells or artificial testis cell organoid with a test reagent and assaying the effect of the test reagent on the artificial testis cells or artificial testis cell organoid.

26. The method of any one of claims 1 to 23, further comprising obtaining stem cells or tissue comprising stem cells from a patient and culturing the stem cells or tissue comprising stem cells from the patient with the artificial testis cells or artificial testis cell organoid to provide differentiated patient stem cells.

27. The method of claim 26, wherein the stem cells are primordial germ cells, pro- spermatogonia or spermatogonial stem cells.

28. The method of claim 27, wherein the primordial germ cell like cells and pro- spermatogonia stem cells differentiate into spermatogonia.

29. The method of any one of claims 26 to 28, further comprising transferring the stem cells or differentiated spermatogonia back to a patient in need thereof.

30. The method of any one of claims 26 to 28, wherein the patient has previously undergone a gonadotoxic treatment and / or has nonobstructive azoospermia or severe oligospermy.

31. The method of claim 30, wherein the gonadotoxic treatment is selected from the group consisting of chemotherapy and radiation.

32. The method of any one of claims 26 to 31 , wherein the stem cells or tissue comprising stem cells are obtained from the patient prior to a gonadotoxic treatment.

33. The method of any one of claims 1 to 18, further comprising on about day 8 coculturing primordial germ cells with the genital ridge cells.

34. The method of claim 33, wherein the primordial germ cells are derived from an embryo.

35. The method of any one of claims 33 to 34, wherein the primordial germ cells are primordial germ cell-like cells.

36. The method of any one of claims 33 to 35, wherein the primordial germ cells differentiate into spermatogonia.

37. The method of claim 36, further comprising isolating the spermatogonia.

38. A cell culture comprising artificial testis cells produced by a method of any one of claims 1 to 23 and 33-37.

39. Isolated artificial testis cells produced by the method of any one of claims 19 to 23.

40. Artificial Leydig cells produced by the method of claim 19.

41. Artificial Sertoli cells produced by the method of claim 19.

42. Artificial Myoid cells produced by the method of claim 19.

43. Artificial stromal progenitor cells produced by the method of claim 19.

44. An artificial testis organoid produced by the method of claim 19.

45. Spermatogonia produced by the method of any one of claims 33 to 37.

46. Differentiated patient stem cells produced by the method of any one of claim 26 to 28 and 30 to 32.

47. An in vitro method for production of artificial Leydig-like cells from vertebrate pluripotent stem cells comprising: deriving genital ridge cells from pluripotent stem cells; and treating the genital ridge cells with a base medium comprising SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growthfactor BB), bFGF2 (Basic fibroblast growth factor), DAPT and LiCL so that the genital ridge cells differentiate into Leydig-like cells.

48. The method of claim 47, wherein the step of deriving genital ridge cells further comprises: providing vertebrate pluripotent stem cells in a maintenance medium comprising a ROCK inhibitor; at day 0, removing the maintenance medium comprising a ROCK inhibitor and culturing the vertebrate pluripotent stem cells with the base medium comprising CHIR99021 so that the vertebrate pluripotent stem cells differentiate into presomatic mesoderm cells; on about day 4, removing the base medium comprising CHIR99021 and culturing the presomatic mesoderm cells in base medium comprising fibroblast growth factor 9 (FGF9) and heparin so that the presomatic mesoderm cells differentiate into intermediate mesoderm cells; and on about day 7 or 8, removing the medium comprising FGF9 and heparin and culturing the cells in a base medium comprising FGF9, insulin and / or IGF1, EGF, RA,PGD2, LH, FSH and / or T or a base medium comprising insulin and / r IGF1, FGF9, RA, and PDG2 so that the cells differentiate into genital ridge cells.

49. The method of any one of claims 47 to 48, wherein the genital ridge cells are treated with the base medium comprising SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), bFGF2 (Basic fibroblast growth factor), DAPT and LiChon about day 8.

50. The method of claim 49, wherein the genital ridge cells are dissociated into single cells.

51. The method of any one of claims 47 to 50, wherein the Leydig-like cells are produced in the culture by about day 16.

52. The method of any one of claims 47 to 51 , wherein the Leydig-like cells demonstrate upregulation or expression of one or more markers selected from the group consisting of STAR and 3BHSD.

53. The method of any one of claims 47 to 52, wherein the vertebrate pluripotent stem cells are human stem cells.

54. The method of claim 53, wherein the human stem cells are human embryonic stem cells.

55. The method of claim 53, wherein the human stem cells are induced pluripotent stem cells.

56. The method of any one of claims 47 to 55, further comprising the step of isolating the Leydig-like cells.

57. The method of claim 56, further comprising transplanting the isolated Leydig-like cells into a mammal.

58. The method of any one of claims 47 to 56, further comprising contacting the Leydig- like cells with a test reagent and assaying the effect of the test reagent on the cells.

59. The method of any one of claims 47 to 56, further comprising obtaining stem cells or tissue comprising stem cells from a patient and culturing the stem cells or tissue comprising stem cells from the patient with the Leydig-like cells.

60. The method of claim 59, further comprising transferring the stem cells or cells differentiated from the stem cells back to a patient in need thereof.

61. Artificial Leydig cells produced by the method of any one of claims 47 to 56.

62. An in vitro method for production of artificial Myoid-like cells from vertebrate pluripotent stem cells comprising: deriving genital ridge cells from pluripotent stem cells; and treating the genital ridge cells with a base medium comprising SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), valproic acid, BMP2 (Bone Morphogenetic Protein 2), BMP4 (Bone Morphogenetic Protein 4), and Activin A so that the genital ridge cells differentiate into Myoid-like cells.

63. The method of claim 62, wherein the step of deriving genital ridge cells further comprises: providing vertebrate pluripotent stem cells in a maintenance medium comprising a ROCK inhibitor;at day 0, removing the maintenance medium comprising a ROCK inhibitor and culturing the vertebrate pluripotent stem cells with the base medium comprising CHIR99021 so that the vertebrate pluripotent stem cells differentiate into presomatic mesoderm cells; on about day 4, removing the base medium comprising CH1R99021 and culturing the presomatic mesoderm cells in base medium comprising fibroblast growth factor 9 (FGF9) and heparin so that the presomatic mesoderm cells differentiate into intermediate mesoderm cells; and on about day 7 or 8, removing the medium comprising FGF9 and heparin and culturing the cells in a base medium comprising FGF9, insulin and / or IGF1, EGF, RA,PGD2, LH, FSH and / or T or a base medium comprising insulin and / r IGF1, FGF9, RA, and PDG2 so that the cells differentiate into genital ridge cells.

64. The method of any one of claims 62 to 63, wherein the genital ridge cells are treated with the base medium comprising SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), valproic acid, BMP2 (Bone Morphogenetic Protein 2), BMP4 (Bone Morphogenetic Protein 4), and Activin A on about day 8.

65. The method of claim 64, wherein the genital ridge cells are dissociated into single cells.

66. The method of any one of claims 62 to 65, wherein the Myoid- like cells are produced in the culture by about day 16.

67. The method of any one of claims 62 to 66, wherein the Myoid-like cells demonstrate upregulation or expression of one or more Myoid cell markers.

68. The method of any one of claims 62 to 67, wherein the vertebrate pluripotent stem cells are human stem cells.

69. The method of claim 68, wherein the human stem cells are human embryonic stem cells.

70. The method of claim 68, wherein the human stem cells are induced pluripotent stem cells.

71. The method of any one of claims 62 to 70, further comprising the step of isolating the Myoid-like cells.1 . The method of claim 71 , further comprising transplanting the isolated Myoid-like cells into a mammal.

73. The method of any one of claims 62 to 71, further comprising contacting the Myoidlike cells with a test reagent and assaying the effect of the test reagent on the cells.

74. The method of any one of claims 62 to 71, further comprising obtaining stem cells or tissue comprising stem cells from a patient and culturing the stem cells or tissue comprising stem cells from the patient with the Leydig-like cells.

75. The method of claim 72, further comprising transferring the stem cells or cells differentiated from the stem cells back to a patient in need thereof.

76. Artificial Myoid cells produced by the method of any one of claims 62 to 71.

77. An in vitro method for production of artificial testis cells from mouse pluripotent stem cells comprising: deriving intermediate mesoderm cells from mouse pluripotent stem cells; and treating the intermediate mesoderm cells with a base medium comprising FGF9, insulin and / or IGF1, PGD2, RA, BMP4 (Bone Morphogenetic Protein 4), and EGF or a base medium comprising FGF9, insulin and / or IGF1, PGD2, RA, BMP4 (Bone Morphogenetic Protein 4), EGF, FSH and LH or Human Chorionic Gonadotropin (HCG) so that the intermediate ridge cells differentiate into testis cells.

78. The method of claim 77, wherein the step of deriving intermediate mesoderm cells further comprises: providing mouse pluripotent stem cells; at day 0, culturing the mouse pluripotent stem cells with the base medium comprising Activin A (AA) and bFGF (Basic Fibroblast Growth Factor); and on about day 2, removing the base medium comprising AA and bFGF and culturing the cells in base medium comprising AA, RA, and BMP4.

79. The method of any one of claims 77 to 78, wherein the cells are treated with the base medium comprising FGF9, insulin and / or IGF1, PGD2, RA, BMP4, and EGF on about day 6 or FGF9, insulin and / or IGF1, PGD2, RA, BMP4, EGF, FSH, and LH or HCG on about day 680. The method of any one of claims 77 to 79, wherein on about day 7 the culture medium is replaced with a Leydig cell differentiation medium comprising a base medium supplemented with SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), bFGF2 (Basic fibroblast growth factor), DAPT and LiCh81. The method of any one of claims 77 to 79, wherein on about day 7 the culture medium is replaced with a Myoid cell differentiation medium comprising a base medium supplemented with SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), valproic acid, BMP2 (Bone Morphogenetic Protein 2), BMP4 (Bone Morphogenetic Protein 4), and Activin A.

82. The method of any one of claims 77 to 81 , wherein testis organoids are formed in the culture by about day 8.

83. The method of claim 82, where the testis organoids comprise one or more of artificial Sertoli cells, artificial Leydig cells, artificial myoid cells and artificial stromal cells.

84. The method of claim 82, wherein the testis organoids comprise two or more of artificial Sertoli cells, artificial Leydig cells, artificial myoid cells and artificial stromal cells.

85. The method of claim 82, wherein the testis organoids comprise three or more of artificial Sertoli cells, artificial Leydig cells, artificial myoid cells and artificial stromal cells.

86. The method of claim 82, wherein the testis organoids comprise artificial Sertoli cells, artificial Leydig cells, artificial myoid cells and artificial stromal cells.

87. The method of any one of claims 82 to 86, wherein the organoids demonstrate upregulation or expression of one or more markers selected from the group consisting of LHX9, PDGRA, COUPTFII, TCF21, SOX9, GATA4, SF1, SMA, DHH, STAR and 3BHSD.

88. The method of any one of claims 77 to 87, wherein the mouse stem cells are mouse embryonic stem cells.

89. The method of any one of claims 77 to 87, wherein the mouse stem cells are induced pluripotent stem cells.

90. The method of any one of claims 77 to 87, further comprising the step of isolating 1) the artificial testis cells from the organoids or 2) the testis organoid.

91. The method of claim 90, wherein the artificial testis cells are Sertoli-like cells.

92. The method of claim 90, wherein the artificial testis cells are Leydig-like cells.

93. The method of claim 90, wherein the artificial testis cells are Myoid-like cells.

94. The method of claim 90, wherein the artificial testis cells are stromal progenitor cells.

95. The method of any one of claims 90 to 92, further comprising transplanting the isolated artificial testis cells or artificial testis organoid into a mammal.

96. The method of any one of claims 77 to 94, further comprising contacting the artificial testis cells or artificial testis cell organoid with a test reagent and assaying the effect of the test reagent on the artificial testis cells or artificial testis cell organoid.

97. A testis organoid produced by the method of any one of claims 77 to 94.

98. Artificial Leydig cells produced by the method of any one of claims 77 to 94.

99. Artificial Sertoli cells produced by the method of any one of claims 77 to 94.

100. Artificial Myoid cells produced by the method of any one of claims 77 to 94.

101. Artificial stromal progenitor cells produced by the method of any one of claims 77 to 94.

102. A method comprising: contacting artificial testis cells or organoids according to any of claims 38-46, 61, 76 and 97-101 with a test reagent; and assaying the effect of the test reagent on the artificial testis cells or organoids.

103. A method comprising: transplanting the artificial testis cells or organoids according to any of claims 38-46, 61, 76 and 97-101 into a subject.

104. A method comprising: obtaining stem cells or tissue comprising stem cells from a patient; andco-culturing the stem cells or tissue comprising stem cells from the patient with artificial testis cells or organoids according to any of claims 38-46, 61, 76 and 97-101.

105. The method of claim 104, wherein the stem cells are selected from the group consisting of primordial germ cell like cells (PGCLC), prospermatogonia, and spermatogonial stem / progenitor cells (SSC / SPCs).

106. The method of claim 105, wherein the pro-spermatogonia stem cells differentiate into spermatogonia.

107. The method of any one of claims 104 to 106, further comprising transferring the spermatogonia back to a patient in need thereof.

108. The method of any one of claims 104 to 107, wherein the patient has previously undergone a gonadotoxic treatment and / or has nonobstructive azoospermia patient or severe oligospermy.

109. The method of claim 108, wherein the gonadotoxic treatment is selected from the group consisting of chemotherapy and radiation.

110. The method of any one of claims 104 to 109, wherein the stem cells or tissue comprising stem cells are obtained from the patient prior to a gonadotoxic treatment.

111. A method of expanding patient or in vitro derived germ cells comprising: co-culturing the patient or in vitro derived germ cells with artificial testis cells or organoids according to any of claims 38-46, 61, 76 and 97-101.

112. The method of claim 111, wherein the patient or in vitro derived germ cells are primordial germ cell-like cells (PGCLCs), prospermatogonia or prospermatogonia-like cells.

113. The method of any one of claims 111 to 112, wherein co-culturing results in licensure of the primordial germ cell-like cells (PGCLCs), prospermatogonia, prospermatogonia-like cells to form spermatogonia.

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