Artificial testicular cells and a method for producing artificial testicular cells

Abstract

The present invention relates to the in vitro method for producing testicular cells (e.g., Sertoli and / or Leydig cells) and related organoids. The testicular cells and testicular-like organoids may be used for therapeutic purposes, including promoting the production of spermatogonial cells from prespermatogonial stem cells.

Description

Detailed description of the invention

[0001] [Cross-reference of related applications] This application claims priority to U.S. Provisional Application 63 / 533,148, filed on 17 August 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] [Field of Invention] This invention relates to the in vitro method for producing testicular cells (e.g., Sertoli and / or Leydig cells) and related organoids.

[0003] [Background of the Invention] Infertility is a rapidly growing global crisis, with approximately 50% of cases attributed to male infertility. One percent of men worldwide of reproductive age (20-50 years) suffer from non-obstructive azoospermia (i.e., a lack of germ cells or mature spermatids). In the United States, approximately 300,000 men fall into this category, most of whom have no known genetic cause. Traditional treatments such as in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) require sperm, leaving these men without treatment options. In addition to adult infertility, approximately 10,000 pre-pubescent boys in the United States develop cancer each year, requiring gonadal toxic treatments such as chemotherapy and radiation therapy (1). Furthermore, approximately 1,000 pediatric patients with hematological and immunological deficiencies, as well as autoimmune diseases, undergo myeloablative conditioning before bone marrow transplantation, which is also gonadal toxic (2). These treatments, involving alkylating chemotherapy, total body irradiation (3), and gonadal radiation (4), damage both the somatic and germ cell populations of the testes, putting patients at significant risk of infertility. However, advances in medicine have cured 85% of these children, and by adulthood they wish to have their own children (5). Since any reproductive therapy requires gametes, developing methods to reconstruct germ cell development in vitro or in vivo for these patients remains a clinical challenge and will continue to be a focus of the reproductive biology community for decades to come. Successful in vitro generation of patient-derived testicular somatic cells would provide novel cell-based therapies for known and unknown hereditary and iatrogenic (i.e., treatment-induced) male infertility. Such therapies could also be applied to restoring endocrine function in aging men, men with hypogonadism, or patients with gender identity disorder. Furthermore, if in vitro-induced cells or cell communities (sometimes called organoids) can replicate the natural process of spermatogenesis, they can be used as a novel experimental system for screening new classes of male contraceptives or environmental reproductive toxicities.

[0004] Currently, the competition in cell-based therapies is focused on the development of germ cell-like progenitor cells known as primordial germ cell-like cells (PGCLCs). PGCLCs have been induced in vitro from mouse and human embryonic stem cells (ESCs), but they cannot be maintained in vitro (6). Only when transplanted into the neonatal testes of mice can these mouse PGCLCs develop into sperm, and these sperm can be used to produce live mouse pups. Furthermore, when female and male mouse PGCLCs were reconstituted in vitro with fetal gonadal tissue, the oogenesis and spermatogenesis programs were successfully reconstituted, but the frequency of live mouse pups obtained from germ cells induced in vitro was approximately 1-3% (6-9). These preliminary findings are exciting and highlight the importance of somatic cells for carrying out the gameteogenesis program. Similar experiments using mouse embryonic gonadal tissue from three experimental groups with human or rhesus macaque PGCLCs showed that when female and male human hPGCLCs or male rhesus macaque PGCLCs were combined with mouse fetal ovarian or testicular tissue, respectively, the PGCLCs initiated differentiation but failed to initiate meiosis (9-11). These observations suggest that the somatic and germ cell sources must be compatible (i.e., allogeneic) to initiate meiosis.

[0005] Using somatic cells derived from human fetal testes to aid the progression of in vitro-induced human PGCLC presents ethical and technical constraints associated with fetal tissue-based research. Therefore, generating testicular somatic cells without relying on fetal tissue is clinically important. To date, attempts have been made to reprogram human fibroblasts into Sertoli-like or Leydig-like cells using carefully selected combinations of transcription factors. Despite expressing several known markers of Sertoli and Leydig cells, it remains difficult to assess how closely in vitro-induced cells resemble in vivo cells or how well they replicate endogenous function. Furthermore, given that gene transfer is necessary for efficient induction and continuous expression of transcription factors is required to maintain the destiny of Sertoli and Leydig cells, the clinical utility of such cells remains unclear.

[0006] [Summary of the Invention] This invention relates to an in vitro method for producing testicular cells and related organoids.

[0007] In a first embodiment, the present invention provides an in vitro method for producing artificial testicular cells from pluripotent stem cells of vertebrates, comprising the steps of: inducing germinal protuberance cells from pluripotent stem cells; and differentiating the germinal protuberance cells into testicular cells by treating them with a basal medium containing fibroblast growth factor 9 (FGF9), insulin and / or insulin-like growth factor 1 (IGF1), and epidermal growth factor (EGF). In some preferred embodiments, the basal medium further comprises prostaglandin D2 (PGD2) and / or retinoic acid (RA). In some preferred embodiments, the basal medium further comprises follicle-stimulating hormone (FSH) and / or luteinizing hormone (LH), or human chorionic gonadotropin (HCG). In some preferred embodiments, the basal medium further comprises testosterone (T). In some preferred embodiments, the basal medium further comprises IWR1.

[0008] In some preferred embodiments, the step of inducing germline cells further includes: providing vertebrate pluripotent stem cells in a maintenance medium containing a ROCK inhibitor; removing the maintenance medium containing the ROCK inhibitor on day 0, and culturing the vertebrate pluripotent stem cells in a basal medium containing CHIR99021 to differentiate the vertebrate pluripotent stem cells into unsegmented mesoderm cells; removing the basal medium containing CHIR99021 on approximately day 4, and adding fibroblast growth factor 9 (FGF9) and A step of culturing the unsegmented mesoderm cells in a basal medium containing valine to differentiate the unsegmented mesoderm cells into intermediate mesoderm cells; a step of removing the medium containing FGF9 and heparin on about the 7th or 8th day; and a step of culturing the cells in a basal medium containing FGF9, insulin and / or IGF1, EGF, RA, PGD2, LH, FSH and / or T, or in a basal medium containing insulin and / or IGF1, FGF9, RA and PDG2 to differentiate the cells into germline cells.

[0009] In some preferred embodiments, the germline cells are treated on about day 8 or 9 with the basal medium containing FGF9, insulin and / or IGF1, EGF, LH, FSH, and / or T to provide gonadal formation-inducing cells.

[0010] In some preferred embodiments, the gonadal formation-inducing cells are cultured to induce organoid formation.

[0011] In some preferred embodiments, the germline cells are dissociated into single cells on approximately day 8.

[0012] In some preferred embodiments, preferably on about 12 days, the method further includes the steps of removing the organoid from the culture and culturing the organoid at the gas-liquid interface to mature the testicular organoid. In some preferred embodiments, the testicular organoid comprises one or more artificial Sertoli cells, artificial Leydig cells, artificial myosophical cells, and artificial stromal cells. In some preferred embodiments, the testicular organoid comprises two or more artificial Sertoli cells, artificial Leydig cells, artificial myosophical cells, and artificial stromal cells. In some preferred embodiments, the testicular organoid comprises three or more artificial Sertoli cells, artificial Leydig cells, artificial myosophical cells, and artificial stromal cells. In some preferred embodiments, the testicular organoid comprises artificial Sertoli cells, artificial Leydig cells, artificial myosophical cells, and artificial stromal cells. In some preferred embodiments, the organoid exhibits 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.

[0013] 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.

[0014] In some preferred embodiments, the method further comprises 1) isolating the artificial testicular cells from the organoid, or 2) isolating the testicular organoid. In some preferred embodiments, the artificial testicular cells are Sertoli-like cells. In some preferred embodiments, the artificial testicular cells are Leydig-like cells. In some preferred embodiments, the artificial testicular cells are myoplasmic cell-like cells. In some preferred embodiments, the artificial testicular cells are stromal progenitor cells.

[0015] In some preferred embodiments, the method further includes the step of transplanting the isolated artificial testicular cells or artificial testicular organoids into a mammal.

[0016] In some preferred embodiments, the method further includes the steps of contacting the artificial testicular cells or artificial testicular cell organoids with a test reagent, and evaluating the effect of the test reagent on the artificial testicular cells or artificial testicular cell organoids.

[0017] In some preferred embodiments, the method further comprises the steps of obtaining stem cells or stem cell-containing tissue from a patient, and culturing the patient-derived stem cells or stem cell-containing tissue with artificial testicular cells or artificial testicular cell organoids 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 spermatogonial cells.

[0018] In some preferred embodiments, the method further includes the step of returning the stem cells or differentiated spermatogonial cells to a patient in need. In some preferred embodiments, the patient has been diagnosed with non-obstructive azoospermia or severe oligozoospermia, or has previously received gonadal toxicity treatment. In some preferred embodiments, the stem cells or stem cell-containing tissue are obtained from the patient prior to gonadal toxicity treatment. In some preferred embodiments, the stem cells or stem cell-containing tissue are obtained from a patient with NOA.

[0019] In some preferred embodiments, the method further comprises the step of co-culturing primordial germ cells with said gonadal ridge cells at about day 8. In some preferred embodiments, said primordial germ cells are derived from an embryo. In some preferred embodiments, said primordial germ cells are primordial germ cell-like cells. In some preferred embodiments, said primordial germ cells differentiate into spermatogonia.

[0020] In some preferred embodiments, the method further comprises the step of isolating said spermatogonia.

[0021] In a second aspect, the present invention provides a cell culture comprising artificial testicular cells produced by the aforementioned method.

[0022] In a third aspect, the present invention provides isolated artificial testicular cells produced by the aforementioned method, preferably isolated artificial Sertoli cells, isolated artificial Leydig cells, isolated artificial myoid cells, and / or isolated artificial interstitial progenitor cells.

[0023] In a fourth aspect, the present invention provides an artificial testicular organoid produced by the aforementioned method.

[0024] In a fifth aspect, the present invention provides spermatogonia produced by the aforementioned method.

[0025] In a sixth aspect, the present invention provides differentiated patient stem cells produced by the aforementioned method.

[0026] In a seventh aspect, the present invention provides an in vitro method for producing artificial Leydig-like cells from pluripotent stem cells of vertebrates, comprising the steps of: inducing germline cells from pluripotent stem cells; and differentiating the germline cells into Leydig-like cells by treating them with a basal medium containing SAG (Smoothened agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), bFGF2 (Basic fibroblast growth factor), DAPT, and LiCl2.

[0027] In some preferred embodiments, the step of inducing germline cells further includes: providing vertebrate pluripotent stem cells in a maintenance medium containing a ROCK inhibitor; on day 0, removing the maintenance medium containing the ROCK inhibitor, and culturing the vertebrate pluripotent stem cells in a basal medium containing CHIR99021 to differentiate the vertebrate pluripotent stem cells into presomatic mesoderm cells; on approximately day 4, removing the basal medium containing CHIR99021, and culturing the presomatic mesoderm cells in a basal medium containing fibroblast growth factor 9 (FGF9) and heparin to differentiate the presomatic mesoderm cells A step of differentiating mesoderm cells into intermediate mesoderm cells; and a step of removing the medium containing FGF9 and heparin on about the 7th or 8th day; and a step of culturing the cells in a basal medium containing FGF9, insulin and / or IGF1, EGF, RA, PGD2, LH, FSH and / or T, or in a basal medium containing insulin and / or IGF1, FGF9, RA and PDG2, to differentiate the cells into germline cells.

[0028] In some preferred embodiments, the germline cells are treated on approximately day 8 with the basal medium containing SAG (Smoothened agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), bFGF2 (Basic fibroblast growth factor), DAPT, and LiCl2. In some preferred embodiments, the germline cells are dissociated into single cells.

[0029] In some preferred embodiments, the Leydig-like cells are produced in the culture by approximately day 16. In some preferred embodiments, the Leydig-like cells exhibit upregulation or expression of one or more markers selected from the group consisting of STAR and 3BHSD.

[0030] 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.

[0031] In some preferred embodiments, the method further includes the step of isolating the Leydig-like cells.

[0032] In some preferred embodiments, the method further includes the step of transplanting the isolated Leydig-like cells into a mammal.

[0033] In some preferred embodiments, the method further includes the steps of contacting the Leydig-like cells with a test reagent and evaluating the effect of the test reagent on the cells.

[0034] In some preferred embodiments, the method further includes the steps of obtaining stem cells or stem cell-containing tissue from a patient, and culturing the patient-derived stem cells or stem cell-containing tissue with Leydig-like cells. In some preferred embodiments, the method further includes the steps of returning the stem cells or cells differentiated from the stem cells to a patient in need.

[0035] In the ninth aspect, the present invention provides artificial Leydig cells produced by the method described above.

[0036] In a tenth embodiment, the present invention provides an in vitro method for producing artificial myoplasmic cell-like cells from pluripotent stem cells of vertebrates, comprising the steps of: inducing germinal protuberance cells from pluripotent stem cells; and differentiating the germinal protuberance cells into myoplasmic cell-like cells by treating them with a basal medium containing 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.

[0037] In some preferred embodiments, the step of inducing germline cells further includes: providing vertebrate pluripotent stem cells in a maintenance medium containing a ROCK inhibitor; on day 0, removing the maintenance medium containing the ROCK inhibitor, and culturing the vertebrate pluripotent stem cells in a basal medium containing CHIR99021 to differentiate the vertebrate pluripotent stem cells into presomatic mesoderm cells; on approximately day 4, removing the basal medium containing CHIR99021, and culturing the presomatic mesoderm cells in a basal medium containing fibroblast growth factor 9 (FGF9) and heparin to differentiate the presomatic mesoderm cells A step of differentiating mesoderm cells into intermediate mesoderm cells; and a step of removing the medium containing FGF9 and heparin on about the 7th or 8th day; and a step of culturing the cells in a basal medium containing FGF9, insulin and / or IGF1, EGF, RA, PGD2, LH, FSH and / or T, or in a basal medium containing insulin and / or IGF1, FGF9, RA and PDG2, to differentiate the cells into germline cells.

[0038] In some preferred embodiments, the germline cells are treated on approximately day 8 with the basal medium containing 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, the germline cells are dissociated into single cells.

[0039] In some preferred embodiments, the myomyocyte-like cells are produced in the culture by approximately day 16. In some preferred embodiments, the myomyocyte-like cells exhibit upregulation or expression of one or more myomyocyte markers.

[0040] 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.

[0041] In some preferred embodiments, the method further includes the step of isolating the myocardium-like cells. In some preferred embodiments, the method further includes the step of transplanting the isolated myocardium-like cells into a mammal.

[0042] In some preferred embodiments, the method further includes the steps of contacting the muscle-like cells with a test reagent and evaluating the effect of the test reagent on the cells.

[0043] In some preferred embodiments, the method further includes the steps of obtaining stem cells or stem cell-containing tissue from a patient, and culturing the patient-derived stem cells or stem cell-containing tissue with Leydig-like cells. In some preferred embodiments, the method further includes the steps of returning the stem cells or cells differentiated from the stem cells to a patient in need.

[0044] In the eleventh embodiment, the present invention provides artificial muscle-like cells produced by the method described above.

[0045] In a twelfth embodiment, the present invention provides an in vitro method for producing artificial testicular cells from mouse pluripotent stem cells, comprising the steps of: inducing intermediate mesoderm cells from mouse pluripotent stem cells; and differentiating the intermediate mesoderm cells into testicular cells by treating them with a basal medium containing FGF9, insulin and / or IGF1, PGD2, RA, BMP4 (bone morphogenetic protein 4), and EGF, or with a basal medium containing FGF9, insulin and / or IGF1, PGD2, RA, BMP4 (bone morphogenetic protein 4), EGF, FSH, and LH or human chorionic gonadotropin (HCG).

[0046] In some preferred embodiments, the step of inducing intermediate mesoderm cells further includes: providing mouse pluripotent stem cells; culturing the mouse pluripotent stem cells on day 0 in the basal medium containing activin A (AA) and bFGF (basic fibroblast growth factor); and removing the basal medium containing AA and bFGF on about day 2, and culturing the cells in the basal medium containing AA, RA, and BMP4. In some preferred embodiments, the cells are treated on about day 6 in the basal medium containing FGF9, insulin and / or IGF1, PGD2, RA, BMP4, and EGF, or on about day 6 or 7 in the basal medium containing FGF9, insulin and / or IGF1, PGD2, RA, BMP4, EGF, FSH, and LH or HCG. In some preferred embodiments, the culture medium is replaced on approximately day 7 with Leydig cell differentiation medium containing a basal 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 LiCl2. In some preferred embodiments, the culture medium is replaced on approximately day 7 with myoform cell differentiation medium containing a basal 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.

[0047] In some preferred embodiments, the testicular organoid is formed in the culture by approximately day 8. In some preferred embodiments, the testicular organoid comprises one or more artificial Sertoli cells, artificial Leydig cells, artificial myosophyll cells, and artificial stromal cells. In some preferred embodiments, the testicular organoid comprises two or more artificial Sertoli cells, artificial Leydig cells, artificial myosophyll cells, and artificial stromal cells. In some preferred embodiments, the testicular organoid comprises three or more artificial Sertoli cells, artificial Leydig cells, artificial myosophyll cells, and artificial stromal cells. In some preferred embodiments, the testicular organoid comprises artificial Sertoli cells, artificial Leydig cells, artificial myosophyll cells, and artificial stromal cells. In some preferred embodiments, the organoid exhibits 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.

[0048] 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.

[0049] In some preferred embodiments, the method further comprises 1) isolating the artificial testicular cells from the organoid, or 2) isolating the testicular organoid. In some preferred embodiments, the artificial testicular cells are Sertoli-like cells. In some preferred embodiments, the artificial testicular cells are Leydig-like cells. In some preferred embodiments, the artificial testicular cells are myoplasmic cell-like cells. In some preferred embodiments, the artificial testicular cells are stromal progenitor cells.

[0050] In some preferred embodiments, the method further includes the step of transplanting the isolated artificial testicular cells or artificial testicular organoids into a mammal.

[0051] In some preferred embodiments, the method further includes the steps of contacting the artificial testicular cells or artificial testicular cell organoids with a test reagent, and evaluating the effect of the test reagent on the artificial testicular cells or artificial testicular cell organoids.

[0052] In a thirteenth aspect, the present invention provides a testicular organoid produced by the method described above.

[0053] In a fourteenth embodiment, the present invention provides artificial Leydig cells produced by the method described above.

[0054] In the fifteenth embodiment, the present invention provides artificial Sertoli cells produced by the method described above.

[0055] In the sixteenth aspect, the present invention provides artificial muscle-like cells produced by the method described above.

[0056] In the seventeenth embodiment, the present invention provides artificial stromal progenitor cells produced by the method described above.

[0057] In the eighteenth aspect, the present invention provides a method comprising the steps of: contacting artificial testicular cells or organoids described in any of the above aspects with a test reagent; and evaluating the effect of the test reagent on the artificial testicular cells or organoids.

[0058] In the nineteenth embodiment, the present invention provides a method comprising the step of transplanting artificial testicular cells or organoids described in any of the above embodiments.

[0059] In a twentieth embodiment, the present invention provides a method comprising the steps of obtaining stem cells or stem cell-containing tissue from a patient; and co-culturing the patient-derived stem cells or stem cell-containing tissue with artificial testicular cells or organoids as described in any of the above embodiments. In some preferred embodiments, the stem cells are selected from the group consisting of primordial germ cell-like cells (PGCLCs), prespermatogonial cells, and spermatogonial stem / progenitor cells (SSCs / SPCs). In some preferred embodiments, the prespermatogonial stem cells differentiate into spermatogonial cells.

[0060] In some preferred embodiments, the method further includes the step of returning the spermatogonial cells to a patient in need.

[0061] In some preferred embodiments, the patient has previously received gonadal toxic therapy and / or has non-obstructive azoospermia or severe oligospermia. In some preferred embodiments, the gonadal toxic therapy is selected from the group consisting of chemotherapy and radiotherapy. In some preferred embodiments, the stem cells or stem cell-containing tissue are obtained from the patient prior to the gonadal toxic therapy.

[0062] In a 21st embodiment, the present invention provides a method for growing patient-derived or in vitro-induced germ cells, comprising the step of co-culturing patient-derived or in vitro-induced germ cells with artificial testicular cells or organoids described in any of the above embodiments. In some preferred embodiments, the primordial germ cells are primordial germ cell-like cells (PGCLCs), prespermatogonia, or spermatogonial cells. In some preferred embodiments, the co-culturing provides the ability of the primordial germ cell-like cells (PGCLCs), prespermatogonia, or prespermatogonia-like cells to form spermatogonial cells.

[0063] [Brief explanation of the drawing] Figure 1. Schematic diagram of the mouse ESC differentiation protocol.

[0064] Figure 2. Fluorescence micrographs showing improved expression of many gonadal markers (WT1, GATA4, SF1, CoupTFII) and Sertoli cell markers (SOX9, GATA3) in the improved mouse protocol compared to the conventional protocol.

[0065] Figure 3. Fluorescence micrograph showing the generation of Leydig-like cells after 15 days of culture. The cells are co-stained with SF1 and 3BHSD.

[0066] Figure 4A-B. Graphs showing increased expression of multiple testicular cell type markers. (A) Sertoli cell markers (blue = old, yellow = new). (B) Peritusparenchymal myoform cells.

[0067] Figure 5. Schematic diagram of the human ESC differentiation protocol.

[0068] Figure 6. Schematic diagram of the human iPSC differentiation protocol.

[0069] Figure 7. Schematic diagram of a protocol combining human ESC-derived testis-like cells and primordial germ cells.

[0070] Figure 8. Schematic diagram of a protocol for inducing peritubular myosoidal cells from human ESCs.

[0071] Figure 9. Schematic diagram of the protocol for inducing Leydig cells from human ESCs.

[0072] Figure 10. Differentiation efficiency of human somatic cell-like cells in the presence of hormones, using the protocol described in PCT US2023 / 13608. A) Modified differentiation scheme. B) qPCR expression of gonads, Sertoli, Leydig, and off-target markers.

[0073] Figure 11. Modified testicular differentiation protocol of the present invention. (A) Schematic diagram and culture medium composition. (B) Various gonad, Sertoli, and Leydig cell markers. D16 Soma+PGC+ / - hormone is the most efficient and refined condition.

[0074] Figure 12. Schematic diagram of the modified mouse ESC differentiation protocol. Briefly, cells were dissociated on day 4 and plated in 25K cells onto a 96-well U-bottom plate. After maintaining the 3D aggregates for 3 days, they were transplanted into ThinCert® cells and harvested on day 17.

[0075] Figures 13A-E. Graph data showing that the differentiation scheme shown in Figure 12 effectively induces testicular progenitor cell markers. Furthermore, it promotes marker expression in Sertoli cells, Leydig cells, and myosoidal cells, and some endothelial cell markers also begin to peak. The two different colors represent two different culture medium compositions for culturing organoids on ThinCert® cell culture inserts. Note: Testicular progenitor cell markers include Tcf21, Pdgfr-alfa, and Nr2f2 (A); Sertoli cell markers include Sox9, Wt1, Gata4, and Inhbb (B); Leydig cell markers include Cyp11a1, Cyp17a1, Hsd3b6, Nr5a1, Cyp21a1, and Cy11b1 (C); myoiform cell markers include Cnn1, Sma-Alfa, and Myh11 (D); and endothelial cell markers include Pecam1, Esam, and Cdh5 (E). The multiplicative changes in gene expression were calculated relative to pre-differentiation ESCs (day 0). Gapdh was used as a housekeeping gene for normalizing Ct values ​​in qPCR analysis.

[0076] Figures 14A-D. Fluorescence micrographs showing complete reconstruction of the testicular microenvironment and tubular structure in organoids prepared by the scheme shown in Figure 12. A) Co-staining of Leydig cells (SF1), peritubular myoid cells (SMA), and Sertoli cells (SOX9). B) Sertoli cells (GATA4+SOX9) form the blood-testis barrier (ZO-1). C) Our in vitro-derived Sertoli cells (SOX9+) produce anti-Müllerian hormone (AMH). D) Leydig cells present in the organoids are mature Leydig cells expressing SF1, HSD3B, and StAR.

[0077] Figure 15. In vitro-induced organoids produce testosterone in response to LH or HCG induction.

[0078] Figure 16. Schematic diagram of mixing germ cells on day 2 postnatology with somatic cells induced in vitro by our method. Aggregation occurs on day 4, according to the differentiation scheme shown in Figure 12.

[0079] Figures 17A-D. Graphs showing the number and percentage of germ cells in organoids on day 1 and day 3 in a 96-well plate. The total number of germ cells is determined by counting the total number of DDX4 or DAZL-positive cells on day 1 and day 3 (A). Percentage and total number of OCT4-GFP-positive prespermatogonia (B), percentage and total number of PLZF-positive undifferentiated spermatogonial cells (C), and percentage and total number of differentiating Stra8-positive spermatogonial cells.

[0080] Figure 18. Experimental scheme for in vitro testicular organoid generation from human stem cells.

[0081] Figure 19. Graph data showing that increasing the EGF1 concentration to 50 ng / ml improves the expression level of Sertoli cell markers by day 22.

[0082] Figure 20A-B. Immunofluorescence micrographs of testicular organoids collected after 22 days of culture. (A) WT1: Marker for testicular stromal cells (Leydig cells and myosoidal cell progenitor cells). (B) Formation of SOX9-positive and Gata4-positive tubules.

[0083] [Definition] As used herein, the term “stem cell” (“SC”) refers to a cell capable of self-renewal and differentiation into multiple lineages. Stem cells are developmentally pluripotent or multipotent cells. Stem cells can divide to produce two daughter stem cells, or one daughter stem cell and one progenitor (“transit”) cell, which then proliferate into mature, fully formed cells of tissue. Stem cells may be, for example, embryonic (“embryonic stem cells”) or adult-derived. For example, U.S. Patent No. 5,843,780 to Thompson describes the production of stem cell lines from human embryos. PCT Publications WO00 / 52145 and WO01 / 00650 describe the use of adult-derived human cells in nuclear transfer methods for producing stem cell lines. Therefore, as used herein, the term "stem cells" includes 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 the ability to differentiate into a variety of cell types, including adipocytes, chondrocytes, osteocytes, muscle cells, bone marrow stromal cells, and thymic stromal cells (mesenchymal stem cells); hepatocytes, vascular cells, and muscle cells (hematopoietic stem cells); muscle cells, hepatocytes, and glial cells (bone marrow stromal cells), and even cells derived from all three germ layers (adult neural stem cells).

[0085] As used herein, the term "pluripotent cell" refers to a cell capable of forming a complete embryo (e.g., a blastocyst).

[0086] As used herein, the terms “pluripotent cell” or “pluripotent stem cell” refer to a cell that possesses full pluripotency, such as a cell capable of developing into any of the approximately 260 different cell types found in mammals. Pluripotent cells are capable of self-renewal and can remain dormant or quiescent within tissues. Unlike totipotent cells (e.g., a fertilized diploid egg cell), pluripotent cells, even pluripotent embryonic stem cells, are not typically able to form new blastocysts.

[0087] As used herein, the term “induced pluripotent stem cell” (“iPSC”) refers to a stem cell derived from somatic cells, such as differentiated somatic cells, that possesses higher potential than those somatic cells. iPS cells are capable of self-renewal and differentiation into mature cells.

[0088] As used herein, the term "multipotent cell" refers to a cell that has the ability to develop into a subset of approximately 260 different cell types found in mammals. Unlike pluripotent cells, multipotent cells do not have the ability to form all types of cells.

[0089] As used herein, the term "progenitor cell" refers to a cell that is expected to differentiate into a specific type of cell or form a specific type of tissue.

[0090] As used herein, the term “embryonic stem cell” (“ES cell” or “ESC”) refers to pluripotent cells derived from the inner cell mass of a blastocyst (e.g., a 4-5 day old human embryo) that have the ability to produce many or all types of cells present in a mature animal.

[0091] As used herein, the term "feeder cells" refers to cells used as growth supports in certain tissue culture systems. Feeder cells may be germinal striatal cells or stromal cells. As used herein, the term "chemically defined medium" refers to a culture medium with a chemical composition known quantitatively and qualitatively, or essentially known. Chemically defined media do not contain any animal products, including serum or serum-derived components (e.g., albumin).

[0092] As used herein, the term "serum-free medium" refers to a culture medium that does not contain serum, but does not necessarily mean that it does not contain other undefined components.

[0093] [Detailed description of the invention] Methods, kits, compositions, and systems are provided for culturing pluripotent stem cells to produce cell populations including artificial testicular cells (such as Sertoli cells and Leydig cells). In particular, culture conditions for generating artificial testicular cells from human pluripotent stem cell starting cultures are provided. Methods for using the cells in various therapeutic applications are also provided.

[0094] These methods overcome the limitations noted in the "Background of the Invention" and leverage genetic, evolutionary, and molecular biological insights gained from scRNAseq data collected by the inventors across developmental stages and multiple species to develop novel, highly efficient, directed somatic cell differentiation protocols. The inventors tuned many parameters of the protocols in mouse ESCs and two human ESC strains and analyzed scRNAseq data to confirm progression through expected cell states along the developmental trajectory. Benchmarking and classification of in vitro-induced cell states relies on the abundant in vivo markers described above during gonadal differentiation.

[0095] Somatic cells of the testes are essential for testicular tissue homeostasis and for male reproduction and overall health. Somatic cells provide a range of unknown growth factors and cytokines necessary to induce germ cell development in vivo, and are required to completely reconstitute female germ cell development in vitro, or to promote the differentiation of male primordial germ cell-like cells (PGCLCs) into spermatogonial cells.

[0096] While it is possible to co-culture PGCLCs induced in vitro with fetal somatic cells or allogeneic embryonic gonads isolated from littermates using mice, this is costly in non-human primates and ethically unacceptable in humans. Therefore, a deeper understanding of somatic cell specification programs and the creation of alternative somatic cell sources are essential for somatic cell reconstruction in petri dishes, genetic / spontaneous gene mutations, in vivo replacement of damaged cells in response to iatrogenic drugs in cancer treatment, rejuvenation of aged gonads, or actually generating germ cells in vitro using organoids. To address this gap, we provide an improved method for producing human artificial testicular cells (Sertoli cells and / or Leydig cells, etc.) from stem cells (e.g., embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs)) using the differentiation scheme described below.

[0097] (pluripotent stem cells) The method systems, systems, and kits of the present invention allow for the use of various pluripotent cells. Preferred 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 yet another particularly preferred embodiment, the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

[0098] In some preferred embodiments, pluripotent stem cells may be genetically modified by methods known in the Art to express one or more exogenous genes.

[0099] (Basal culture medium) A concern in culturing human ES cells is to remove as many undefined and animal-derived components as possible from the ES cell culture conditions. Standardizing culture conditions minimizes normal variations in the biomaterials to which cells are exposed. Furthermore, avoiding the use of animal-derived materials, cells, exudates, or components avoids the possibility of cross-species viral infection through the culture system. Thus, using chemically defined media (CDMs) that avoid the use of animal products provides baseline culture conditions to which differentiation factors can be added, yielding predictable effects.

[0100] 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 differentiation protocols, mTeSR media such as mTeSR1™ from StemCell Technologies may be used. In some embodiments, the maintenance medium preferably contains a ROCK inhibitor such as Y27632. For mouse stem cells, the maintenance medium is preferably GMEM from ThermoFisher Scientific, preferably supplemented with LIF (leukemia suppressor) and optionally knockout serum. The basal differentiation medium can be any of the many commercially available media. In some preferred embodiments, a combination of Dulbecco's Modified Eagle Medium and Hams F12 Medium (DMEM / F12; Invitrogen), which is sold as a mixture, may be used. In other preferred embodiments, APEL medium, such as STEMdiff™ APEL™ medium from StemCell Technologies, may be used. STEMdiff™ APEL™ medium is a serum-free and animal component-free medium specifically developed to support the differentiation of hPSCs. This medium was initially described for inducing hematopoietic cells when supplemented with VEGF, BMP-4, SCF, and activin A, but it has also proven to be an effective basal medium for hPSC differentiation into other lineages, including cardiomyocytes. In other preferred embodiments, mTeSR medium may be used for stem cell maintenance.

[0101] (Differentiation into artificial testicular cells) This invention provides a method and reagents for producing artificial testicular cells (e.g., Sertoli cells, Leydig cells, myosoidal cells, and / or stromal cells) from pluripotent stem cells. This invention is not limited to the use of any specific pluripotent stem cells or chemically defined culture media. The methods herein for producing artificial testicular cells are described in relation to events occurring at various points in time. It will be recognized that the methods may be modified by changing the described time schedule. As used herein, “Day 0” refers to the day and time when the pluripotent stem cells are removed from the maintenance medium and exposed to the differentiation medium. The differentiation timeline is therefore defined with Day 0 as the starting point. Where the term “approximately” Day X is used, this refers to a number of days plus or minus 12 hours from the start of Day 0. For example, “approximately Day 4” means 96 hours (i.e., 4 days) plus or minus 12 hours from the start of Day 0. If the start time of Day 0 was 9:00 AM, then “approximately Day 4” would therefore refer to 96 hours plus or minus 12 hours from that point.

[0102] The first step of the method for producing human artificial testicular cells according to the present invention includes supplying pluripotent hESCs or iPSCs as described above. In some preferred embodiments, the pluripotent stem cells are supplied in a stem cell maintenance medium. In some preferred embodiments, the stem cell maintenance medium is a chemically defined medium such as mTeSR medium. In some preferred embodiments, the stem cell maintenance medium contains a ROCK inhibitor. In some preferred embodiments, the ROCK inhibitor is Y27632.

[0103] The second step of the method of the present invention includes the steps of removing pluripotent stem cells from maintenance medium and culturing the pluripotent stem cells in basal medium supplemented with a drug suitable for inducing the pluripotent stem cells into 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 APEL medium, such as STEMdiff® APEL® medium from StemCell Technologies. In some particularly preferred embodiments, the basal medium is supplemented with CHIR99021 in a concentration of 0.5 to 15 μM (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 μM, as well as values ​​and ranges within that range). This step is defined as day 0. The basal culture medium, including supplements, should preferably be replaced daily.

[0104] A third step of the method of the present invention includes culturing the unsegmented mesoderm cells produced in the second step in a basal medium supplemented with a drug for inducing the unsegmented mesoderm cells to form intermediate mesoderm, on about the fourth day or the fourth day. In some preferred embodiments, the basal medium is a chemically defined medium. In some particularly preferred embodiments, the basal medium is APEL medium, such as STEMdiff® APEL® medium from StemCell Technologies. In some particularly preferred embodiments, the basal medium is supplemented with FGF9 at a concentration of 20 to 500 ng / ml (50, 100, 150, 200, 250, 300, 350, 400, 450 ng / ml, and values ​​and ranges within that range). In some particularly preferred embodiments, the basal medium is further supplemented with heparin at a concentration of 0.1 to 10 μg / 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 μg / ml, and values ​​and ranges within that range). The basal medium containing the supplement may preferably be replaced every two days.

[0105] A fourth step of the method of the present invention includes culturing the intermediate mesoderm cells produced in the third step in a basal medium on about seven days or seven days to induce the intermediate mesoderm cells to form germline cells. In some preferred embodiments, the basal medium is a chemically defined medium. In some particularly preferred embodiments, the basal medium is APEL medium, such as STEMdiff® APEL® medium from StemCell Technologies. The basal medium may preferably be replaced every two days. In some particularly preferred embodiments, the basal medium is supplemented with IGF1 in a concentration of 5 to 100 nM (e.g., 10, 17, 20, 30, 40, 50, 60, 70, 80, 90 nM, and values ​​and ranges within that range). In some particularly preferred embodiments, the basal medium is further supplemented with insulin in a concentration of 10 to 500 nM (e.g., 20, 50, 100, 200, 300, 400 nM, and values ​​and ranges within that range). In some embodiments, the basal medium may be further supplemented with retinoic acid (RA) in concentrations of 0.01 to 10 μM (e.g., 0.05, 0.1, 1.0, 5.0, 8.0 μM, and values ​​and ranges within that range). In some embodiments, the basal medium may be further supplemented with PGD2 in concentrations of 50 to 1000 ng / ml (e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900 ng / ml, and values ​​and ranges within that range). In some embodiments, the basal medium may be further supplemented with FGF9 in concentrations of 50 to 500 ng / ml (e.g., 100, 200, 300, 400 ng / ml, and values ​​and ranges within that range). In some more preferred embodiments, the basal medium used in step 5 may be further supplemented with 5 to 100 ng / ml of bone morphogenetic protein 4 (BMP4) (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90 ng / ml, and values ​​and ranges within that range). In some more preferred embodiments, the basal medium used in step 4 may be further supplemented with 5 to 200 ng / ml of epidermal growth factor (EGF) (e.g., 10, 20, 30, 50, 70, 100, 150 ng / ml, and values ​​and ranges within that range).In some more preferred embodiments, the basal medium used in step 5 may be further supplemented with 0.5–10 μM (e.g., 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 μM, and values ​​and ranges within that range) of IWR1. In some preferred embodiments, the basal medium used in step 4 is further supplemented with 5–100 ng / ml (10, 20, 30, 40, 50, 60, 70, 80, 90 ng / ml, and values ​​and ranges within that range) of luteinizing hormone (LH). In another embodiment, human chorionic gonadotropin (HCG) is used instead of LH in amounts of 1–10 units (1, 2, 3, 5, or 10 units, and values ​​and ranges within that range). In yet another embodiment, cells are first treated with HCG in the ranges described above until about 15 days, and then treated with LH in the ranges described above from about 15 days onward. In some preferred embodiments, the basal medium used in the fourth step is further supplemented with follicle-stimulating hormone (FSH) in a concentration of 5–300 ng / ml (10, 20, 30, 40, 50, 100, 150, 200, 250 ng / ml, as well as values ​​and ranges within that range). In some preferred embodiments, a combination of insulin and IGF1 within the above range is used. In some preferred embodiments, a combination of insulin, IGF1, and FGF9 within the above range is used. In some preferred embodiments, a combination of insulin, IGF1, FGF9, RA, EGF, and PGD2 within the above range is used.

[0106] The fifth step of the method of the present invention includes culturing the germline cells produced in the fourth step in a basal medium supplemented with a drug for inducing germline cells to form artificial testicular cells, on about eight days or eight days. In some preferred embodiments, the cells are dissociated and plated onto Aggrewell® plates or U-bottom plates. In some preferred embodiments, 10,000 to about 50,000, most preferably about 30,000, dissociated cells are transferred to the 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 StemCell Technologies' STEMdiff® APEL® medium. In some particularly preferred embodiments, the basal medium is supplemented with IGF1 in a range of 5 to 100 nM (e.g., 10, 17, 20, 30, 40, 50, 60, 70, 80, 90 nM, and values ​​and ranges within that range). In some particularly preferred embodiments, the basal medium is further supplemented with insulin at a concentration of 10–500 nM (e.g., 20, 50, 100, 200, 300, 400 nM, and values ​​and ranges within that range). In some embodiments, the basal medium may be further supplemented with retinoic acid (RA) at a concentration of 0.01–10 μM (e.g., 0.05, 0.1, 1.0, 5.0, 8.0 μM, and values ​​and ranges within that range). In some embodiments, the basal medium may be further supplemented with PGD2 at a concentration of 50–1000 ng / ml (e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900 ng / ml, and values ​​and ranges within that range). In some embodiments, the basal medium may be further supplemented with 50–500 ng / ml (e.g., 100, 200, 300, 400 ng / ml, and values ​​and ranges within that range) of FGF9. In some more preferred embodiments, the basal medium used in the fifth step may be further supplemented with 5–100 ng / ml (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90 ng / ml, and values ​​and ranges within that range) of bone morphogenetic protein 4 (BMP4).In some more preferred embodiments, the basal medium used in step 5 may be further supplemented with epidermal growth factor (EGF) in a range of 1 to 200 ng / ml (e.g., 5, 10, 20, 30, 50, 70, 100, 150 ng / ml, and values ​​and ranges within that range). In some even more preferred embodiments, the basal medium used in step 5 may be further supplemented with IWR1 in a range of 0.1 to 10 μM (e.g., 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 μM, and values ​​and ranges within that range). In some preferred embodiments, combinations of insulin and IGF1 within the above ranges are utilized. In some preferred embodiments, combinations of insulin, IGF1, and FGF9 within the above ranges are utilized. In some preferred embodiments, combinations of insulin, IGF1, FGF9, RA, and PGD2 within the above ranges are utilized. In some preferred embodiments, combinations of insulin, IGF1, FGF9, EGF, and IWR1 within the above range are used. In some preferred embodiments, combinations of insulin, IGF1, FGF9, EGF, RA, IWR1, and PGD2 within the above range are used. The basal medium containing the supplement may preferably be replaced every two days.

[0107] In some more preferred embodiments, the basal medium used in step 5 is further supplemented with one or more hormones (FSH, LH, or HCG). In some more preferred embodiments, one or more hormones are added on approximately day 9. In some preferred embodiments, the basal medium (preferably containing one or more of the above ranges of insulin, IGF1, FGF9, EGF, RA, IWR1, and PGD2) is further supplemented with luteinizing hormone (LH) in amounts of 5 to 100 ng / ml (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90 ng / ml, and values ​​and ranges within that range). In another embodiment, human chorionic gonadotropin (HCG) is used instead of LH in amounts of 1 to 10 units (1, 2, 3, 5, or 10 units, and values ​​and ranges within that range). In some preferred embodiments, the basal medium (preferably comprising one or more of the above-mentioned ranges of insulin, IGF1, FGF9, EGF, RA, IWR1, and PGD2) is further supplemented with 20–300 ng / ml of follicle-stimulating hormone (FSH) (e.g., 50, 100, 150, 200, 250 ng / ml, and values ​​and ranges within that range). In some preferred embodiments, the basal medium (preferably comprising one or more of the above-mentioned ranges of insulin, IGF1, FGF9, BMP4, EGF, RA, IWR1, and PGD2) is further supplemented with 0.01–10 μM of testosterone (e.g., 0.05, 0.1, 1.0, 5.0, 8.0 μM, and values ​​and ranges within that range). In some preferred embodiments, the basal medium (preferably comprising one or more of the above-described ranges of insulin, IGF1, FGF9, EGF, RA, IWR1, and PGD2) comprises LH, FSH, and testosterone within the ranges described herein.

[0108] In some preferred embodiments, step 6 involves maintaining the hESC culture from step 5 for approximately 12 days to allow organoid formation. The organoid is then transferred to a culture system for gas-liquid interface culture. Preferably, the basal medium is supplemented with the same factors described for step 5. In some preferred embodiments, the gas-liquid interface culture system utilizes a ThinCert® cell culture insert in a Transwell® plate. In some preferred embodiments, organoids containing one or more Sertoli cells, Leydig cells, myoid cells, and stromal cells are produced by approximately 22 days. At this point, the organoid may be harvested for use, or it may be dissociated to isolate, for example, Leydig cells, Sertoli cells, myoid cells, or stromal cells.

[0109] In some embodiments, the present invention provides a method for producing artificial Leydig cells. In these embodiments, the above steps for inducing testicular cells are carried out until around day 8 (i.e., steps 1-4). Around day 8, it is preferable to dissociate the germinal ridge cells and culture them in a basal medium supplemented with a drug to induce the germinal ridge cells to form artificial Leydig cells. In some preferred embodiments, the basal medium is supplemented with one or more of the following: SAG (Smoothened agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), bFGF2 (Basic fibroblast growth factor), DAPT, and LiCl2. In some preferred embodiments, SAG is included in the basal medium at a concentration of 0.1 to 5.0 μM (e.g., 0.2, 0.5, 1.0, 2.0, 2.0, 4.0 μM, and within that range and value). In some preferred embodiments, PDGF-AA is included in the basal medium at a concentration of 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml, and within that range and value). In some preferred embodiments, PDGF-BB is included in the basal medium at a concentration of 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml, and within that range and value). In some preferred embodiments, bFGF is included in the basal medium at a concentration of 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml, and within that range and value). In some preferred embodiments, DAPT is included in the basal medium in a concentration of 1.0 to 20.0 μM (e.g., 2.0, 5.0, 10.0, 15.0 μM, and within that range and value). In some preferred embodiments, LiCl2 is included in the basal medium in a concentration of 1.0 to 20.0 mM (e.g., 2.0, 5.0, 10.0, 15.0 mM, and within that range and value).In some preferred embodiments, Leydig cells, characterized by the expression of Leydig cell markers as described in the examples, are produced and harvested around day 16.

[0110] In some embodiments, the present invention provides a method for producing artificial muscle-like cells. In these embodiments, the above steps for inducing testicular cells are carried out until around day 8 (i.e., steps 1-4). Around day 8, it is preferable to dissociate the germinal ridge cells and culture them in a basal medium supplemented with a drug to induce the germinal ridge cells to form artificial muscle-like 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, and the germinal ridge cells differentiate into muscle-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 a concentration of 0.1 to 5.0 μM (e.g., 0.2, 0.5, 1.0, 2.0, 2.0, 4.0 μM, and within that range and value). In some preferred embodiments, PDGF-AA is included in the basal medium at a concentration of 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml, and within that range and value). In some preferred embodiments, PDGF-BB is included in the basal medium at a concentration of 1 to 50 ng / ml (1, 5, 10, 15, 20, 30, 40 ng / ml, and within that range and value). In some preferred embodiments, BMP2 is included in the basal medium at a concentration of 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml, and within that range and value). In some preferred embodiments, BMP4 is included in the basal medium at a concentration of 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml, and within that range and value).In some preferred embodiments, activin A is included in the basal medium at a concentration of 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml, and within that range and value). In some preferred embodiments, valpro is included in the basal medium at a concentration of 1.0 to 50.0 nM (e.g., 2.0, 10.0, 20.0, 30.0, 40.0 nM, and within that range and value). 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 harvested around day 16.

[0111] In some embodiments, the present invention provides a method for growing primordial germ cells, such as primordial germ cell-like cells (PGCLCs). In these embodiments, the above steps for inducing testicular cells are carried out until around day 8 (i.e., steps 1-4). Around day 8, germline cells are dissociated and combined with primordial germ cells. In some preferred embodiments, the basal medium is supplemented with the same drugs in the same concentration range as described above for the production of artificial testicular cells and organoids. In some preferred embodiments, the dissociated germline cells are mixed with primordial germ cells in a ratio of approximately 9:1. Around day 9, the medium is replaced with a basal medium containing insulin, FGF9, RA, PDG2, EGF, FSH, LH, and testosterone in the same concentration range as described above for the induction of artificial testicular cells and organoids. As recognized, this culture medium does not contain IWR1. Similar to the artificial testicular cell protocol, in some preferred embodiments, the organoids are formed by about day 12 and are preferably cultured at the gas-liquid interface as described above around day 12. Preferably, the organoids may be harvested at about day 16 for further use.

[0112] The present invention provides a method for producing artificial testicular cells and organoids from pluripotent mouse cells such as mESCs. In some preferred embodiments, pluripotent stem cells are provided in stem cell maintenance medium. In some preferred embodiments, the stem cell maintenance medium is GMEM supplemented with LIF and serum.

[0113] The second step of the method of the present invention includes the steps of removing pluripotent stem cells from maintenance medium and culturing the pluripotent stem cells in basal medium supplemented with a drug suitable for inducing the pluripotent stem cells to an epiblast. This step 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 2:1 to 1:2, most preferably 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 called priming medium. In some particularly preferred embodiments, the basal medium is supplemented with N2 supplement at a concentration of 1 to 10 μl / ml (e.g., 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 μl / ml, and within that range and value). In some particularly preferred embodiments, the basal medium is supplemented with B-27 supplement at a concentration of 1 to 20 μl / ml (e.g., 1.0, 5.0, 10.0, 20.0 μl / ml, and within that range and value). In some particularly preferred embodiments, the basal medium is supplemented with KSR at a concentration of 1 to 20 μl / ml (e.g., 1.0, 5.0, 10.0, 20.0 μl / ml, and within that range and value). In some particularly preferred embodiments, the basal medium is supplemented with activin A at a concentration of 1 to 20 ng / ml (e.g., 1.0, 5.0, 10.0, 20.0 ng / ml, and within that range and value). In some particularly preferred embodiments, the basal medium is supplemented with bFGF at a concentration of 1 to 20 ng / ml (e.g., 1.0, 5.0, 10.0, 20.0 ng / ml, and within that range and value).

[0114] A third step of the method of the present invention includes the steps of removing the epiblast from the priming medium used in the second step on approximately two days, and culturing the epiblast in a basal medium supplemented with a drug suitable for inducing epiblast cells to form an 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 called differentiation medium. In some particularly preferred embodiments, the basal medium is supplemented with activin A at a concentration of 1 to 20 ng / ml (1.0, 5.0, 10.0, 20.0 ng / ml, and ranges and values ​​within that range). In some particularly preferred embodiments, the basal medium is supplemented with RA at a concentration of 10 to 200 nM (e.g., 10.0, 50.0, 100.0, 200.0 nM, and ranges and values ​​within that range). In some particularly preferred embodiments, the basal medium is supplemented with BMP4 at a concentration of 1 to 20 ng / ml (e.g., 1.0, 2.5, 5.0, 10.0, 20.0 ng / ml, as well as ranges and values ​​within that range).

[0115] In some preferred embodiments, anterior intermediate mesoderm cells are dissociated on approximately day 4 and transferred to a U-bottom 96-well plate. In some embodiments, 10,000 to 50,000, most preferably about 25,000, dissociated cells are transferred to the plate. In another embodiment, about 100,000 to 500,000, most preferably about 300,000, dissociated cells are transferred to an Aggrewell® plate.

[0116] In some embodiments, intermediate mesoderm cells are dissociated on day 4, re-aggregated in Aggrewell or U-bottom dishes, and cultured in progenitor cell medium containing RA, IGF1, insulin, FGF9, PDG2, BMP4, EGF1, and / or y27632. In some particularly preferred embodiments, the basal medium is supplemented with IGF1 at a concentration of 5–100 nM (e.g., 10, 17, 20, 30, 40, 50, 60, 70, 80, 90 nM, and values ​​and ranges within that range). In some particularly preferred embodiments, the basal medium is further supplemented with insulin at a concentration of 10–500 nM (e.g., 20, 50, 100, 200, 300, 400 nM, and values ​​and ranges within that range). In some preferred embodiments, the basal medium is supplemented with RA at a concentration of 10.0–200 nM (e.g., 10.0, 20.0, 50.0, 100.0, 200.0 nM, and values ​​and ranges within that range). In some embodiments, the basal medium may be further supplemented with PGD2 at a concentration of 50–1000 ng / ml (e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900 ng / ml, and values ​​and ranges within that range). In some embodiments, the basal medium may be further supplemented with FGF9 at a concentration of 5–40 ng / ml (10, 20, 30 ng / ml, and values ​​and ranges within that range). In some preferred embodiments, the basal medium may be further supplemented with 1 to 10 μM (e.g., 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 μM, and within that range and value). In some even more preferred embodiments, the basal medium used in step 5 may be further supplemented with 5 to 100 ng / ml (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90 ng / ml, and within that range and value). In some even more preferred embodiments, the basal medium used in step 5 may be further supplemented with 1 to 200 ng / ml (e.g., 2, 5, 10, 20, 30, 50, 70, 100, 150 ng / ml, and within that range and value).In some preferred embodiments, the basal medium used in the fourth step is further supplemented with 5–100 ng / ml (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90 ng / ml, and values ​​and ranges within that range) of luteinizing hormone (LH). In another embodiment, instead of LH, human chorionic gonadotropin (HCG) is used in amounts of 1–10 units (1, 2, 3, 5, or 10 units, and values ​​and ranges within that range). In some preferred embodiments, the basal medium used in the fourth step is further supplemented with 5–300 ng / ml (e.g., 10, 20, 30, 40, 50, 100, 150, 200, 250 ng / ml, and values ​​and ranges within that range).

[0117] In some embodiments, cells are transferred to ThinCert® cell culture inserts approximately 7 days or 3 days after being cultured in Aggrewell® or U-bottom plates.

[0118] The basal medium containing supplements may preferably be replaced every two days. If the culture is maintained with progenitor cells for about eight days, testicular organoids will form.

[0119] In other preferred embodiments, after about 24 hours, the progenitor cell medium is replaced with myomorphic cell differentiation medium to enhance the production of myomorphic cells within the organoids. 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 to differentiate germline cells into myomorphic cell-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 a concentration of 0.1 to 5.0 μM (e.g., 0.2, 0.5, 1.0, 2.0, 2.0, 4.0 μM, and within that range and value). In some preferred embodiments, PDGF-AA is included in the basal medium at a concentration of 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml, and within that range and value). In some preferred embodiments, PDGF-BB is included in the basal medium at a concentration of 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml, and within that range and value). In some preferred embodiments, BMP2 is included in the basal medium at a concentration of 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml, and within that range and value). In some preferred embodiments, BMP4 is included in the basal medium at a concentration of 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml, and within that range and value). In some preferred embodiments, activin A is included in the basal medium at a concentration of 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml, and within that range and value).In some preferred embodiments, Valpro is included in the basal medium at a concentration of 1.0 to 50.0 nM (e.g., 2.0, 10.0, 20.0, 30.0, 40.0 nM, and within that range). 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 harvested around day 8.

[0120] In other preferred embodiments, after about 24 hours, the progenitor cell medium is replaced with Leydig differentiation medium to enhance the production of myoform cells within the organoids. In some preferred embodiments, the basal medium is supplemented with one or more of the following: SAG (Smoothened agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), bFGF2 (Basic fibroblast growth factor), DAPT, and LiCl2. In some preferred embodiments, SAG is included in the basal medium in a concentration of 0.1 to 5.0 μM (e.g., 0.2, 0.5, 1.0, 2.0, 2.0, 4.0 μM, and within that range and value). In some preferred embodiments, PDGF-AA is included in the basal medium at a concentration of 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml, and within that range and value). In some preferred embodiments, PDGF-BB is included in the basal medium at a concentration of 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml, and within that range and value). In some preferred embodiments, bFGF is included in the basal medium at a concentration of 1 to 50 ng / ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng / ml, and within that range and value). In some preferred embodiments, DAPT is included in the basal medium at a concentration of 1.0 to 20.0 μM (e.g., 2.0, 5.0, 10.0, 15.0 μM, and within that range and value). In some preferred embodiments, LiCl2 is included in the basal medium at a concentration of 1.0–20.0 mM (e.g., 2.0, 5.0, 10.0, 15.0 mM, and within that range). In some preferred embodiments, Leydig cells characterized by the expression of Leydig cell markers as described in the examples are produced and harvested around day 8.

[0121] In some preferred embodiments, artificial testicular 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, artificial testicular cells produced by the method described above express at least one marker selected from the group consisting of EMX2, WT, SOX9, LHX9, DHH, STAR, and 3BHSD. In some preferred embodiments, artificial testicular cells express at least two markers selected from the group consisting of EMX2, WT, SOX9, LHX9, DHH, STAR, and 3BHSD. In some preferred embodiments, artificial testicular cells express at least three markers selected from the group consisting of EMX2, WT, SOX9, LHX9, DHH, STAR, and 3BHSD. In some preferred embodiments, artificial testicular cells express the markers EMX2, WT, SOX9, LHX9, DHH, STAR, and 3BHSD.

[0122] In some preferred embodiments, the culture is maintained until organoids are formed. The organoids are preferably three-dimensional organoids having a substantially spherical shape. In some preferred embodiments, the organoids are characterized by including tubular structures. In some preferred embodiments, the organoids are characterized by including smooth muscle actin.

[0123] In some preferred embodiments, artificial testicular cells or organoids may be harvested or isolated from the culture for further use.

[0124] (Use of artificial testicular cells and organoids) In several preferred embodiments, the methods, reagents, and kits described herein, as well as the artificial testicular cells and organoids produced using them, are found to have use in a variety of research, diagnostic, clinical, and therapeutic applications. In some embodiments, the artificial testicular cells or organoids are used for direct transplantation into a subject. In some preferred embodiments, the artificial testicular cells or organoids are used in somatic cell replacement therapy in a subject requiring it. In some embodiments, the artificial testicular cells produced by the methods herein are useful for diagnostic, prognostic, and / or therapeutic applications.

[0125] In some embodiments, isolated artificial testicular cells or organoids may be directly transplanted into a subject. Where appropriate, the cells are co-administered with one or more pharmaceutical or bioactive agents that promote the survival and function of the transplanted cells.

[0126] In some embodiments, human organoids are transplanted into mice for further differentiation and / or maturation of cells within the organoids. In other embodiments, the organoids are combined with germ cells, preferably induced in vivo or in vitro, to achieve proliferation of human germline stem cells and further promote differentiation. These methods are expected to produce haploid round or elongated sperm, which may find adjunctive applications in reproductive medical technologies such as IVF / ICSI.

[0127] In some embodiments, the testicular cells or organoids of the present invention may be used in co-culture of gamete stem cells such as patient-derived primordial germ cell-like cells, progenitor spermatogonial cells, or spermatogonial stem / progenitor cells (SSC / SPC). In some preferred embodiments, the cells are cultured so that the patient-derived stem cells differentiate into spermatogonial cells. In some preferred embodiments, the gamete stem cells, or cells derived from gamete stem cells such as spermatogonial cells, are returned to the patient or a patient in need thereof. In some preferred embodiments, the patient has non-obstructive azoospermia. In some preferred embodiments, the patient has previously received gonadal toxic therapy, including but not limited to chemotherapy and / or radiotherapy. In some preferred embodiments, the stem cells or stem cell-containing tissue are obtained from the patient prior to gonadal toxic therapy. In some preferred embodiments, the testicular tissue or somatic cells of interest are preserved prior to gonadal toxic therapy (chemotherapy and radiotherapy), and germline stem cells may be produced or isolated in the future. In some preferred embodiments, the patient was born with a genetic disorder that affects fertility or causes them to become infertile. In some preferred embodiments, germline stem cells obtained from tissue can be proliferated using cells induced in vitro according to the present invention. In some preferred embodiments, the method described above further includes the step of collecting fibroblast tissue from a patient to be reprogrammed into induced pluripotent stem cells that can be used to produce autologous artificial testicular cells. In some preferred embodiments, these differentiated cells can be combined with germline stem cells: primordial germ cell-like cells (PGCLCs), prospertoire cells (proSSCs), or neonatal / adult spermatogonial stem / progenitor cells (SSCs / SPCs). In some preferred embodiments, the stem cells can proliferate or differentiate into spermatogonial cells or later germ cell stages. In some preferred embodiments, the method further includes the step of returning the proliferated stem cells or differentiated spermatogonial cells to a patient in need.

[0128] In some embodiments, artificial testicular cells or organoids may be supplied on a support material. Supports suitable for use for the purposes of the present invention include tissue templates, conduits, partitions, and reservoirs useful for tissue repair. In particular, synthetic and natural materials in the form of foams, sponges, gels, hydrogels, textiles, and nonwoven structures have been used in vitro and in vivo to reconstruct or regenerate biological tissues and to deliver chemotactic agents to induce tissue growth, and these materials are suitable for use in carrying out the methods of the present invention. For example, see the materials described in U.S. Patent Nos. 5,770,417, 6,022,743, 5,567,612, 5,759,830, 6,626,950, 6,534,084, 6,306,424, 6,365,149, 6,599,323, 6,656,488, U.S. Patent Publication No. 2004 / 0062753 Al, 4,557,264, and 6,333,029.

[0129] Cells prepared using the methods and reagents described herein may be implanted as dispersed cells or formed into implantable clusters. In some embodiments, the cells are provided in a biocompatible, degradable polymer support; encapsulated in a porous, permeable, or semipermeable non-degradable device; or in an encapsulated form (for example, to protect the implanted cells from host immune responses, etc.). The cells may be implanted in a suitable site in the recipient. Suitable implantation sites include, for example, the testes or subcutaneous tissue.

[0130] In some embodiments, cells or cell clusters are encapsulated for transplantation into a target. Encapsulation techniques are generally classified into microencapsulation, which involves small spherical vehicles, and macroencapsulation, which involves larger plate membranes and hollow fiber membranes (Uludag, H. et al. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64, the whole of which is incorporated herein by reference). Methods for preparing microcapsules include those described below: Lu MZ, et al. Biotechnol Bioeng. 2000, 70: 479-83; Chang TM and Prakash S, Mol Biotechnol. 2001, 17: 249-60; and Lu MZ, et al, J. Microencapsul. 2000, 17: 245-51; the whole of which is incorporated herein by reference. For example, microcapsules may be prepared by compounding modified collagen with a terpolymer shell of 2-hydroxyethyl methyl acrylate (HEMA), methacrylic acid (MAA), and methyl methacrylate (MMA), resulting in a capsule thickness of 2–5 μm. Such microcapsules may be further encapsulated with an additional 2–5 μm terpolymer shell to impart a negatively charged, smooth surface and minimize adsorption of plasma proteins (see Chia, SM et al. Multi-layered microcapsules for cell encapsulation Biomaterials. 2002 23: 849-56; the whole is incorporated herein by reference). In some embodiments, the microcapsules are based on alginates, which are marine polysaccharides (see Sambanis, Diabetes Technol. Ther. 2003, 5: 665-8; the whole is incorporated herein by reference), or derivatives thereof.For example, microcapsules can be prepared by polymer electrolyte complex formation in the presence of calcium chloride, using polyanions such as sodium alginate and sodium cellulose sulfate, and a polycation such as poly(methylene-co-guanidine) hydrochloride.

[0131] In some embodiments, cells prepared using the methods and reagents described herein are microencapsulated for transplantation into a target (e.g., to prevent immune destruction of the cells). Microencapsulation of cells (e.g., pancreatic lineage cells, β-like cells, etc.) provides local protection of the implanted / transplanted cells from immune attack (e.g., with or without the use of systemic immunosuppressants). In some embodiments, cells and / or cell clusters are made from polymers, hydrogels, or other suitable materials: poly(orthoesters), poly(anhydride), poly(phosphoesters), poly(phosphazenes), polysaccharides, polyesters, poly(lactic acid), poly(L-lysine), poly(glycolic acid), poly(lactic acid-coglycolic acid), poly(lactic acid-co-lysine), poly(lactic acid-grafted-lysine), polyanhydride, poly(fatty acid dimer), poly(fumaric acid), poly(sebacic acid), poly(carboxyphenoxypropane), poly(carboxyphenoxyhexane), poly(anhydride-co-imide), poly(a Poly(iminocarbonate), poly(urethane), poly(organophazene), poly(phosphate), poly(ethylene vinyl acetate), poly(caprolactone), poly(carbonate), poly(amino acid), poly(acrylate), polyacetal, poly(cyanoacrylate), poly(styrene), poly(vinyl chloride), poly(vinyl fluoride), poly(vinylimidazole), chlorosulfonated polyolefin, polyethylene oxide, polystyrene, polysaccharides, alginates, hydroxypropylcellulose (HPC), N-isopropylacrylamide (NIP A), polyethylene glycol, polyvinyl alcohol (PVA), polyethyleneimine, chitosan (CS), chitin, dextran sulfate, heparin, chondroitin sulfate, gelatin, etc., and their derivatives, copolymers, and mixtures thereof (but not limited to these). In some embodiments, cells are microencapsulated in an encapsulation material containing or consisting of alginate.Cells may be embedded in a material or within particles (e.g., nanoparticles, microparticles, etc.) or other structures (e.g., matrices, nanotubes, vesicles, globules, etc.). In some embodiments, the microencapsulation structure is modified with immunomodulatory or immunosuppressive compounds to reduce or prevent the immune response against the encapsulated cells. For example, pancreatic cell lineage cells are encapsulated within an encapsulation material (e.g., alginate hydrogel) modified by adhering an immunomodulator (e.g., CXCL12 (also known as SDF-1), which is an immunomodulatory chemokine). In some embodiments, such immunomodulators are T cell chemokines and / or pro-survivability factors.

[0132] In some embodiments, cells prepared using the methods and reagents described herein are macroencapsulated for transplantation into a subject. For example, macroencapsulation of cells in a permeable or semipermeable chamber provides local protection of implanted / transplanted cells from immune attack (e.g., with or without the use of systemic immunosuppressants), prevents the spread of cells to other tissues or areas of the body, and / or allows for efficient removal of cells. Suitable devices for macroencapsulation are described, for example, in U.S. Patent No. 5,914,262; Uludag, et al, Advanced Drug Delivery Reviews, 2000, pp. 29-64, vol. 42, which is incorporated herein by reference in its entirety.

[0133] Other encapsulation (micro or macro) devices and methods may find applications in the embodiments described herein. For example, the methods and devices described in U.S. Patent Application Publication No. 20130209421 and U.S. Patent No. 8,785,185 (each of which is incorporated herein by reference in whole) are within the scope of the embodiments described herein.

[0134] In some embodiments, the testicular cells or organoids of the present invention may be used in hormone therapy. In some preferred embodiments, the organoids are encapsulated and implanted subcutaneously into a subject.

[0135] In some embodiments, the testicular cells or organoids of the present invention may be used to restore fertility. In some preferred embodiments, endogenous defective somatic cells in the testis are combined with or replaced by the testicular cells or organoids of the present invention. In other preferred embodiments, the organoids are implanted in a subject so that spermatogenesis occurs in an ectopic location other than the testis (e.g., subcutaneously).

[0136] In further embodiments, populations of artificial testicular cells and organoids may be used to prepare antibody and cDNA libraries that are specific to differentiated phenotypes. Common techniques used in antibody production (raising), purification, and modification, as well as common techniques used in their use in immunoassays and immunoisolation methods, The following resources describe the preparation of mRNA and cDNA libraries: 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 for preparing mRNA and cDNA libraries are described in RA Methodologies: A Laboratory Guide for Isolation and Characterization (RE 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 suitable for use in drug screening and therapeutic applications.

[0137] In some embodiments, artificial testicular cells and organoids prepared by the methods provided herein are used to screen for drugs (e.g., small molecule drugs, peptides, polynucleotides, etc.) or environmental conditions (e.g., culture conditions or operations) that affect cells. In particular, screening applications relate to testing pharmaceutical compounds in drug research and drugs used for cryopreservation of gametes, including sperm. Evaluating the activity of candidate pharmaceutical compounds generally involves combining cells with the candidate compound, determining changes in cell morphology, marker phenotype, or metabolic activity (compared to untreated cells or cells treated with an inactive compound) attributable to the compound, and then correlating the effects of the compound with the observed changes. Any suitable assay for detecting changes related to the test drug may be used in such embodiments. Screening may be performed, for example, because the compound is designed to have a pharmacological effect on testicular cell types, because a compound designed to have an effect elsewhere may have unintended side effects, or because the compound is part of a library screening for a desired effect. Two or more drugs may be tested in combination (simultaneously or sequentially, in combination with cells) to detect potential drug-drug interaction effects. Depending on the application, compounds are screened for cytotoxicity.

[0138] In some embodiments, methods and systems are provided for evaluating the safety and efficacy of drugs that act on testicular cells, or drugs that may be used for other purposes but may have unintended effects on testicular cells. In some embodiments, the cells described herein are used in high-throughput screening (HTS) applications. In some embodiments, a large number (e.g., 1 × 10) are used. 3 , l×10 4 , l×10 5 , l×10 6An HTS screening platform (e.g., cells and plates) is provided that enables rapid testing of drugs (e.g., small molecule compounds, peptides, etc.) of 1,000 mg or more. In some embodiments, artificial testicular cells or organoids prepared using the methods and reagents described herein are used for therapeutic delivery to a target. The cells may be in direct contact with the target tissue, or they may be sealed or encapsulated (e.g., to avoid direct contact). In embodiments where the cells are encapsulated, exchange of nutrients, gases, etc., between the encapsulated cells and the target tissue is permitted. In some embodiments, the cells are implanted / transplanted onto a matrix or other delivery platform.

[0139] In some embodiments, the methods and kits described herein are useful for identifying additional factors, reagents, and methods for producing artificial testicular cells or other cell types. The methods used herein may be used to screen factors, reagents, and / or conditions for differentiation effects. In some embodiments, any screening performed in this embodiment or other embodiments discussed herein may be high-throughput screening.

[0140] [Examples] [Example 1] The following examples describe reagents and protocols for producing artificial testicular cells derived from mouse embryonic stem cells (mESCs) according to the present invention.

[0141] [mESC culture] (Reagents / Materials) 1. Mouse embryonic stem cells (TG2A) 2- GMEM(ThermoFisher scientific Cat #1171035) 3. Type B gelatin solution (Sigma Aldrich Cat #G1393 - 100ML) 4-MEM non-essential amino acids (ThermoFisher Scientific Cat #11140050) 5-2-mercaptoethanol (ThermoFisher Scientific Cat #21985023) 6. Sodium pyruvate solution (Sigma Aldrich Cat #S8636-100ML) 7. Embryonic stem cell fetal bovine serum, qualified product (ThermoFisher Scientific Cat #16141079) 8- TrypLE Express Enzyme(ThermoFisher scientific Cat #12604013) 9- Leukemia inhibitory factor (Sigma Aldrich Cat # L5283-10UG) 10-Penicillin-Streptomycin (5,000 U / mL) (ThermoFisher Scientific Cat #15070063) 11- PBS- / -(ThermoFisher scientific Cat #10010023) 12-Dimethyl sulfoxide (Sigma-Aldrich Cat #D8418-100ML) 13-6 well cell culture plate (ThermoFisher Scientific Cat #140675) Growth medium - A small amount of culture medium can be prepared and stored at 4°C for up to one month. Composition of 100 ml of growth medium - Glasgow MEM (GMEM) 92 ml, fetal bovine serum ES cells (qualified) 10 ml, non-essential amino acids 1 ml, sodium pyruvate 1 ml, penicillin-streptomycin 1 ml, 2-mercaptoethanol 100 μl, LIF 100 μl.

[0142] 2X freeze buffer - 20% dimethyl sulfoxide and 80% fetal bovine serum ES cells (qualified) were prepared and stored in a freezer at -20°C.

[0143] Cell culture: (1) Before seeding the cells, add 2 ml of 0.1% gelatin solution per well and incubate overnight at room temperature.

[0144] (2) Thawing TG2A cells Remove 1 ml vial of frozen cells (frozen in 1:1 freezing buffer and growth medium) from liquid nitrogen and immediately thaw in a 37°C water bath.

[0145] (3) Add 2 ml of growth medium to the thawed cells to wash them, and then centrifuge the cells at 1000 RPM for 5 minutes.

[0146] (4) Aspirate the culture medium and dilute the pelleted cells with 2 ml of growth medium.

[0147] (5) Aspirate the gelatin from the plate and seed approximately 500,000 cells in 2 ml of growth medium per well of the 6-well plate.

[0148] (6) Replace the entire culture medium every 24 hours.

[0149] (7) When the cells reach approximately 80% confluence, pass the cells onto a new plate.

[0150] (a) To subculture the cells, aspirate the culture medium and rinse the cells with 1 ml of PBS per well.

[0151] (b) Aspirate PBS.

[0152] (c) Add 300 μl of TrypLE Express Enzyme to each well and incubate in a CO2 incubator at 37°C for 4 minutes.

[0153] (d) Remove the cells from the incubator and add 2 mL of growth medium to quench the TrypLE Express Enzyme.

[0154] (e) Centrifuge the cells at 1000 RPM for 5 minutes. Aspirate the liquid and dilute the cells with 2 ml of growth medium.

[0155] (f) Transfer approximately 500,000 cells per well to a gelatin-coated 6-well cell culture plate with 2 ml of growth medium.

[0156] (g) Replace the entire culture medium every 24 hours.

[0157] (h) After reaching approximately 80% confluence, repeat the same process as in point 7.

[0158] [Differentiation of TG2A cells into anterior intermediate mesoderm-like cells] (Reagents / Materials) 1. Neurobasal medium (ThermoFisher Scientific Cat #21103049) 2. DMEM-F12 medium (ThermoFisher Scientific Cat #11320033) 3-2-mercaptoethanol (ThermoFisher Scientific Cat #21985023) 4. Sodium pyruvate solution (Sigma Aldrich Cat #S8636-100ML) 5-MEM non-essential amino acids (ThermoFisher Scientific Cat #11140050) 6- N-2 Refill (ThermoFisher scientific Cat #17502048) 7- B-27 Refill (ThermoFisher scientific Cat #17504044) 8- Glutamax(100X)(ThermoFisher scientific Cat #35050061) 9. Knockout serum replacer (ThermoFisher Scientific Cat #10828010) 10-Penicillin-Streptomycin (5,000 U / mL) (ThermoFisher Scientific Cat #15070063) 11- TrypLE Express Enzyme(ThermoFisher scientific Cat #12604013) 12- Activin A (R&D systems Cat #338-AC-010) 13- bFGF(Proteintech Cat #HZ-1285) 14- Retinoic acid (Sigma-Aldrich Cat #R2625) 15- BMP4(R and D systems Cat #314-BP-500) 16-6 well cell culture plate (ThermoFisher Scientific Cat #140675) 17- ROCK inhibitor Y-27632 (Enzo Life Sciences Cat #ALX-270-333) 18- PBS- / -(ThermoFisher scientific Cat #10010023) Priming medium A small amount of basal priming medium can be prepared and stored at 4°C for up to one month.

[0159] Composition of 100 ml of priming medium: Add 46.9 ml of Neurobasal medium, 46.9 ml of DMEM-F12 medium, 500 μl of N2 supplement, 1 ml of B27 supplement, 500 μl of Glutamax, 100 μl of 2-mercaptoethanol, 1 ml of sodium pyruvate, 1 ml of knockout serum replacer, 1 ml of non-essential amino acids, and 1 ml of penicillin-streptomycin. Add additional growth factors, 10 ng / ml of activin A, and 10 ng / ml of bFGF, and mix them into the medium immediately before adding it to the cells.

[0160] AIM differentiation medium A small amount of basal AIM differentiation medium can be prepared and stored at 4°C for up to one month.

[0161] Composition of 100 ml of AIM differentiation 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 μl of 2-mercaptoethanol, and 1 ml of penicillin-streptomycin. Add additional growth factors, 10 ng / ml of activin A, 100 nM of RA, and 2.5 ng / ml of BMP4, to the medium immediately before adding it to the cells.

[0162] (1) Preparation of starting cells for differentiation To initiate differentiation, starting cells must be undifferentiated and in the proliferation phase. To ensure that starting cells are consistently in the same proliferation phase across experiments, cells are harvested from an 80% confluent plate (at least one passage after thawing).

[0163] (a) Aspirate the culture medium and wash the cells with 1 ml of PBS- / - in a 6-well plate.

[0164] (b) Aspirate PBS- / - and incubate the cells in a CO2 incubator for 5 minutes with 300 μl of TrypLE Express per well of a 6-well plate.

[0165] (c) Remove the cells from the incubator and add 2 mL of growth medium to quench the TrypLE Express Enzyme.

[0166] (d) Centrifuge the cells at 1000 RPM for 5 minutes, aspirate the liquid, and dilute the pelleted cells with growth medium.

[0167] (e) Seed approximately 300,000 cells per well in 2 ml of growth medium in a gelatin-coated 6-well plate.

[0168] (f) Replace the complete culture medium with fresh growth medium every 24 hours.

[0169] (g) 48 hours after seeding, the cells can be prepared as starting cells for differentiation.

[0170] (2) Initiation of differentiation (a) After 48 hours, aspirate the growth medium from the cells that have grown and wash with 1 ml of PBS- / - per well of a 6-well plate.

[0171] (b) Aspirate PBS- / - and incubate the cells in a CO2 incubator for 5 minutes with 300 μl of TrypLE Express per well of a 6-well plate.

[0172] (c) Remove the cells from the incubator and add 2 mL of growth medium to quench the TrypLE Express Enzyme.

[0173] (d) Centrifuge the cells at 1000 RPM for 5 minutes, aspirate the liquid, and dilute the paletted cells with priming medium.

[0174] (e) Seed approximately 300,000 cells per well in 2 ml of priming medium in a gelatin-coated 6-well plate, and incubate the cells in a CO2 incubator.

[0175] (f) After 24 hours of incubation, replace the priming complete medium. Since the cells are loosely adhered to the surface, the medium was replaced very carefully to avoid disturbing the plate too much.

[0176] (g) After 48 hours of total incubation in priming medium, replace the complete medium with 2 ml of AIM differentiation medium per well of a 6-well plate. Since the cells are loosely adhered to the surface, the medium was replaced very carefully to avoid disturbing the plate too much.

[0177] (h) After 72 hours of total incubation, replace the complete medium with AIM differentiation medium containing 3 ml of 5 μM Y-27632 per well of a 6-well plate.

[0178] (i) After a total incubation period of 96 hours, the cells can be prepared as starting cells for organoid differentiation.

[0179] Cell samples were collected daily starting two days after differentiation for RNA isolation. qPCR was performed for multiple AIM markers, gonadal markers, the pluripotency marker Oct4, and the posterior intermediate mesoderm marker HoxD11. Data were confirmed by immunostaining for AIM, as well as gonadal markers Wt1 and Sox9.

[0180] [Differentiation of peritubular muscle-like cells from AIM cells differentiated for 4 days.] (material) 1. DMEM-F12 medium (ThermoFisher Scientific Cat #11320033) 2-2-mercaptoethanol (ThermoFisher Scientific Cat #21985023) 3. Sodium pyruvate solution (Sigma Aldrich Cat #S8636-100ML) 4-MEM non-essential amino acids (ThermoFisher Scientific Cat #11140050) 5- Glutamax(ThermoFisher scientific Cat #35050061) 6. Knockout serum replacer (ThermoFisher Scientific Cat #10828010) 7- Penicillin-Streptomycin (5,000 U / mL) (ThermoFisher Scientific Cat #15070063) 8- FGF9(R&D systems Cat #273-F9-025) 9- BMP4(R and D systems Cat #314-BP-500) 10- EGF1 11- PDGFAA(Sigma Aldrich Cat #SRP3228) 12- PDGFBB(Sigma Aldrich Cat #SRP3229) 13 - Smoothed Agonist (SAG) (Millipore Cat #566661) 14- Valproic acid (Sigma Aldrich Cat #P4543-10G) 15- BMP2 (R&D Systems Cat # 355-BM-010) 16- Activin A (R&D systems Cat #338-AC-010) 17- Retinoic acid (Sigma-Aldrich Cat ##R2625) 18- IGF1(Sigma-Aldrich Cat #I3769) 19- Insulin (Sigma-Aldrich Cat #I9278) 20- Prostaglandin D2 (Cayman Chemical Cat #12010) 21- ROCK inhibitor Y-27632 (Enzo Life Sciences Cat #ALX-270-333) 22- Insulin, Transferrin, Selenium (ITS) (100X) (ThermoFisher Scientific Cat #41400045) 23- Matrigel basement membrane matrix (Corning Cat #354234) 24- 10% bovine serum albumin (BSA) solution in DPBS (Sigma Aldrich Cat #A1595) 25- TrypLE Express Enzyme(ThermoFisher scientific Cat #12604013) [Prepare two types of culture media.] 1. Progenitor cell medium - Composition of 100 ml of progenitor cell 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 μl of 2-mercaptoethanol, and 1 ml of penicillin-streptomycin. Additional factors, 1% Matrigel, 100 nM retinoic acid, 17 nM IGF1, 100 nM insulin, 10 ng / ml FGF9, 500 ng / ml PGD2, 20 ng / ml BMP4, 50 ng / ml EGF1, and 5 μM Y-27632, were mixed immediately before adding the medium to the cells.

[0181] 2. Myoid cell differentiation medium - Composition of 100 ml of myoid cell differentiation medium: Add 98.9 ml of DMEM-F12 medium, 100 μl of ITS, and 1 ml of BSA. Additional factors, 0.5 μM SAG, 10 ng / ml PDGFRAA, 10 ng / ml PDGFRBB, 10 nm valproic acid, 10 ng / ml BMP2, 10 ng / ml BMP4, and 10 ng / ml activin A, were mixed immediately before adding the medium to the cells.

[0182] 1. Preparation of plates for muscle-like cell differentiation - Six hours before seeding the cells onto the plate, the plate needs to be coated with Matrigel. Add 56 μL of Matrigel to 12 ml of DMEM / F12 medium and add 1 ml to each well of a 12-well plate. Incubate the plate in a CO2 incubator for approximately 6 hours.

[0183] 2. Seeding of 4-day AIM cells onto Matrigel-coated plates.

[0184] (a) From the differentiated AIM cells on day 4, aspirate the AIM differentiation medium and wash the cells three times with 2 ml of PBS- / - per well in a 6-well plate.

[0185] (b) Add 500 μl of TrypLE Express Enzyme to each well of a 6-well plate and incubate in a CO2 incubator for 5 minutes.

[0186] (c) Remove the cells from the incubator and add 2 mL of growth medium per well of a 6-well plate to quench the TrypLE Express Enzyme.

[0187] (d) Centrifuge the cells at 1000 RPM for 5 minutes, aspirate the liquid, and dilute the paletted cells with progenitor cell medium.

[0188] (e) Approximately 50,000 cells were mixed in 1 ml of progenitor cell medium per well of a 12-well plate.

[0189] (f) Aspirate the Matrigel coating solution from the plate after approximately 6 hours have passed since it was added to the plate.

[0190] (g) Seed cells onto a plate and incubated in a CO2 incubator for 24 hours.

[0191] (h) Aspirate the progenitor cell medium and add 1 ml of muscle-like cell differentiation medium. Incubate the plate in a CO2 incubator.

[0192] (i) Replace the muscle-like cell differentiation medium daily.

[0193] (j) Samples are collected on the 3rd and 5th day after seeding.

[0194] [Differentiation of peritubular Leydig cells from AIM cells differentiated for 4 days.] (material) 1. DMEM-F12 medium (ThermoFisher Scientific Cat #11320033) 2-2-mercaptoethanol (ThermoFisher Scientific Cat #21985023) 3. Sodium pyruvate solution (Sigma Aldrich Cat #S8636-100ML) 4-MEM non-essential amino acids (ThermoFisher Scientific Cat #11140050) 5- Glutamax(ThermoFisher scientific Cat #35050061) 6. Knockout serum replacer (ThermoFisher Scientific Cat #10828010) 7- Penicillin-Streptomycin (5,000 U / mL) (ThermoFisher Scientific Cat #15070063) 8- FGF9(R&D systems Cat #273-F9-025) 9- BMP4(R and D systems Cat #314-BP-500) 10- EGF1 11- PDGFAA(Sigma Aldrich Cat #SRP3228) 12- PDGFBB(Sigma Aldrich Cat #SRP3229) 13 - Smoothed Agonist (SAG) (Millipore Cat #566661) 14- DAPT(Stem cells Cat #72082) 15- Retinoic acid (Sigma-Aldrich Cat ##R2625) 16- IGF1(Sigma-Aldrich cat #I3769) 17- Insulin (Sigma-Aldrich cat #I9278) 18- Prostaglandin D2 (Cayman Chemical Cat #12010) 19- ROCK inhibitor Y-27632 (Enzo Life Sciences Cat #ALX-270-333) 20- Insulin, Transferrin, Selenium (ITS) (100X) (ThermoFisher Scientific Cat #41400045) 21- Matrigel basement membrane matrix (Corning Cat #354234) 22- 10% bovine serum albumin (BSA) solution in DPBS (Sigma Aldrich Cat #A1595) 23- TrypLE Express Enzyme(ThermoFisher scientific Cat #12604013) twenty four- [Prepare three types of culture media.] 1. Progenitor cell medium - Composition of 100 ml of progenitor cell 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 μl of 2-mercaptoethanol, and 1 ml of penicillin-streptomycin. Additional factors, 1% Matrigel, 100 nM retinoic acid, 17 nM IGF1, 100 nM insulin, 10 ng / ml FGF9, 500 ng / ml PGD2, 20 ng / ml BMP4, 50 ng / ml EGF1, and 5 μM Y-27632, were mixed immediately before adding the medium to the cells.

[0195] 2. Leydig cell basal medium - 100 ml of Leydig cell basal medium composition: 98.9 ml of DMEM-F12 medium, 100 μl of ITS, and 1 ml of BSA were added. Additional factors, 0.5 μM SAG, 10 ng / ml PDGFRAA, 10 ng / ml PDGFRBB, and 10 ng / ml FGF2, were mixed immediately before adding the medium to the cells.

[0196] 3. Leydig cell differentiation medium - 100 ml of muscle-like cell differentiation medium: Add 98.9 ml of DMEM-F12 medium, 100 μl of ITS, and 1 ml of BSA. Additional factors, 0.5 μM SAG, 10 ng / ml PDGFRAA, 10 ng / ml PDGFRBB, 5 mM LiCl2, 10 ng / ml FGF2, and 10 μM DAPT, were mixed immediately before adding the medium to the cells.

[0197] 1. Preparation of Leydig cell differentiation plates - Two hours before seeding the cells onto the plate, the plate needs to be coated with Matrigel. Add 56 μL of Matrigel to 12 ml of DMEM / F12 medium and add 1 ml per well to a 12-well plate. Incubate the plate in a CO2 incubator for approximately 6 hours.

[0198] 2. Seeding of 4-day AIM cells onto Matrigel-coated plates.

[0199] (k) From the differentiated AIM cells on day 4, aspirate the AIM differentiation medium and wash the cells three times with 2 ml of PBS- / - per well in a 6-well plate.

[0200] (l) Add 500 μl of TrypLE Express Enzyme to each well of a 6-well plate and incubate in a CO2 incubator for 5 minutes.

[0201] (m) Remove the cells from the incubator and add 2 mL of growth medium per well of a 6-well plate to quench the TrypLE Express Enzyme.

[0202] (n) Centrifuge the cells at 1000 RPM for 5 minutes, aspirate the liquid, and dilute the paletted cells with progenitor cell medium.

[0203] (o) Approximately 50,000 cells were mixed in 1 ml of progenitor cell medium per well of a 12-well plate.

[0204] (p) The Matrigel coating solution, which has been added to the plate for approximately 6 hours, is aspirated from the plate.

[0205] (q) Seed the cells onto a plate and incubate in a CO2 incubator for 24 hours.

[0206] (r) Aspirate the progenitor cell medium and add 1 ml of Leydig cell basal medium. Incubate the plate in a CO2 incubator.

[0207] (s) Replace the Leydig cell basal medium daily for up to 3 days.

[0208] (t) After culturing in Leydig cell basal medium for 3 days, replace the Leydig cell basal medium with Leydig cell differentiation medium.

[0209] (u) Replace the Leydig cell differentiation medium daily.

[0210] (v) Collect samples on day 4, day 8, and day 15 after seeding.

[0211] [Development of testicular organoids from AIM cells differentiated over 4 days.] (material) 1. DMEM-F12 medium (ThermoFisher Scientific Cat #11320033) 2,2-Mercaptoethanol (ThermoFisher Scientific Cat #21985023) 3. Sodium pyruvate solution (Sigma Aldrich Cat #S8636-100ML) 4. MEM non-essential amino acids (ThermoFisher Scientific Cat #11140050) 5.Glutamax(ThermoFisher scientific Cat #35050061) 6. Knockout serum replacer (ThermoFisher Scientific Cat #10828010) 7. Penicillin-Streptomycin (5,000 U / mL) (ThermoFisher Scientific Cat #15070063) 8.FGF9(R&D systems Cat #273-F9-025) 9.BMP4(R and D systems Cat #314-BP-500) 10. EGF1 11. Retinoic acid (Sigma-Aldrich Cat ##R2625) 12.IGF1(Sigma-Aldrich cat #I3769) 13. Insulin (Sigma-Aldrich cat #I9278) 14. Prostaglandin D2 (Cayman Chemical Cat #12010) 15. ROCK inhibitor Y-27632 (Enzo Life Sciences Cat #ALX-270-333) 16. Matrigel basement membrane matrix (Corning Cat #354234) 17.TrypLE Express Enzyme(ThermoFisher scientific Cat #12604013) 18. Aggrewell® 400, 24-well plates (Stemcell Technologies Cat #34421) 19. Anti-adhesion cleaning solution (Stemcell Technologies Cat #07010) Organoid differentiation medium A small amount of basal organoid differentiation medium can be prepared and stored at 4°C for up to one month.

[0212] Organoid differentiation medium composition for 100 ml: 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 μl of 2-mercaptoethanol, and 1 ml of penicillin-streptomycin were added. Additional factors, 1% Matrigel, 100 nM retinoic acid, 17 nM IGF1, 100 nM insulin, 10 ng / ml FGF9, 500 ng / ml PGD2, 20 ng / ml BMP4, 50 ng / ml EGF1, and 5 μM Y-27632 were mixed immediately before adding the medium to the cells.

[0213] 1. Preparation of plates for organoid differentiation Before initiating organoid differentiation, prepare an Aggrewell® 400-24 plate or a 96-well U-bottom plate using the following procedure.

[0214] a- Add 500 μl of anti-adhesion washing solution to each well of an Aggrewell® 400-24 well plate and incubate at room temperature for 30 minutes.

[0215] b- Aspirate the anti-adhesion washing solution and wash thoroughly with 1 ml of PBS- / - per well.

[0216] Aspirate c-PBS- / - and add 500 μl of organoid differentiation medium to each well of an Aggrewell® 400-24 well plate. Add 100 μl of organoid differentiation medium to a 96-well U-bottom plate.

[0217] d- Centrifuge the plate at 500 RCF for 5 minutes to remove any trapped air in the wells. This prepares the plate for cell seeding.

[0218] 2. Differentiation of organoids a- Aspirate the AIM differentiation medium and wash the cells, which have been differentiated for 4 days, three times with 2 ml of PBS- / - per well in a 6-well plate.

[0219] b- Add 500 μl of TrypLE Express Enzyme to each well of a 6-well plate and incubate in a CO2 incubator for 5 minutes.

[0220] c- Remove the cells from the incubator and quench the TrypLE Express Enzyme by adding 2 mL of growth medium per well in a 6-well plate.

[0221] The d- cells were centrifuged at 1000 RPM for 5 minutes, the liquid was aspirated, and the paletted cells were diluted with organoid differentiation medium.

[0222] e- Approximately 300,000 cells were mixed in 500 μl of organoid differentiation medium and added to each well of a pre-prepared Aggrewell® plate. When using a 96-well plate, approximately 25,000 cells from 100 μl of organoid differentiation medium were transferred to a nonstick 96u bottom-well plate.

[0223] Incubate the plate in an f-CO2 incubator for 10 minutes to allow the cells to settle in the wells.

[0224] Centrifuge the g-Aggrewell® plate or 96-well plate again at 500 RCF for 5 minutes.

[0225] Incubate the cells in an h-CO2 incubator.

[0226] i- After leaving the organoids being formed for two days, half of the culture medium was replaced daily with fresh organoid differentiation medium.

[0227] j- For RNA isolation and qPCR analysis of terminal differentiation markers, organoids were collected from Aggrewell® plates on days 6 and 8 of differentiation. On day 7, organoids were collected from or transferred to 96-well plates. 96-well plates are a better method! The data was confirmed by whole-mount immunostaining of the k-progenitor cell marker CoupTf2, Sertoli cell markers Gata4, Sox9, and Wt1, and Leydig cell markers 3BHSD, STAR, and Cyp17a1.

[0228] [Extended culture of organoids in trans-wells] (material) 1. DMEM-F12 medium (ThermoFisher Scientific Cat #11320033) 2-2-mercaptoethanol (ThermoFisher Scientific Cat #21985023) 3. Sodium pyruvate solution (Sigma Aldrich Cat #S8636-100ML) 4-MEM non-essential amino acids (ThermoFisher Scientific Cat #11140050) 5- Glutamax(ThermoFisher scientific Cat #35050061) 6. Knockout serum replacer (ThermoFisher Scientific Cat #10828010) 7- Penicillin-Streptomycin (5,000 U / mL) (ThermoFisher Scientific Cat #15070063) 8- FGF9(R&D systems Cat #273-F9-025) 9- BMP4(R and D systems Cat #314-BP-500) 10- EGF1 11- Retinoic acid (Sigma-Aldrich Cat #R2625) 12- IGF1(Sigma-Aldrich cat #I3769) 13- Insulin (Sigma-Aldrich cat #I9278) 14- Prostaglandin D2 (Cayman Chemical Cat #12010) 15- ROCK inhibitor Y-27632 (Enzo Life Sciences Cat #ALX-270-333) 16- Matrigel basement membrane matrix (Corning 354234) 17. Follicle-stimulating hormone (FSH) (Sigma-Aldrich Cat #F4021) 18- Luteinizing Hormone (LH) (Sigma-Aldrich Cat #L6220) 19- Testosterone 20- TrypLE Express Enzyme(ThermoFisher scientific Cat #12604013) ThinCert® Tissue Culture Insert (Greiner Bio Cat #10443845) [2. Extended culture including the step of transferring organoids from a 96-well plate or Aggrewell® plate onto a trans-well] a) Before implanting the organoids into the trans-wells, ThinCert® cell culture inserts were immersed overnight in 700 μl of organoid reaggregation medium in a 24-well plate.

[0229] b) Using a large-diameter pipette tip for 200 μl, the organoid was pipetted with approximately 10 μl of culture medium and implanted in the center of the trans well.

[0230] c) The plates were incubated in a CO2 incubator.

[0231] d) Half of the culture medium was replaced with fresh re-aggregating medium every other day.

[0232] e) On days 7, 14, and 17, the implants were collected and fixed with 4% PFA for 10 minutes.

[0233] f) For immunohistochemical staining, the grafts were embedded using OCT and frozen sections were prepared.

[0234] [Results and Discussion] An overview of the differentiation protocol is shown in Figure 1. This protocol is an improved version of the protocol described in PCT US2023 / 13608, the entire content of which is incorporated herein by reference. Modifications to the protocol include the addition of BMP4 on days 2–4, the reduction of FGF9 concentration in ALLGF, and the addition of BMP4 and EGF1. These modifications resulted in improved expression of many gonadal and Sertoli cell markers (see Figure 2).

[0235] Furthermore, extending culture of the organoid at the gas-liquid interface to day 8 improves Leydig cell production. Specifically, on day 17, Leydig cells that are double positive for SF1 and 3βHSD are detected. These Leydig cells are abundantly present within clusters, similar to that observed in mouse and human testes (see Figure 3). Under improved culture conditions, in addition to identification of Leydig cells, further maturation of Sertoli cells (Figure 4A) was detected, and detection of peritubular myoid cell markers (Figure 4B) was also confirmed.

[0236] Pure populations of Leydig cells and peritubular myoid cells are obtained by first culturing to day 8 according to the above protocol, then isolating the cells, and continuing culture of gonadal progenitor cells on Matrigel for an additional 3 to 8 days using established differentiation methods into Leydig cells and myoid cells. Simply applying existing culture media for Leydig cells and myoid cells to ESCs is insufficient to generate myoid cells and Leydig cells.

[0237] Finally, by transferring day 4 anterior intermediate mesoderm cells to non-stick 96U-bottom plates, then transferring the organoids to Transwell® plates, and subsequently adding FSH+HCG or FSH+LH, in vitro differentiation of testicular somatic cells and tubule formation were significantly improved (Figures 12-17). Indeed, these organoids produce testosterone and corticosterone.

[0238] [Example 2] The following examples describe reagents and protocols for producing artificial testicular cells from human embryonic stem cells (hESCs) or iPSCs according to the present invention.

[0239] [Directed differentiation of human ESCs into multiple testis-like somatic cells.] A schematic diagram of this protocol is shown in Figure 5.

[0240] (Reagents) Media and supplements 1. STEMdiff APEL medium (Stem Cell Technologies, cat.no.05270 or no.05275) 2.DMEM / F-12(ThermoFisher scientific cat.no.11320-082) 3.DMSO (Sigma Aldrich, cat.no.D5879) 4. Dulbecco's phosphate-buffered saline (DPBS) (ThermoFisher Scientific, Cat. no. 14190-144) 5. Accutase (Cat.no.07920) 6.0.5M EDTA stock solution (Lonza 51201) 7.Quick-RNA Miniprep Kit (Zymo Research, Cat.No.R1055) 8. Anti-adhesion cleaning solution (Stem cell Technologies Cat.No.07010) 9. Primary antibody solution 1 (EMD Millipore, Cat.No.KP31812-200ml) 10.Secondary antibody solution 2 (EMD Millipore, Cat.No.KP31855-200ml) 11.0.5M EDTA stock (Lonza 51201) 12. Cleaning solution 13. CHIR99021 (R&D, cat.no.9902): Stock solution (10mM) Gently centrifuge the tube before opening. Reconstitute 10mg of CHIR99021 in 2.149ml of DMSO to make a 10mM stock solution. Dispense into 20μL aliquots, label with "Ch", and store at -20°C. Thawed tubes can be stored at 4°C for 24 hours.

[0241] 14. Y27632 ROCK inhibitor (Enzo, Cat. no. ALX-270-333): Dissolve 1 mg of stock solution (5 mM) in 625 μL of sterile TC water. Dispense into 20 μL aliquots and label with "Y". Store in a stem cell core at -20°C. Dilute with 10 mL of culture medium to a final concentration of 10 μM. Discard the tubing after use.

[0242] 15. Heparin (Sigma Aldrich, cat.no.: H4784-250mg): Stock solution (1mg ml) -1 ) 1 mg ml in ultrapure water -1 Reconstitute the solution and filter it through a 0.22 μm polyethersulfone (PES) filter (syringe-driven filter unit). Dispense into 40 μL portions and store at 4°C for up to 12 months.

[0243] 16. FGF9 (R&D, cat.no.273-F9-025): Stock solution FGF9 (100 μg / ml). Gently centrifuge before opening the tube. Reconstitute into 100 μg / ml filtered DPBS containing 0.1% (wt / vol) bovine serum albumin. Dispense into 20 μl portions and store at -80°C for up to 6 months. Store the stock at -80°C. Thawed FGF9 aliquots can be stored at 4°C for up to 2 weeks.

[0244] 17. RA (Sigma, cat.no. R2625-5mg): Stock solution (0.5mm) Dissolve 1mg of RA in 3ml of DMSO to prepare a 1.11mM stock solution. Dilute the stock solution to 0.5mM and prepare 50μl or 100μl dispensing solutions. This working solution can be stored at -20°C. Use the 0.5mM stock solution in the cell culture medium at the concentration required for the experiment. Discard the tube after use.

[0245] 18. Insulin (Sigma, cat.no.I9278): Stock solution (1.7mM) obtained from the vendor.

[0246] 19. IGF1-50μg (Sigma, Cat.no.I3769): Stock solution (10 mM) Dissolve 50 μg of IGF1 in 658 μl of 0.2% acetic acid to make a 10 mM stock solution. Dispense into 10 μl aliquots and store at -80°C. Dilute the stock solution to 100 μM with 0.2% acetic acid and add to the cell culture medium according to the concentration required for the experiment. Thawed tubes can be stored at 4°C for up to 1 month.

[0247] 20. PGD2 (Cayman, cat.no.12010): Stock solution (2 mg / ml) Dissolve PGD2 in 200 μl of PBS (pH 7.4) to make a stock concentration of 2 mg / ml. Dispense into 5 μl aliquots and store at -80°C. Discard the tubing after use.

[0248] 21. Matrigel (Corning, cat.no.354234, Lot no.9133008): Stock solution (2 mg / ml or 1 mg / ml) Dilute Matrigel with DMEM / F12 medium to the desired concentration (2 mg / ml or 1 mg / ml), dispense, and store at -20°C until use.

[0249] 22. Testosterone (Sigma, cat.no.T1500-5g): Stock solution (5mM) 6g of testosterone was dissolved in 1ml of DMSO and serially diluted to a concentration of 5mM.

[0250] 23. Follicle-stimulating hormone (Sigma: cat.no.F4021-25ug) - Stock solution (100 μg / ml): Dissolve 25 ug of FSH in 250 μl of ultrapure water, dispense, and store at -20°C until use.

[0251] 24. Luteinizing hormone (Sigma: cat.no.L6420-10μg): Stock solution (100μg / ml) Dissolve 25μg of LH in 100μl of ultrapure water, dispense, and store at -20°C until use.

[0252] 25. Epithelial growth factor (EGF) derived from mouse submandibular gland (Sigma, cat.no.E4127-.1mg): Stock solution (100ug / ml) 0.1mg of EGF was dissolved in 10ml of 0.1% BSA / PBS, then aliquoted into 1ml portions and stored at -20°C until use.

[0253] 26. Dissolve 10 mg / ml of IWR1 (Sigma: cat no. 681669-10 mg) in DMSO, dispense, and store at -20°C until use.

[0254] (Materials) 1. Nunc™ 4-well dish for IVF (Fisher, cat. no. 144444) 2. Nunc™ cell culture-treated multi-dish - 24-well (Fisher, cat. no. 142475) 3. Ultra-low attachment U-bottom microplate (Corning, cat. no. 12-456-721) 4. Greiner Bio-One ThinCert® tissue culture insert (Greiner Bio-One, cat. no. 10443845) 5. Costar 24-well plate (Corning, Cat. No. 3526) 6. 12 mm coverslip (Hampton Research, Cat. No. HR3-277) 7. Benchtop centrifuge (Eppendrof, 5417c) 8. Biological safety cabinet 9. CO₂ incubator (Heraeus) 10. 15 mL conical tube (Falcon, cat. no. 352096) 11. 50 mL conical tube (Falcon, cat. no. 352070) 12. Cryocontainer (Nalgene, Mr. Frosty) 13. Inverted phase contrast tissue culture microscope (KL1500CD, Leica) 14. Laser scanning confocal microscope (Nikon Eclipse Ti2) 15. Pipette boy (Drummond) 16. Pipette (Gilson) 17. Automatic cell counter (BioRad, TC20) 18. Serological pipette (Fisher, Cat. No. 13-678-11D, 13-676-10J, 13-676-10R) 19. Stericup® 0.22 μm filter unit (Millipore, 03290) 20. Sterile filter pipette tips (Genesee Scientific, 24-815, 24-804, 24-830, 26-401) 21. Sterile microcentrifuge tube (Fisher, Cat. No. 05-408-120) 22. Aggrewell® 800 24-well, 5-pack (Stem cell technologies, Cat. No. 34815) Coverslip cleaning method: Shake the coverslip in xylene in a 50 mL conical tube for 2-3 hours.

[0255] Remove xylene and rinse with acetone. Remove the acetone, add fresh acetone to a 50 mL conical tube, and rinse for 2-3 hours.

[0256] Discard the acetone. If desired, autoclave in water using a liquid cycle. Rinse the coverslip twice with 100% ethanol. Store the coverslip in 100% ethanol at 4°C.

[0257] [Day 1: ESC plating in single-cell suspension to improve differentiation reproducibility] (Cell pellet (RNA-seq analysis) or plate coating for coverslip staining) Dry the coverslips at room temperature in a biological safety cabinet hood under sterile conditions until the alcohol has completely evaporated. After drying, transfer the coverslips to a 24-well or 4-well plate using forceps.

[0258] • Before plating the cells, coat the 24-well or 4-well plate with Matrigel for 1 hour.

[0259] (Single-cell suspension for monolayer use) Remove any differentiated colonies from the cultured colonies on a 60mm Petri dish.

[0260] Wash with 2 ml of DPBS buffer.

[0261] Add 0.5 ml of 1x Accutase and incubate for 5-10 minutes.

[0262] Add 2-5 ml of DMEM / F12 medium to stop dissociation.

[0263] Centrifuge at 1000 rpm for 3 minutes to pelletize the cells.

[0264] Resuspend the pellet in 5 ml of mTeSR medium containing 10 μM (Y27632) Rock inhibitor and count the cells.

[0265] • Plate 10,000 cells per well into a 24-well plate coated with Matrigel.

[0266] (ESC cells collected on day 0 as a control): • ES cells were collected on day 0.

[0267] Each well in a 24-well plate was washed with 0.5 ml of DPBS buffer.

[0268] • 0.5 ml of 1x Accutase was added and the mixture was left to stand in an incubator for 3-5 minutes.

[0269] Add 1 ml of DMEM / F12 medium to stop the digestion reaction.

[0270] • Centrifuge at 10,000g for 2 minutes.

[0271] • Aspirate the culture medium.

[0272] • Instantly freeze with liquid nitrogen and store at -80°C until use.

[0273] • Fix the cells in 4% PFA for 20 minutes, wash three times with DPBS, and store in DPBS at 4°C until use.

[0274] (Cells used to initiate differentiation) Remove the mTeSR medium containing the Rock inhibitor and add 0.5 ml of APEL2 medium + CHIR 3 μM.

[0275] • Replace the culture medium every two days.

[0276] (Day 2) Replace the culture medium with APEL2 medium + CHIR 3μM.

[0277] (Day 4: Unsegmented mesoderm) • Collect cells from the required number of wells.

[0278] Add 0.5 ml of 1x Accutase, incubate in an incubator for 3-5 minutes, and then add 1 ml of DMEM / F12 medium to stop the reaction.

[0279] • Centrifuge at 10,000g for 2 minutes.

[0280] • Aspirate the culture medium.

[0281] • Flash-freeze with liquid nitrogen and store at -80°C until use (unsegmented mesoderm).

[0282] • Fix the cells in 4% PFA for 20 minutes, wash three times with DPBS, and store in DPBS at 4°C until use.

[0283] (Start FGF9 phase) Replace the culture medium with 200 ng / ml FGF9 + 1 μg / ml heparin.

[0284] • Replace the culture medium every two days.

[0285] (Day 6) Replace the culture medium with 200 ng / ml FGF9 + 1 μg / ml heparin.

[0286] (Day 7 (Intermediate Mesoderm)) On day 7, collect the cells from the required number of wells.

[0287] Wash the cells in each well of the 24-well plate with 0.5 ml of DPBS buffer.

[0288] Add 0.5 ml of 1x Accutase, incubate in an incubator for 3-5 minutes, and then add 1 ml of DMEM / F12 medium to stop the reaction.

[0289] • Centrifuge at 10,000g for 2 minutes.

[0290] • Aspirate the culture medium.

[0291] • Flash-freeze with liquid nitrogen and store at -80°C until use (intermediate mesoderm).

[0292] • Fix the cells in 4% PFA for 20 minutes, wash three times with DPBS, and store in DPBS at 4°C until use.

[0293] However, the differentiation protocol is continued for the remaining wells: these wells are filled with APEL ± 0.1 μM RA + 100 nM insulin + 17 nM IGF1 + 500 ng / ml PGD2 + 200 ng / ml FGF9.

[0294] (Day 8) (Seed the dissociated gonadal cells into Aggrewell® plates or 96-well u-bottom plates.) (Preparation of Aggrewell® plates for receiving cells.) Add 0.5 ml of surface cleaning solution to each well. Ensure there are no air bubbles in the microwells. Centrifuge at 200 g for 5 minutes to remove air bubbles from each microwell.

[0295] Observe under a bright-field microscope to confirm that there are no air bubbles. If air bubbles remain, centrifuge again at 200g for 5 minutes.

[0296] Incubate the plate at room temperature or 37°C for 30 minutes. After incubation, remove the washing solution and wash with 0.5 ml of DBPS buffer. Ensure the wells are not dry before plating the cells.

[0297] Add 0.5 ml of APEL medium containing the corresponding growth factor, and centrifuge at 200 g for 5 minutes to remove air bubbles. Observe under a bright-field microscope to confirm that no air bubbles are present.

[0298] For the 96-well plate, add APEL medium (0.1 ml) containing the corresponding growth factor, and centrifuge at 200 g for 5 minutes to remove air bubbles. Observe under a bright-field microscope to confirm that no air bubbles are present.

[0299] (Cell plating preparation) • Dissociate the wells containing differentiated cells into single cells.

[0300] Wash the cells in each well of the 24-well plate with 0.5 ml of DPBS buffer.

[0301] Add 0.5 ml of 1x Accutase, incubate in an incubator for 3-5 minutes, and then add 1 ml of DMEM / F12 medium to stop the reaction.

[0302] • Centrifuge at 1000 rpm for 3 minutes.

[0303] • Aspirate the culture medium.

[0304] Resuspend the cells in APEL medium containing the corresponding growth factor and 10 μm of (Y27632) Rock inhibitor, and count the number of cells.

[0305] • Add 0.5 ml (1 × 10) per well to APEL medium containing the corresponding growth factor. 6 ~2×10 6Add the cells (1) and mix gently from top to bottom, then centrifuge at 500g for 5 minutes. Observe under a microscope to confirm that all cells have formed clusters. If using a 96-well plate, seed 30K cells in 100 μl.

[0306] Replace half of the culture medium every other day by gently removing 0.5 ml of old medium by touching the side of the well and adding 0.5 ml of fresh medium.

[0307] (Day 9) - Replace half of the medium every other day by gently touching the side of the well to remove 1 ml of old medium and adding 1 ml of fresh medium. The fresh medium should contain: APEL ± 0.1 μM RA + 100 nM insulin + 17 nM IGF + 500 ng / ml PGD2 ± 200 ng / ml FGF9 ± 1 μM testosterone ± 100 ng / ml follicle-stimulating hormone ± 200 ng / ml luteinizing hormone, or 2 U HCG ± 1 μM IWR1 + 50 ng / ml EGF.

[0308] (10th day (genital ridge)) On day 10, collect the organoids from the required number of wells. To collect the cells, use an uncut tip and pipette up and down to detach and suspend the organoids, then collect them in a 0.5 ml tube. Flash freeze in liquid nitrogen and store at -80°C until use.

[0309] • Fix some of the organoid wells with 4% PFA for 10 minutes, then wash three times with DPBS.

[0310] Incubate in 30% sucrose at 4°C for 72 hours. After incubation, remove the sucrose and wash three times with DBPS buffer for 5 minutes each. Store in DPBS buffer at 4°C until staining.

[0311] • Inject various combinations of growth factors into the remaining wells; change the culture medium every two days.

[0312] APEL ±0.1 μM RA + 100 nM insulin + 17 nM IGF1 + 500 ng / ml PGD2 ±200 ng / ml FGF9 + 1 μM testosterone ±100 ng / ml follicle-stimulating hormone ±200 ng / ml luteinizing hormone ±1 μM IWR1 + 50 ng / ml EGF.

[0313] (Day 12) Transfer the organoids to a ThinCert® cell culture insert and add 700 μl of culture medium to the lower chamber (APEL ±0.1 μM RA + 100 nM insulin + 17 nM IGF1 + 500 ng / ml PGD2 ±200 ng / ml FGF9 + 1 μM testosterone ±100 ng / ml follicle-stimulating hormone ±200 ng / ml luteinizing hormone ±1 μM IWR1 + 50 ng / ml EGF).

[0314] (Day 14) • Replace the culture medium.

[0315] APEL ±0.1 μM RA + 100 nM insulin + 17 nM IGF1 + 500 ng / ml PGD2 ±200 ng / ml FGF9 + 1 μM testosterone ±100 ng / ml follicle-stimulating hormone ±200 ng / ml luteinizing hormone ±1 μM IWR1 + 50 ng / ml EGF.

[0316] (Day 16) On day 16, collect the organoids from the required number of wells. To collect the cells, use an uncut tip and pipette up and down to detach and suspend the organoids, then collect them in a 0.5 ml tube. Flash freeze in liquid nitrogen and store at -80°C until use.

[0317] • Fix some of the organoid wells with 4% PFA for 10 minutes, then wash three times with DPBS.

[0318] Incubate in 30% sucrose at 4°C for 72 hours. After incubation, remove the sucrose and wash three times with DBPS buffer for 5 minutes each. Store in DPBS buffer at 4°C until staining.

[0319] • Inject various combinations of growth factors into the remaining wells; change the culture medium every two days.

[0320] APEL ±0.1 μM RA + 100 nM insulin + 17 nM IGF1 + 500 ng / ml PGD2 ±200 ng / ml FGF9 + 1 μM testosterone ±100 ng / ml follicle-stimulating hormone ±200 ng / ml luteinizing hormone + ±1 μM IWR1 + 50 ng / ml EGF.

[0321] (Day 18) • Change the culture medium and add growth factors every two days.

[0322] APEL ±0.1 μM RA + 100 nM insulin + 17 nM IGF1 + 500 ng / ml PGD2 ±200 ng / ml FGF9 + 1 μM testosterone ±100 ng / ml follicle-stimulating hormone ±200 ng / ml luteinizing hormone ±1 μM IWR1 + 50 ng / ml EGF.

[0323] (Day 20) • Change the culture medium and add growth factors every two days.

[0324] APEL ±0.1 μM RA + 100 nM insulin + 17 nM IGF1 + 500 ng / ml PGD2 ±200 ng / ml FGF9 + 1 μM testosterone ±100 ng / ml follicle-stimulating hormone ±200 ng / ml luteinizing hormone ±1 μM IWR1 + 50 ng / ml EGF.

[0325] (Day 22) On day 22 (testicular organoids), collect organoids from the required number of wells. To collect cells, use an uncut tip and pipette up and down to detach and suspend the organoids, then collect them in a 0.5 ml tube. Flash freeze with liquid nitrogen and store at -80°C until use.

[0326] • Fix some of the organoid wells with 4% PFA for 10 minutes, then wash three times with DPBS.

[0327] • Incubate in 30% sucrose at 4°C for 72 hours. After incubation, remove the sucrose and wash three times with DPBS buffer for 5 minutes each. Store in DBPS buffer at 4°C until staining. These samples are prepared for immunohistochemistry.

[0328] (Coverslip staining protocol d4 / d7 monolayer cells.) Fix the cells with 4% PFA for 20 minutes.

[0329] Wash three times in PBS- / - for 5 minutes each, then store in PBS- / - at 4°C until staining.

[0330] • Rinse quickly three times with PBS- / - before staining.

[0331] Blocking buffer PBS+ / +, 0.1% Triton-x 100, 10% donkey serum or goat serum, and 10% Na-Azide (8.85 ml PBS+ / +, 50 μl Triton-X 100 + 1 ml donkey or goat serum + 100 μl Na-Azide).

[0332] Add 200 μl of blocking buffer to a 24-well plate and incubate for 1 hour in a lidded, humidified chamber at room temperature.

[0333] Add 200 μl of primary antibody to a 24-well plate and incubate overnight in a lidded, humidified chamber at 4°C.

[0334] • Dilute the blocking buffer to 50% as the primary antibody buffer (PBS+ / +, 0.25% Triton-X, 2.5% donkey or goat serum + 50 μl of NA-Azide).

[0335] The following day, wash the slides three times each with PBS- / - and 0.1% Triton-X for 10 minutes each.

[0336] Add 200 μl of secondary Ab along with DAPI and allow to stand for 1.5 hours in a lidded humidified chamber at room temperature.

[0337] (8.85 ml of PBS+ / +, 1 ml of donkey or goat serum + 100 ml of Na-Azide) Wash with PBS- / - three times for 10 minutes each.

[0338] Mount the image onto a coverslip using Vectashield or Prolong gold, dry it, and store it at 4°C until imaging.

[0339] (Organoid whole-mount staining protocol) Fix the cells with 4% PFA for 10 minutes.

[0340] Rinse with PBS- / - for 5 minutes, three times.

[0341] Incubate in 30% sucrose at 4°C for 72 hours. After incubation, remove the sucrose and wash three times with DPBS buffer for 5 minutes each. Store in DBPS buffer at 4°C until staining.

[0342] • Rinse quickly three times with PBS- / - before staining.

[0343] Blocking buffer PBS+ / +, 0.1% Triton-x 100, 10% donkey serum or goat serum, and 10% Na-Azide (8.85 ml PBS+ / +, 50 μl Triton-X 100 + 1 ml donkey or goat serum + 100 μl Na-Azide).

[0344] Add 50-100 μl of blocking buffer to a 96-well plate or 0.5 ml tube and incubate on a shaker at room temperature for 2-3 hours.

[0345] Add 50-100 μl of primary antibody to a 96-well plate or 0.5 ml tube and incubate overnight on a shaker at 4°C.

[0346] Dilute the blocking buffer to 50% using either primary antibody buffer (PBS+ / +, 0.25% Triton-X, 2.5% donkey or goat serum + 50 μl NA-Azide) or Solution 1.

[0347] The following day, wash the slides six times each with PBS- / - and 0.1% Triton-X for 10 minutes each.

[0348] Add 50-100 μl of secondary antibody along with DAPI to a 96-well plate or 0.5 ml tube, and incubate overnight on a shaker at 4°C.

[0349] (8.85 ml PBS+ / +, 1 ml donkey serum or goat serum + 100 ml Na-Azide) or Solution 2.

[0350] Wash with PBS- / - three times for 10 minutes each.

[0351] Mount the image onto a coverslip using Vectashield or Prolong gold, dry it, and store it at 4°C until imaging.

[0352] [Targeted differentiation of human iPSCs into multiple testicular-like cells.] A schematic diagram of this protocol is shown in Figure 6.

[0353] (reagent) Culture medium and supplements 1. STEMdiff APEL medium (Stem Cell Technologies, cat.no.05270 or no.05275) 2.DMEM / F-12(ThermoFisher scientific cat.no.11320-082) 3.DMSO (Sigma Aldrich, cat.no.D5879) 4. Dulbecco's phosphate-buffered saline (DPBS) (ThermoFisher Scientific, Cat. no. 14190-144) 5. Accutase (Cat.no.07920) 6.0.5M EDTA stock solution (Lonza 51201) 7.Quick-RNA Miniprep Kit (Zymo Research, Cat.No.R1055) 8. Anti-adhesion cleaning solution (Stem cell Technologies Cat.No.07010) 9. Primary antibody solution 1 (EMD Millipore, Cat.No.KP31812-200ml) 10.Secondary antibody solution 2 (EMD Millipore, Cat.No.KP31855-200ml) 11.0.5M EDTA stock (Lonza 51201) 12. Cleaning solution 13. CHIR99021 (R&D, cat.no.9902): Stock solution (10mM) Gently centrifuge the tube before opening. Reconstitute 10mg of CHIR99021 in 2.149ml of DMSO to make a 10mM stock solution. Dispense into 20μL aliquots, label with "Ch", and store at -20°C. Thawed tubes can be stored at 4°C for 24 hours.

[0354] 14. Y27632 ROCK Inhibitor (Enzo, Cat.no.ALX-270-333): Stock solution (5 mM) Dissolve 1 mg in 625 μL of sterile TC water. Aliquot into 20 μL portions, label as "Y", and store at -20°C in the stem cell core. Dilute with 10 mL of culture medium to a final concentration of 10 μM. Discard the tube after use.

[0355] 15. Heparin (Sigma Aldrich, cat.no.: H4784-250mg): Stock solution (1 mg / ml -1 ) Reconstitute to 1 mg / ml -1 with ultrapure water, and filter through a 0.22 μm polyethersulfone (PES) filter (syringe-driven filter unit). Aliquot into 40 μL portions and store at 4°C for up to 12 months.

[0356] 16. FGF9 (R&D, cat.no. 273-F9-025): Stock solution (100 μg / ml) Centrifuge briefly before opening the tube. Reconstitute to 100 μg / ml in filtered DPBS containing 0.1% (wt / vol) bovine serum albumin. Aliquot into 20 μl portions and store at -80°C for up to 6 months. Store stock at -80°C. Thawed FGF9 aliquots can be stored at 4°C for up to 2 weeks.

[0357] 17. RA (Sigma, cat.no. R2625-5mg): Stock solution (0.5 mM) Dissolve 1 mg of RA in 3 mL of DMSO to prepare a 1.11 mM stock solution. Dilute the stock solution to 0.5 mM and prepare 50 μl or 100 μl aliquots. This working solution can be stored at -20°C. Use the 0.5 mM stock solution in cell culture medium according to the desired concentration for the experiment. Discard the tube after use.

[0358] 18. Insulin (Sigma, cat.no. I9278): Obtained from supplier as stock solution (1.7 mM).

[0359] 19. IGF1-50μg (Sigma, Cat.no.I3769): Stock solution (10 mM) Dissolve 50 μg of IGF1 in 658 μl of 0.2% acetic acid to make a 10 mM stock solution. Dispense into 10 μl aliquots and store at -80°C. Dilute the stock solution to 100 μM with 0.2% acetic acid and add to the cell culture medium according to the concentration required for the experiment. Thawed tubes can be stored at 4°C for up to 1 month.

[0360] 20. PGD2 (Cayman, cat.no.12010): Stock solution (2 mg / ml) Dissolve PGD2 in 200 μl of PBS (pH 7.4) to make a stock concentration of 2 mg / ml. Dispense into 5 μl aliquots and store at -80°C. Discard the tubing after use.

[0361] 21. Matrigel (Corning, cat.no.354234, Lot no.9133008): Stock solution (2 mg / ml or 1 mg / ml) Dilute Matrigel with DMEM / F12 medium to the desired concentration (2 mg / ml or 1 mg / ml), dispense, and store at -20°C until use.

[0362] 22. Testosterone (Sigma, cat.no.T1500-5g): Stock solution (5mM) 6g of testosterone was dissolved in 1ml of DMSO and serially diluted to a concentration of 5mM.

[0363] 23. Follicle-stimulating hormone (Sigma: cat.no.F4021-25ug) - Stock solution (100 μg / ml): Dissolve 25 ug of FSH in 250 μl of ultrapure water, dispense, and store at -20°C until use.

[0364] 24. Luteinizing hormone (Sigma: cat.no.L6420-10μg): Stock solution (100μg / ml) Dissolve 25μg of LH in 100μl of ultrapure water, dispense, and store at -20°C until use.

[0365] 25. Epithelial growth factor (EGF) derived from mouse submandibular gland (Sigma, cat.no.E4127-.1mg): Stock solution (100ug / ml) 0.1mg of EGF was dissolved in 10ml of 0.1% BSA / PBS, then aliquoted into 1ml portions and stored at -20°C until use.

[0366] 26. IWR1 (Sigma: cat no. 681669-10mg) - Stock solution: Dissolve 10mg / ml in DMSO, aliquot, and store at -20°C until use.

[0367] (material) 1. Nunc® 4 Welldish for IVF (Fisher, cat.no.144444) 2. Nunc® Cell Culture-Treated Multi-Dish - 24 Wells (Fisher, Cat. No. 142475) 3. Ultra-low adsorption U-shaped bottom microplate (Corning, cat. no. 12-456-721) 4. Greiner Bio-One ThinCert® Tissue Culture Insert (Greiner Bio-One, cat.no.10443845) 5. Costar 24-well plate (Corning, Cat. No. 3526) 6. Coverslip 12mm (Hampton Research, Cat. No. HR3-277) 7. Benchtop centrifuge (Eppendrof, 5417c) 8. Biological safety cabinet 9. CO2 Incubator (Heraeus) 10. Conical tube 15ml (Falcon, cat.no.352096) 11. Conical tube 50ml (Falcon, cat.no.352070) 12. Freezer container (Nalgene, Mr. Frosty) 13. Inverted phase-contrast tissue culture microscope (KL1500CD, Leica) 14. Laser confocal microscope (Nikon Eclipse Ti2) 15. Pipette Boy (Drummond) 16. Pipette (Gilson) 17. Automatic cell counter (BioRad, TC20) 18. Serum pipettes (Fisher, Cat. No. 13-678-11D, 13-676-10J, 13-676-10R) 19. Stericup (registered trademark) 0.22 μm filter unit (Millipore, 03290) 20. Sterile filter pipette tips (Genesee Scientific, 24-815, 24-804, 24-830, 26-401) 21. Sterile microcentrifuge tube (Fisher, Cat. No. 05-408-120) 22. Aggrewell® 800 24-well, 5-pack (Stem cell technologies, Cat. No. 34815) Coverslip cleaning method: Shake the coverslip in xylene in a 50 mL conical tube for 2-3 hours.

[0368] Remove xylene and rinse with acetone. Remove the acetone, add fresh acetone to a 50 mL conical tube, and rinse for 2-3 hours.

[0369] Discard the acetone. If desired, autoclave in water using a liquid cycle. Rinse the coverslip twice with 100% ethanol. Store the coverslip in 100% ethanol at 4°C.

[0370] [Day 1: IPSC plating in single-cell suspension to improve differentiation reproducibility] (Cell pellet (RNA-seq analysis) or plate coating for coverslip staining) Dry the coverslips at room temperature in a biological safety cabinet hood under sterile conditions until the alcohol has completely evaporated. After drying, transfer the coverslips to a 24-well or 4-well plate using forceps.

[0371] • Before plating the cells, coat the 24-well or 4-well plate with Matrigel for 1 hour.

[0372] (Single-cell suspension for monolayer use) Remove any differentiated colonies from the cultured colonies on a 60mm Petri dish.

[0373] Wash with 2 ml of DPBS buffer.

[0374] Add 0.5 ml of 1x Accutase and incubate for 5-10 minutes.

[0375] Add 2-5 ml of DMEM / F12 medium to stop dissociation.

[0376] Centrifuge at 1000 rpm for 3 minutes to pelletize the cells.

[0377] Resuspend the pellet in 5 ml of mTeSR medium containing 10 μM (Y27632) Rock inhibitor and count the cells.

[0378] • Plate 10,000 cells per well into a 24-well plate coated with Matrigel.

[0379] (As a control, iPSC cells were collected on day 0): • IPSC cells were collected on day 0.

[0380] Each well in a 24-well plate was washed with 0.5 ml of DPBS buffer.

[0381] • 0.5 ml of 1x Accutase was added and the mixture was left to stand in an incubator for 3-5 minutes.

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

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

[0384] • Aspirate the culture medium.

[0385] • Instantly freeze with liquid nitrogen and store at -80°C until use.

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

[0387] (Cells used to initiate differentiation) Remove the mTeSR medium containing the Rock inhibitor and add 0.5 ml of APEL2 medium + CHIR 8 μM.

[0388] The culture medium should be changed every two days.

[0389] (Day 2) Remove the culture medium and add APEL2 medium + CHIR 8μM.

[0390] (Day 4: Unsegmented mesoderm) • Collect cells from the required number of wells.

[0391] Add 0.5 ml of 1x Accutase, incubate in an incubator for 3-5 minutes, and then add 1 ml of DMEM / F12 medium to stop the reaction.

[0392] • Centrifuge at 10,000g for 2 minutes.

[0393] • Aspirate the culture medium.

[0394] • Flash-freeze with liquid nitrogen and store at -80°C until use (unsegmented mesoderm).

[0395] • Fix the cells in 4% PFA for 20 minutes, wash three times with DPBS, and store in DPBS at 4°C until use.

[0396] (Start FGF9 phase) Replace the culture medium with 200 ng / ml FGF9 + 1 μg / ml heparin.

[0397] • Replace the culture medium every two days.

[0398] (Day 6) Replace the culture medium with 200 ng / ml FGF9 + 1 μg / ml heparin.

[0399] (Day 7 (Intermediate Mesoderm)) On day 7, collect the cells from the required number of wells.

[0400] Wash the cells in each well of the 24-well plate with 0.5 ml of DPBS buffer.

[0401] Add 0.5 ml of 1x Accutase, incubate in an incubator for 3-5 minutes, and then add 1 ml of DMEM / F12 medium to stop the reaction.

[0402] • Centrifuge at 10,000g for 2 minutes.

[0403] • Aspirate the culture medium.

[0404] • Flash-freeze with liquid nitrogen and store at -80°C until use (intermediate mesoderm).

[0405] • Fix the cells in 4% PFA for 20 minutes, wash three times with DPBS, and store in DPBS at 4°C until use.

[0406] • Continue the differentiation protocol for the remaining wells: These wells will be fed the following culture medium: APEL ± 0.1 μM RA + 100 nM insulin + 17 nM IGF1 + 500 ng / ml PGD2 + 200 ng / ml FGF9 + 50 ng / ml EGF1.

[0407] (Day 8) (Preparation of gonadal organoids in Aggrewell® plates or 96-well plates) (Preparation of Aggrewell® plates for receiving cells.) Add 0.5 ml of surface cleaning solution to each well. Ensure there are no air bubbles in the microwells. Centrifuge at 200 g for 5 minutes to remove air bubbles from each microwell.

[0408] Observe under a bright-field microscope to confirm that there are no air bubbles. If air bubbles remain, centrifuge again at 200g for 5 minutes.

[0409] Incubate the plate at room temperature or 37°C for 30 minutes. After incubation, remove the washing solution and wash with 0.5 ml of DBPS buffer. Ensure the wells are not dry before plating the cells.

[0410] Add 0.5 ml of APEL medium containing the corresponding growth factor, and centrifuge at 200 g for 5 minutes to remove air bubbles. Observe under a bright-field microscope to confirm that no air bubbles are present.

[0411] • When using a 96-well plate, add 0.1 ml of APEL medium containing the corresponding growth factor, and centrifuge at 200 g for 5 minutes to remove air bubbles. Observe under a bright-field microscope to confirm that no air bubbles are present.

[0412] (Cell plating preparation) • Dissociate the wells containing differentiated cells into single cells.

[0413] Wash the cells in each well of the 24-well plate with 0.5 ml of DPBS buffer.

[0414] Add 0.5 ml of 1x Accutase, incubate in an incubator for 3-5 minutes, and then add 1 ml of DMEM / F12 medium to stop the reaction.

[0415] • Centrifuge at 1000 rpm for 3 minutes.

[0416] • Aspirate the culture medium.

[0417] Resuspend the cells in APEL medium containing the corresponding growth factor and 10 μm of (Y27632) Rock inhibitor, and count the number of cells.

[0418] • Add 0.5 ml (1 × 10) per well to APEL medium containing the corresponding growth factor. 6 ~2×10 6 Add the cells (1) and mix slowly from top to bottom, then centrifuge at 500g for 5 minutes. Observe under a microscope to confirm that all cells have formed clusters.

[0419] • When using a 96-well plate, add 30K cells to 100 μl of differentiation medium. Mix the cells, stirring gently from top to bottom, and centrifuge at 500 g for 5 minutes. Observe under a microscope to confirm that all cells have formed clusters.

[0420] Replace half of the culture medium every other day by gently removing 0.5 ml of old medium by touching the side of the well and adding 0.5 ml of fresh medium.

[0421] (Day 9) - Replace half of the medium every other day by gently touching the side of the well to remove 1 ml of old medium and adding 1 ml of fresh medium. The fresh medium shall contain: APEL ±0.1 μM RA + 100 nM insulin + 17 nM IGF1 + 500 ng / ml PGD2 ±200 ng / ml FGF9 ±1 μM testosterone ±100 ng / ml follicle-stimulating hormone ±200 ng / ml luteinizing hormone ±1 μM IWR1.

[0422] (10th day (genital ridge)) On day 10, collect the organoids from the required number of wells. To collect the cells, use an uncut tip and pipette up and down to detach and suspend the organoids, then collect them in a 0.5 ml tube. Flash freeze in liquid nitrogen and store at -80°C until use.

[0423] • Fix some of the organoid wells with 4% PFA for 10 minutes, then wash three times with DPBS.

[0424] Incubate in 30% sucrose at 4°C for 72 hours. After incubation, remove the sucrose and wash three times with DPBS buffer for 5 minutes each. Store in DBPS buffer at 4°C until staining.

[0425] • Inject various combinations of growth factors into the remaining wells; change the culture medium every two days.

[0426] APEL ±0.1 μM RA + 100 nM insulin + 17 nM IGF1 + 500 ng / ml PGD2 ±200 ng / ml FGF9 + 1 μM testosterone ±100 ng / ml follicle-stimulating hormone ±200 ng / ml luteinizing hormone ±1 μM IWR1.

[0427] (Day 12) Transfer the organoids to a ThinCert® cell culture insert and add 700 μl of culture medium (APEL ± 0.1 μM RA + 100 nM insulin + 17 nM IGF1 + 500 ng / ml PGD2 ± 200 ng / ml FGF9 + 1 μM testosterone ± 100 ng / ml follicle-stimulating hormone ± 200 ng / ml luteinizing hormone ± 1 μM IWR1) to the bottom well.

[0428] (Day 14) • Replace the culture medium.

[0429] APEL ±0.1 μM RA + 100 nM insulin + 17 nM IGF1 + 500 ng / ml PGD2 ±200 ng / ml FGF9 + 1 μM testosterone ±100 ng / ml follicle-stimulating hormone ±200 ng / ml luteinizing hormone ±1 μM IWR1.

[0430] (Day 16) On day 16, collect the organoids from the required number of wells. To collect the cells, use an uncut tip and pipette up and down to detach and suspend the organoids, then collect them in a 0.5 ml tube. Flash freeze in liquid nitrogen and store at -80°C until use.

[0431] • Fix some of the organoid wells with 4% PFA for 10 minutes, then wash three times with DPBS.

[0432] Incubate in 30% sucrose at 4°C for 72 hours. After incubation, remove the sucrose and wash three times with DPBS buffer for 5 minutes each. Store in DBPS buffer at 4°C until staining.

[0433] • Inject various combinations of growth factors into the remaining wells; change the culture medium every two days.

[0434] APEL ±0.1 μM RA + 100 nM insulin + 17 nM IGF1 + 500 ng / ml PGD2 ±200 ng / ml FGF9 + 1 μM testosterone ±100 ng / ml follicle-stimulating hormone ±200 ng / ml luteinizing hormone + ±1 μM IWR1.

[0435] (Day 18) • Change the culture medium and add growth factors every two days.

[0436] APEL ±0.1 μM RA + 100 nM insulin + 17 nM IGF1 + 500 ng / ml PGD2 ±200 ng / ml FGF9 + 1 μM testosterone ±100 ng / ml follicle-stimulating hormone ±200 ng / ml luteinizing hormone ±1 μM IWR1.

[0437] (Day 20) • Change the culture medium and add growth factors every two days.

[0438] APEL ±0.1 μM RA + 100 nM insulin + 17 nM IGF1 + 500 ng / ml PGD2 ±200 ng / ml FGF9 + 1 μM testosterone ±100 ng / ml follicle-stimulating hormone ±200 ng / ml luteinizing hormone ±1 μM IWR1.

[0439] (Day 22) On day 22 (testicular organoids), collect organoids from the required number of wells. To collect cells, use an uncut tip and pipette up and down to detach and suspend the organoids, then collect them in a 0.5 ml tube. Flash freeze with liquid nitrogen and store at -80°C until use.

[0440] • Fix some of the organoid wells with 4% PFA for 10 minutes, then wash three times with DPBS.

[0441] Incubate in 30% sucrose at 4°C for 72 hours. After incubation, remove the sucrose and wash three times with DPBS buffer for 5 minutes each. Store in DBPS buffer at 4°C until staining.

[0442] (Single-layer coverslip staining protocol) Fix the cells with 4% PFA for 20 minutes.

[0443] Wash three times in PBS- / - for 5 minutes each, then store in PBS- / - at 4°C until staining.

[0444] • Rinse quickly three times with PBS- / - before staining.

[0445] Blocking buffer PBS+ / +, 0.1% Triton-x 100, 10% donkey serum or goat serum, and 10% Na-Azide (8.85 ml PBS+ / +, 50 μl Triton-X 100 + 1 ml donkey or goat serum + 100 μl Na-Azide).

[0446] Add 200 μl of blocking buffer to a 24-well plate and incubate for 1 hour in a lidded, humidified chamber at room temperature.

[0447] Add 200 μl of primary antibody to a 24-well plate and incubate overnight in a lidded, humidified chamber at 4°C.

[0448] • Dilute the blocking buffer to 50% as the primary antibody buffer (PBS+ / +, 0.25% Triton-X, 2.5% donkey or goat serum + 50 μl of NA-Azide).

[0449] The following day, wash the slides three times each with PBS- / - and 0.1% Triton-X for 10 minutes each.

[0450] Add 200 μl of secondary Ab along with DAPI and allow to stand for 1.5 hours in a lidded humidified chamber at room temperature.

[0451] (8.85 ml of PBS+ / +, 1 ml of donkey or goat serum + 100 ml of Na-Azide) Wash with PBS- / - three times for 10 minutes each.

[0452] Mount the image onto a coverslip using Vectashield or Prolong gold, dry it, and store it at 4°C until imaging.

[0453] (Organoid whole-mount staining protocol) Fix the cells with 4% PFA for 10 minutes.

[0454] Rinse with PBS- / - for 5 minutes, three times.

[0455] Incubate in 30% sucrose at 4°C for 72 hours. After incubation, remove the sucrose and wash three times with DPBS buffer for 5 minutes each. Store in DBPS buffer at 4°C until staining.

[0456] • Rinse quickly three times with PBS- / - before staining.

[0457] Blocking buffer PBS+ / +, 0.1% Triton-x 100, 10% donkey serum or goat serum, and 10% Na-Azide (8.85 ml PBS+ / +, 50 μl Triton-X 100 + 1 ml donkey or goat serum + 100 μl Na-Azide).

[0458] Add 50-100 μl of blocking buffer to a 96-well plate or 0.5 ml tube and incubate on a shaker at room temperature for 2-3 hours.

[0459] Add 50-100 μl of primary antibody to a 96-well plate or 0.5 ml tube and incubate overnight on a shaker at 4°C.

[0460] Dilute the blocking buffer to 50% using either primary antibody buffer (PBS+ / +, 0.25% Triton-X, 2.5% donkey or goat serum + 50 μl NA-Azide) or Solution 1.

[0461] The following day, wash the slides six times each with PBS- / - and 0.1% Triton-X for 10 minutes each.

[0462] Add 50-100 μl of secondary antibody along with DAPI to a 96-well plate or 0.5 ml tube, and incubate overnight on a shaker at 4°C.

[0463] (8.85 ml PBS+ / +, 1 ml donkey serum or goat serum + 100 ml Na-Azide) or Solution 2.

[0464] Wash with PBS- / - three times for 10 minutes each.

[0465] Mount the image onto a coverslip using Vectashield or Prolong gold, dry it, and store it at 4°C until imaging.

[0466] [A combination of ESC-derived testicular somatic cell-like cells and primordial germ cell-like cells that promotes PGCLC maturation.] A schematic diagram of this protocol is shown in Figure 7.

[0467] (reagent) Culture medium and supplements 1. STEMdiff APEL medium (Stem Cell Technologies, cat.no.05270 or no.05275) 2.DMEM / F-12(ThermoFisher scientific cat.no.11320-082) 3.DMSO (Sigma Aldrich, cat.no.D5879) 4. Dulbecco's phosphate-buffered saline (DPBS) (ThermoFisher Scientific, Cat. no. 14190-144) 5. Accutase (Cat.no.07920) 6.0.5M EDTA stock solution (Lonza 51201) 7.Quick-RNA Miniprep Kit (Zymo Research, Cat.No.R1055) 8. Anti-adhesion cleaning solution (Stem cell Technologies Cat.No.07010) 9. Primary antibody solution 1 (EMD Millipore, Cat.No.KP31812-200ml) 10.Secondary antibody solution 2 (EMD Millipore, Cat.No.KP31855-200ml) 11.0.5M EDTA stock (Lonza 51201) 12. Cleaning solution 13. CHIR99021 (R&D, cat.no.9902): Stock solution (10mM) Gently centrifuge the tube before opening. Reconstitute 10mg of CHIR99021 in 2.149ml of DMSO to make a 10mM stock solution. Dispense into 20μL aliquots, label with "Ch", and store at -20°C. Thawed tubes can be stored at 4°C for 24 hours.

[0468] 14. Y27632 ROCK inhibitor (Enzo, Cat. no. ALX-270-333): Dissolve 1 mg of stock solution (5 mM) in 625 μL of sterile TC water. Dispense into 20 μL aliquots and label with "Y". Store in a stem cell core at -20°C. Dilute with 10 mL of culture medium to a final concentration of 10 μM. Discard the tubing after use.

[0469] 15. Heparin (Sigma Aldrich, cat.no.: H4784-250mg): Stock solution (1mg ml) -1 ) 1 mg ml in ultrapure water -1 Reconstitute the solution and filter it through a 0.22 μm polyethersulfone (PES) filter (syringe-driven filter unit). Dispense into 40 μL portions and store at 4°C for up to 12 months.

[0470] 16. FGF9 (R&D, cat.no.273-F9-025): Stock solution FGF9 (100 μg / ml). Gently centrifuge before opening the tube. Reconstitute into 100 μg / ml filtered DPBS containing 0.1% (wt / vol) bovine serum albumin. Dispense into 20 μl portions and store at -80°C for up to 6 months. Store the stock at -80°C. Thawed FGF9 aliquots can be stored at 4°C for up to 2 weeks.

[0471] 17. RA (Sigma, cat.no. R2625-5mg): Stock solution (0.5mm) Dissolve 1mg of RA in 3ml of DMSO to prepare a 1.11mM stock solution. Dilute the stock solution to 0.5mM and prepare 50μl or 100μl dispensing solutions. This working solution can be stored at -20°C. Use the 0.5mM stock solution in the cell culture medium at the concentration required for the experiment. Discard the tube after use.

[0472] 18. Insulin (Sigma, cat.no.I9278): Stock solution (1.7mM) obtained from the vendor.

[0473] 19. IGF1-50μg (Sigma, Cat.no.I3769): Stock solution (10 mM) Dissolve 50 μg of IGF1 in 658 μl of 0.2% acetic acid to make a 10 mM stock solution. Dispense into 10 μl aliquots and store at -80°C. Dilute the stock solution to 100 μM with 0.2% acetic acid and add to the cell culture medium according to the concentration required for the experiment. Thawed tubes can be stored at 4°C for up to 1 month.

[0474] 20. PGD2 (Cayman, cat.no.12010): Stock solution (2 mg / ml) Dissolve PGD2 in 200 μl of PBS (pH 7.4) to make a stock concentration of 2 mg / ml. Dispense into 5 μl aliquots and store at -80°C. Discard the tubing after use.

[0475] 21. Matrigel (Corning, cat.no.354234, Lot no.9133008): Stock solution (2 mg / ml or 1 mg / ml) Dilute Matrigel with DMEM / F12 medium to the desired concentration (2 mg / ml or 1 mg / ml), dispense, and store at -20°C until use.

[0476] 22. Testosterone (Sigma, cat.no.T1500-5g): Stock solution (5mM) 6g of testosterone was dissolved in 1ml of DMSO and serially diluted to a concentration of 5mM.

[0477] 23. Follicle-stimulating hormone (Sigma: cat.no.F4021-25ug) - Stock solution (100 μg / ml): Dissolve 25 ug of FSH in 250 μl of ultrapure water, dispense, and store at -20°C until use.

[0478] 24. Luteinizing hormone (Sigma: cat.no.L6420-10μg): Stock solution (100μg / ml) Dissolve 25μg of LH in 100μl of ultrapure water, dispense, and store at -20°C until use.

[0479] 25. Epithelial growth factor (EGF) derived from mouse submandibular gland (Sigma, cat.no.E4127-.1mg): Stock solution (100ug / ml) 0.1mg of EGF was dissolved in 10ml of 0.1% BSA / PBS, then aliquoted into 1ml portions and stored at -20°C until use.

[0480] 26. IWR1 (Sigma: cat no. 681669-10mg) - Stock solution: Dissolve 10mg / ml in DMSO, aliquot, and store at -20°C until use.

[0481] (material) 1. Nunc® 4 Welldish for IVF (Fisher, cat.no.144444) 2. Nunc® Cell Culture-Treated Multi-Dish - 24 Wells (Fisher, Cat. No. 142475) 3. Ultra-low adsorption U-shaped bottom microplate (Corning, cat. no. 12-456-721) 4. Greiner Bio-One ThinCert® Tissue Culture Insert (Greiner Bio-One, cat.no.10443845) 5. Costar 24-well plate (Corning, Cat. No. 3526) 6. Coverslip 12mm (Hampton Research, Cat. No. HR3-277) 7. Benchtop centrifuge (Eppendrof, 5417c) 8. Biological safety cabinet 9. CO2 Incubator (Heraeus) 10. Conical tube 15ml (Falcon, cat.no.352096) 11. Conical tube 50ml (Falcon, cat.no.352070) 12. Freezer container (Nalgene, Mr. Frosty) 13. Inverted phase-contrast tissue culture microscope (KL1500CD, Leica) 14. Laser confocal microscope (Nikon Eclipse Ti2) 15. Pipette Boy (Drummond) 16. Pipette (Gilson) 17. Automatic cell counter (BioRad, TC20) 18. Serum pipettes (Fisher, Cat. No. 13-678-11D, 13-676-10J, 13-676-10R) 19. Stericup (registered trademark) 0.22 μm filter unit (Millipore, 03290) 20. Sterile filter pipette tips (Genesee Scientific, 24-815, 24-804, 24-830, 26-401) 21. Sterile microcentrifuge tube (Fisher, Cat. No. 05-408-120) 22. Aggrewell® 800 24-well, 5-pack (Stem cell technologies, Cat. No. 34815) Coverslip cleaning method: Shake the coverslip in xylene in a 50 mL conical tube for 2-3 hours.

[0482] Remove xylene and rinse with acetone. Remove the acetone, add fresh acetone to a 50 mL conical tube, and rinse for 2-3 hours.

[0483] Discard the acetone. If desired, autoclave in water using a liquid cycle. Rinse the coverslip twice with 100% ethanol. Store the coverslip in 100% ethanol at 4°C.

[0484] [Day 1: ESC plating in single-cell suspension to improve differentiation reproducibility] (Cell pellet (RNA-seq analysis) or plate coating for coverslip staining) Dry the coverslips at room temperature in a biological safety cabinet hood under sterile conditions until the alcohol has completely evaporated. After drying, transfer the coverslips to a 24-well or 4-well plate using forceps.

[0485] • Before plating the cells, coat the 24-well or 4-well plate with Matrigel for 1 hour.

[0486] (Single-cell suspension for monolayer use) Remove any differentiated colonies from the cultured colonies on a 60mm Petri dish.

[0487] Wash with 2 ml of DPBS buffer.

[0488] Add 0.5 ml of 1x Accutase and incubate for 5-10 minutes.

[0489] Add 2-5 ml of DMEM / F12 medium to stop dissociation.

[0490] Centrifuge at 1000 rpm for 3 minutes to pelletize the cells.

[0491] Resuspend the pellet in 5 ml of mTeSR medium containing 10 μM (Y27632) Rock inhibitor and count the cells.

[0492] • Plate 10,000 cells per well into a 24-well plate coated with Matrigel.

[0493] (The following morning, differentiation can be initiated using the cells.) Remove the mTeSR medium containing the Rock inhibitor and add 0.5 ml of APEL2 medium + CHIR 3 μM.

[0494] (Day 2) Replace the culture medium with APEL2 medium + CHIR 3μM.

[0495] (Day 4: Unsegmented mesoderm) • Collect cells from the required number of wells.

[0496] Add 0.5 ml of 1x Accutase, incubate in an incubator for 3-5 minutes, and then add 1 ml of DMEM / F12 medium to stop the reaction.

[0497] • Centrifuge at 10,000g for 2 minutes.

[0498] • Aspirate the culture medium.

[0499] • Flash-freeze with liquid nitrogen and store at -80°C until use (unsegmented mesoderm).

[0500] • Fix the cells in 4% PFA for 20 minutes, wash three times with DPBS, and store in DPBS at 4°C until use.

[0501] (Start FGF9 phase) Replace the culture medium with 200 ng / ml FGF9 + 1 μg / ml heparin.

[0502] • Replace the culture medium every two days.

[0503] (Day 6) Replace the culture medium with 200 ng / ml FGF9 + 1 μg / ml heparin.

[0504] (Day 7 (Intermediate Mesoderm)) Replace the culture medium with (APEL ± 0.1 μM RA + 100 nM insulin + 17 nM IGF1 + 500 ng / ml PGD2 ± 200 ng / ml FGF9).

[0505] (Day 8: Mixture of germ cells and somatic cells.) (Induction of differentiated cells into gonadal organoids using a U-bottom microplate.) (Preparation of cells for plating into U-bottom microplates.) • In vitro-induced testicular cells are dissociated into single cells.

[0506] Wash the cells in each well of the 24-well plate with 0.5 ml of DPBS buffer.

[0507] Add 0.5 ml of 1x Accutase, incubate in an incubator for 3-5 minutes, and then add 1 ml of DMEM / F12 medium to stop the reaction.

[0508] • Centrifuge at 1000 rpm for 3 minutes.

[0509] • Aspirate the culture medium.

[0510] Resuspend the cells in APEL medium containing the corresponding growth factor and 10 μm of (Y27632) Rock inhibitor, and count the number of cells.

[0511] Add 100 μl of cells (90% somatic cells, 10% germ cells, totaling 30,000 cells) per well to APEL medium containing the corresponding growth factor, and gently mix from top to bottom to transfer the cells to a U-bottom microplate.

[0512] • Centrifuge the U-bottom microplate at 200g for 3 minutes.

[0513] Observe under a microscope to confirm that all cells are forming clusters.

[0514] (Day 9) - Replace half of the medium every other day by gently touching the side of the well to remove 1 ml of old medium and adding 1 ml of fresh medium. The fresh medium should contain: APEL ± 0.1 μM RA + 100 nM insulin + 17 nM IGF + 500 ng / ml PGD2 ± 200 ng / ml FGF9 ± 1 μM testosterone ± 100 ng / ml follicle-stimulating hormone ± 200 ng / ml luteinizing hormone, or 2 U HCG ± 1 μM IWR1 + 50 ng / ml EGF.

[0515] (Day 10) - The culture medium is changed and IWR1 is removed, but the composition of the medium remains the same.

[0516] (Day 11) On day 11, collect the organoids from the required number of wells and transfer them to a ThinCert® plate.

[0517] (Combine at least five organoids and transfer them to a ThinCert® plate using a 200 μl wide pipette.) The lower chamber contains at least 700 μl of the following medium: APEL ± 0.1 μM RA + 100 nM insulin + 17 nM IGF1 + 500 ng / ml PGD2 ± 200 ng / ml FGF9, 1 μM testosterone ± 100 ng / ml follicle-stimulating hormone ± 200 ng / ml luteinizing hormone ± + 50 ng / ml EGF.

[0518] (Day 13) The upper chamber contains at least 50 μl of culture medium, allowing for various combinations of growth factors; the medium is replaced every two days.

[0519] The lower chamber contains at least 700 μl of culture medium, allowing for various combinations of growth factors; the medium is replaced every two days.

[0520] APEL ±0.1 μM RA + 100 nM insulin + 17 nM IGF1 + 500 ng / ml PGD2 ±200 ng / ml FGF9 + 1 μM testosterone ±100 ng / ml follicle-stimulating hormone ±200 ng / ml luteinizing hormone ±50 ng / ml EGF.

[0521] (Day 16) On day 16, collect the organoids from the required number of wells for RNA. Flash freeze them in liquid nitrogen and store at -80°C until use.

[0522] • Fix some of the organoid wells with 4% PFA for 20 minutes and wash three times with DPBS.

[0523] Incubate in 30% sucrose at 4°C for 72 hours. After incubation, remove the sucrose and wash three times with DPBS buffer for 5 minutes each. Store in DBPS buffer at 4°C until staining.

[0524] (Organoid whole-mount staining protocol) Fix the cells with 4% PFA for 10 minutes.

[0525] Rinse with PBS- / - for 5 minutes, three times.

[0526] Incubate in 30% sucrose at 4°C for 72 hours. After incubation, remove the sucrose and wash three times with DPBS buffer for 5 minutes each. Store in DBPS buffer at 4°C until staining.

[0527] • Rinse quickly three times with PBS- / - before staining.

[0528] Blocking buffer PBS+ / +, 0.1% Triton-x 100, 10% donkey serum or goat serum, and 10% Na-Azide (8.85 ml PBS+ / +, 50 μl Triton-X 100 + 1 ml donkey or goat serum + 100 μl Na-Azide).

[0529] Add 50-100 μl of blocking buffer to a 96-well plate or 0.5 ml tube and incubate on a shaker at room temperature for 2-3 hours.

[0530] Add 50-100 μl of primary antibody to a 96-well plate or 0.5 ml tube and incubate overnight on a shaker at 4°C.

[0531] Dilute the blocking buffer to 50% using either primary antibody buffer (PBS+ / +, 0.25% Triton-X, 2.5% donkey or goat serum + 50 μl NA-Azide) or Solution 1.

[0532] The following day, wash the slides six times each with PBS- / - and 0.1% Triton-X for 10 minutes each.

[0533] Add 50-100 μl of secondary antibody along with DAPI to a 96-well plate or 0.5 ml tube, and incubate overnight on a shaker at 4°C.

[0534] (8.85 ml PBS+ / +, 1 ml donkey serum or goat serum + 100 ml Na-Azide) or Solution 2.

[0535] Wash with PBS- / - three times for 10 minutes each.

[0536] Mount the image onto a coverslip using Vectashield or Prolong gold, dry it, and store it at 4°C until imaging.

[0537] (ICC protocol for OCT sections of tissue and organoids) Section the organoids into 8-12 μm sections and freeze them at -80°C.

[0538] Remove the slides from the freezer and let them sit for 10 minutes.

[0539] • (On the slide) Fix the slide with 4% PFA for 10 minutes.

[0540] Wash the slides with PBS for 5 minutes, three times.

[0541] • Draw a circle on the slide.

[0542] • Inoculate the tissue with 0.1% Triton X-100 / PBS for 15 minutes.

[0543] Block the slides for 1 hour in blocking solution (200 μl 2.5 M glycine / 900 μl PBS / 3% BSA).

[0544] • Dilute the primary antibody with solution I, and incubate the slides overnight at 4°C using this antibody.

[0545] Wash the slides with PBST for 15 minutes, four times.

[0546] • Incubate the slides at room temperature for 2 hours using Solution II secondary antibody.

[0547] Wash with PBS for 15 minutes, four times.

[0548] Mount using Vectashield or Prolong gold.

[0549] [A combination of iPSC-derived testicular somatic cell-like cells and primordial germ cell-like cells that promotes PGCL maturation.] (reagent) Culture medium and supplements 1. STEMdiff APEL medium (Stem Cell Technologies, cat.no.05270 or no.05275) 2.DMEM / F-12(ThermoFisher scientific cat.no.11320-082) 3.DMSO (Sigma Aldrich, cat.no.D5879) 4. Dulbecco's phosphate-buffered saline (DPBS) (ThermoFisher Scientific, Cat. no. 14190-144) 5. Accutase (Cat.no.07920) 6.0.5M EDTA stock solution (Lonza 51201) 7.Quick-RNA Miniprep Kit (Zymo Research, Cat.No.R1055) 8. Anti-adhesion cleaning solution (Stem cell Technologies Cat.No.07010) 9. Primary antibody solution 1 (EMD Millipore, Cat.No.KP31812-200ml) 10.Secondary antibody solution 2 (EMD Millipore, Cat.No.KP31855-200ml) 11.0.5M EDTA stock (Lonza 51201) 12. Cleaning solution 13. CHIR99021 (R&D, cat.no.9902): Stock solution (10mM) Gently centrifuge the tube before opening. Reconstitute 10mg of CHIR99021 in 2.149ml of DMSO to make a 10mM stock solution. Dispense into 20μL aliquots, label with "Ch", and store at -20°C. Thawed tubes can be stored at 4°C for 24 hours.

[0550] 14. Y27632 ROCK inhibitor (Enzo, Cat. no. ALX-270-333): Dissolve 1 mg of stock solution (5 mM) in 625 μL of sterile TC water. Dispense into 20 μL aliquots and label with "Y". Store in a stem cell core at -20°C. Dilute with 10 mL of culture medium to a final concentration of 10 μM. Discard the tubing after use.

[0551] 15. Heparin (Sigma Aldrich, cat.no.: H4784-250mg): Stock solution (1mg ml) -1 ) 1 mg ml in ultrapure water -1Reconstitute the solution and filter it through a 0.22 μm polyethersulfone (PES) filter (syringe-driven filter unit). Dispense into 40 μL portions and store at 4°C for up to 12 months.

[0552] 16. FGF9 (R&D, cat.no.273-F9-025): Stock solution FGF9 (100 μg / ml). Gently centrifuge before opening the tube. Reconstitute into 100 μg / ml filtered DPBS containing 0.1% (wt / vol) bovine serum albumin. Dispense into 20 μl portions and store at -80°C for up to 6 months. Store the stock at -80°C. Thawed FGF9 aliquots can be stored at 4°C for up to 2 weeks.

[0553] 17. RA (Sigma, cat.no. R2625-5mg): Stock solution (0.5mm) Dissolve 1mg of RA in 3ml of DMSO to prepare a 1.11mM stock solution. Dilute the stock solution to 0.5mM and prepare 50μl or 100μl dispensing solutions. This working solution can be stored at -20°C. Use the 0.5mM stock solution in the cell culture medium at the concentration required for the experiment. Discard the tube after use.

[0554] 18. Insulin (Sigma, cat.no.I9278): Stock solution (1.7mM) obtained from the vendor.

[0555] 19. IGF1-50μg (Sigma, Cat.no.I3769): Stock solution (10 mM) Dissolve 50 μg of IGF1 in 658 μl of 0.2% acetic acid to make a 10 mM stock solution. Dispense into 10 μl aliquots and store at -80°C. Dilute the stock solution to 100 μM with 0.2% acetic acid and add to the cell culture medium according to the concentration required for the experiment. Thawed tubes can be stored at 4°C for up to 1 month.

[0556] 20. PGD2 (Cayman, cat.no.12010): Stock solution (2 mg / ml) Dissolve PGD2 in 200 μl of PBS (pH 7.4) to make a stock concentration of 2 mg / ml. Dispense into 5 μl aliquots and store at -80°C. Discard the tubing after use.

[0557] 21. Matrigel (Corning, cat.no.354234, Lot no.9133008): Stock solution (2 mg / ml or 1 mg / ml) Dilute Matrigel with DMEM / F12 medium to the desired concentration (2 mg / ml or 1 mg / ml), dispense, and store at -20°C until use.

[0558] 22. Testosterone (Sigma, cat.no.T1500-5g): Stock solution (5mM) 6g of testosterone was dissolved in 1ml of DMSO and serially diluted to a concentration of 5mM.

[0559] 23. Follicle-stimulating hormone (Sigma: cat.no.F4021-25ug) - Stock solution (100 μg / ml): Dissolve 25 ug of FSH in 250 μl of ultrapure water, dispense, and store at -20°C until use.

[0560] 24. Luteinizing hormone (Sigma: cat.no.L6420-10μg): Stock solution (100μg / ml) Dissolve 25μg of LH in 100μl of ultrapure water, dispense, and store at -20°C until use.

[0561] 25. Epithelial growth factor (EGF) derived from mouse submandibular gland (Sigma, cat.no.E4127-.1mg): Stock solution (100ug / ml) 0.1mg of EGF was dissolved in 10ml of 0.1% BSA / PBS, then aliquoted into 1ml portions and stored at -20°C until use.

[0562] 26. IWR1 (Sigma: cat no. 681669-10mg) - Stock solution: Dissolve 10mg / ml in DMSO, aliquot, and store at -20°C until use.

[0563] (material) 1. Nunc® 4 Welldish for IVF (Fisher, cat.no.144444) 2. Nunc® Cell Culture-Treated Multi-Dish - 24 Wells (Fisher, Cat. No. 142475) 3. Ultra-low adsorption U-shaped bottom microplate (Corning, cat. no. 12-456-721) 4. Greiner Bio-One ThinCert® Tissue Culture Insert (Greiner Bio-One, cat.no.10443845) 5. Costar 24-well plate (Corning, Cat. No. 3526) 6. Coverslip 12mm (Hampton Research, Cat. No. HR3-277) 7. Benchtop centrifuge (Eppendrof, 5417c) 8. Biological safety cabinet 9. CO2 Incubator (Heraeus) 10. Conical tube 15ml (Falcon, cat.no.352096) 11. Conical tube 50ml (Falcon, cat.no.352070) 12. Freezer container (Nalgene, Mr. Frosty) 13. Inverted phase-contrast tissue culture microscope (KL1500CD, Leica) 14. Laser confocal microscope (Nikon Eclipse Ti2) 15. Pipette Boy (Drummond) 16. Pipette (Gilson) 17. Automatic cell counter (BioRad, TC20) 18. Serum pipettes (Fisher, Cat. No. 13-678-11D, 13-676-10J, 13-676-10R) 19. Stericup (registered trademark) 0.22 μm filter unit (Millipore, 03290) 20. Sterile filter pipette tips (Genesee Scientific, 24-815, 24-804, 24-830, 26-401) 21. Sterile microcentrifuge tube (Fisher, Cat. No. 05-408-120) 22. Aggrewell® 800 24-well, 5-pack (Stem cell technologies, Cat. No. 34815) Coverslip cleaning method: Shake the coverslip in xylene in a 50 mL conical tube for 2-3 hours.

[0564] Remove xylene and rinse with acetone. Remove the acetone, add fresh acetone to a 50 mL conical tube, and rinse for 2-3 hours.

[0565] Discard the acetone. If desired, autoclave in water using a liquid cycle. Rinse the coverslip twice with 100% ethanol. Store the coverslip in 100% ethanol at 4°C.

[0566] [Day 1: ESC plating in single-cell suspension to improve differentiation reproducibility] (Cell pellet (RNA-seq analysis) or plate coating for coverslip staining) Dry the coverslips at room temperature in a biological safety cabinet hood under sterile conditions until the alcohol has completely evaporated. After drying, transfer the coverslips to a 24-well or 4-well plate using forceps.

[0567] • Before plating the cells, coat the 24-well or 4-well plate with Matrigel for 1 hour.

[0568] (Single-cell suspension for monolayer use) Remove any differentiated colonies from the cultured colonies on a 60mm Petri dish.

[0569] Wash with 2 ml of DPBS buffer.

[0570] Add 0.5 ml of 1x Accutase and incubate for 5-10 minutes.

[0571] Add 2-5 ml of DMEM / F12 medium to stop dissociation.

[0572] Centrifuge at 1000 rpm for 3 minutes to pelletize the cells.

[0573] Resuspend the pellet in 5 ml of mTeSR medium containing 10 μM (Y27632) Rock inhibitor and count the cells.

[0574] • Plate 10,000 cells per well into a 24-well plate coated with Matrigel.

[0575] (ESC cells collected on day 0 as a control): • Cells used to initiate differentiation.

[0576] Remove the mTeSR medium containing the Rock inhibitor and add 0.5 ml of APEL2 medium + CHIR 8 μM.

[0577] The culture medium should be changed every two days.

[0578] (Day 2) Replace the culture medium with APEL2 medium + CHIR 8μM.

[0579] (Day 4: Unsegmented mesoderm) (Start FGF9 phase) Replace the culture medium with 200 ng / ml FGF9 + 1 μg / ml heparin.

[0580] • Replace the culture medium every two days.

[0581] (Day 6) Replace the culture medium with 200 ng / ml FGF9 + 1 μg / ml heparin.

[0582] (Day 7 (Intermediate Mesoderm)) Replace the culture medium with (APEL ± 0.1 μM RA + 100 nM insulin + 17 nM IGF1 + 500 ng / ml PGD2 ± 200 ng / ml FGF9).

[0583] (Day 8) • Differentiated cells are dissociated into single cells in vitro.

[0584] Wash the cells in each well of the 24-well plate with 0.5 ml of DPBS buffer.

[0585] Add 0.5 ml of 1x Accutase, incubate in an incubator for 3-5 minutes, and then add 1 ml of DMEM / F12 medium to stop the reaction.

[0586] • Centrifuge at 1000 rpm for 3 minutes.

[0587] • Aspirate the culture medium.

[0588] Resuspend the cells in APEL medium containing the corresponding growth factor and 10 μm of (Y27632) Rock inhibitor, and count the number of cells.

[0589] Add 100 μl of cells (90% somatic cells, 10% germ cells, totaling 30,000 cells) per well to APEL medium containing the corresponding growth factors, mix slowly from top to bottom, and place the cells into a U-bottom microplate.

[0590] • Centrifuge the U-bottom microplate at 200g for 3 minutes.

[0591] Observe under a microscope to confirm that all cells are forming clusters.

[0592] (Day 9) Replace the culture medium with (APEL ±0.1 μM RA + 100 nM insulin + 17 nM IGF1 + 500 ng / ml PGD2 ±200 ng / ml FGF9 ±1 μM testosterone ±100 ng / ml follicle-stimulating hormone ±200 ng / ml luteinizing hormone ±50 ng / ml EGF). Replace the culture medium every other day.

[0593] (Day 12: Transfer organoids to ThinCert® plates) (Combine at least five organoids and transfer them to a ThinCert® plate using a 200 μl wide pipette.) The upper chamber contains at least 50 μl of culture medium, allowing for various combinations of growth factors; the medium is replaced every two days.

[0594] The lower chamber contains at least 700 μl of culture medium, allowing for various combinations of growth factors; the medium is replaced every two days.

[0595] APEL ±0.1 μM RA + 100 nM insulin + 17 nM IGF1 + 500 ng / ml PGD2 ±200 ng / ml FGF9 + 1 μM testosterone ±100 ng / ml follicle-stimulating hormone ±200 ng / ml luteinizing hormone ±1 μM + 50 ng / ml EGF.

[0596] (Day 13) The upper chamber contains at least 50 μl of culture medium, allowing for various combinations of growth factors; the medium is replaced every two days.

[0597] The lower chamber contains at least 700 μl of culture medium, allowing for various combinations of growth factors; the medium is replaced every two days.

[0598] APEL ±0.1 μM RA + 100 nM insulin + 17 nM IGF1 + 500 ng / ml PGD2 ±200 ng / ml FGF9 + 1 μM testosterone ±100 ng / ml follicle-stimulating hormone ±200 ng / ml luteinizing hormone ±50 ng / ml EGF.

[0599] (Day 16) On day 16, collect the organoids from the required number of wells for RNA. Flash freeze them in liquid nitrogen and store at -80°C until use.

[0600] • Fix some of the organoid wells with 4% PFA for 20 minutes and wash three times with DPBS.

[0601] Incubate in 30% sucrose at 4°C for 72 hours. After incubation, remove the sucrose and wash three times with DPBS buffer for 5 minutes each. Store in DBPS buffer at 4°C until staining.

[0602] (Organoid whole-mount staining protocol) Fix the cells with 4% PFA for 10 minutes.

[0603] Rinse with PBS- / - for 5 minutes, three times.

[0604] Incubate in 30% sucrose at 4°C for 72 hours. After incubation, remove the sucrose and wash three times with DPBS buffer for 5 minutes each. Store in DBPS buffer at 4°C until staining.

[0605] • Rinse quickly three times with PBS- / - before staining.

[0606] Blocking buffer PBS+ / +, 0.1% Triton-x 100, 10% donkey serum or goat serum, and 10% Na-Azide (8.85 ml PBS+ / +, 50 μl Triton-X 100 + 1 ml donkey or goat serum + 100 μl Na-Azide).

[0607] Add 50-100 μl of blocking buffer to a 96-well plate or 0.5 ml tube and incubate on a shaker at room temperature for 2-3 hours.

[0608] Add 50-100 μl of primary antibody to a 96-well plate or 0.5 ml tube and incubate overnight on a shaker at 4°C.

[0609] Dilute the blocking buffer to 50% using either primary antibody buffer (PBS+ / +, 0.25% Triton-X, 2.5% donkey or goat serum + 50 μl NA-Azide) or Solution 1.

[0610] The following day, wash the slides six times each with PBS- / - and 0.1% Triton-X for 10 minutes each.

[0611] Add 50-100 μl of secondary antibody along with DAPI to a 96-well plate or 0.5 ml tube, and incubate overnight on a shaker at 4°C.

[0612] (8.85 ml PBS+ / +, 1 ml donkey serum or goat serum + 100 ml Na-Azide) or Solution 2.

[0613] Wash with PBS- / - three times for 10 minutes each.

[0614] Mount the image onto a coverslip using Vectashield or Prolong gold, dry it, and store it at 4°C until imaging.

[0615] (ICC protocol for OCT sections of tissue and organoids) Section the organoids into 8-12 μm sections and freeze at -80°C.

[0616] Remove the slides from the freezer and let them sit for 10 minutes.

[0617] • (On the slide) Fix the slide with 4% PFA for 10 minutes.

[0618] Wash the slides with PBS for 5 minutes, three times.

[0619] • Draw a circle on the slide.

[0620] • Inoculate the tissue with 0.1% Triton X-100 / PBS for 15 minutes.

[0621] Block the slides for 1 hour in blocking solution (200 μl 2.5 M glycine / 900 μl PBS / 3% BSA).

[0622] • Dilute the primary antibody with solution I, and incubate the slides overnight at 4°C using this antibody.

[0623] Wash the slides with PBST for 15 minutes, four times.

[0624] • Incubate the slides at room temperature for 2 hours using Solution II secondary antibody.

[0625] Wash with PBS for 15 minutes, four times.

[0626] Mount using Vectashield or Prolong gold.

[0627] [Differentiation into presomatic mesoderm, intermediate mesoderm, and peri-semispermal muscle-like cells.] A schematic diagram of this protocol is shown in Figure 8.

[0628] (reagent) Culture medium and supplements 1. STEMdiff APEL medium (Stem Cell Technologies, cat.no.05270 or no.05275) 2.DMEM / F-12(ThermoFisher scientific cat.no.11320-082) 3.DMSO (Sigma Aldrich, cat.no.D5879) 4. Dulbecco's phosphate-buffered saline (DPBS) (ThermoFisher Scientific, Cat. no. 14190-144) 5. Accutase (Cat.no.07920) 6.0.5M EDTA stock solution (Lonza 51201) 7.Quick-RNA Miniprep Kit (Zymo Research, Cat.No.R1055) 8. Anti-adhesion cleaning solution (Stem cell Technologies Cat.No.07010) 9. Primary antibody solution 1 (EMD Millipore, Cat.No.KP31812-200ml) 10.Secondary antibody solution 2 (EMD Millipore, Cat.No.KP31855-200ml) 11.0.5M EDTA stock (Lonza 51201) 12. Cleaning solution 13. CHIR99021 (R&D, cat.no.9902): Stock solution (10mM) Gently centrifuge the tube before opening. Reconstitute 10mg of CHIR99021 in 2.149ml of DMSO to make a 10mM stock solution. Dispense into 20μL aliquots, label with "Ch", and store at -20°C. Thawed tubes can be stored at 4°C for 24 hours.

[0629] 14. Y27632 ROCK inhibitor (Enzo, Cat. no. ALX-270-333): Dissolve 1 mg of stock solution (5 mM) in 625 μL of sterile TC water. Dispense into 20 μL aliquots and label with "Y". Store in a stem cell core at -20°C. Dilute with 10 mL of culture medium to a final concentration of 10 μM. Discard the tubing after use.

[0630] 15. Heparin (Sigma Aldrich, cat.no.: H4784-250mg): Stock solution (1mg ml) -1 ) 1 mg ml in ultrapure water -1 Reconstitute the solution and filter it through a 0.22 μm polyethersulfone (PES) filter (syringe-driven filter unit). Dispense into 40 μL portions and store at 4°C for up to 12 months.

[0631] 16. FGF9 (R&D, cat.no.273-F9-025): Stock solution FGF9 (100 μg / ml). Gently centrifuge before opening the tube. Reconstitute into 100 μg / ml filtered DPBS containing 0.1% (wt / vol) bovine serum albumin. Dispense into 20 μl portions and store at -80°C for up to 6 months. Store the stock at -80°C. Thawed FGF9 aliquots can be stored at 4°C for up to 2 weeks.

[0632] 17. RA (Sigma, cat.no. R2625-5mg): Stock solution (0.5mm) Dissolve 1mg of RA in 3ml of DMSO to prepare a 1.11mM stock solution. Dilute the stock solution to 0.5mM and prepare 50μl or 100μl dispensing solutions. This working solution can be stored at -20°C. Use the 0.5mM stock solution in the cell culture medium at the concentration required for the experiment. Discard the tube after use.

[0633] 18. Insulin (Sigma, cat.no.I9278): Stock solution (1.7mM) obtained from the vendor.

[0634] 19. IGF1-50μg (Sigma, Cat.no.I3769): Stock solution (10 mM) Dissolve 50 μg of IGF1 in 658 μl of 0.2% acetic acid to make a 10 mM stock solution. Dispense into 10 μl aliquots and store at -80°C. Dilute the stock solution to 100 μM with 0.2% acetic acid and add to the cell culture medium according to the concentration required for the experiment. Thawed tubes can be stored at 4°C for up to 1 month.

[0635] 20. PGD2 (Cayman, cat.no.12010): Stock solution (2 mg / ml) Dissolve PGD2 in 200 μl of PBS (pH 7.4) to make a stock concentration of 2 mg / ml. Dispense into 5 μl aliquots and store at -80°C. Discard the tubing after use.

[0636] 21. Matrigel (Corning, cat.no.354234, Lot no.9133008): Stock solution (2 mg / ml or 1 mg / ml) Dilute Matrigel with DMEM / F12 medium to the desired concentration (2 mg / ml or 1 mg / ml), dispense, and store at -20°C until use.

[0637] 22. SAG (Millipore, cat.no.566661): Stock solution (0.5 mM). Prepare 5 µl of dispensed solution and store at -20°C.

[0638] 23. PDGFAA (Sigma, cat.no. SRP3228-10ug): Stock solution (10ug / ml) - Dissolve 10ug in 1ml of 0.1% BSA. Prepare 50ul of dispensed solution and store at -20°C.

[0639] 24. PDGFBB (Sigma, cat.no. SRP3229-10ug): Stock solution (10ug / ml) - Dissolve 10ug in 1ml of 0.1% BSA. Prepare 50ul of dispensed solution and store at -20°C.

[0640] 25. Valproic acid (Sigma, cat.no. P4543-10G): Dissolve 1.6619 mg of stock solution (1 mM) in 1 ml of sterile water. Prepare 50 µl of dispensing solution and store at -80°C.

[0641] 26. BMP2 (R&D, cat.no.355-BM-050): Stock solution (50 ug / ml) - Dissolve 50 ug in 1 ml of sterile 4 mM HCl containing 0.1% BSA, and store 50 ug of dispensed solution at 80°C.

[0642] 27. BMP4 (R&D: cat.no.314-BP-500): Stock solution (200 μg / ml) - Dissolve 200 μg / mL in 1 ml of sterile 4 mM HCl containing 0.1% BSA, and store 50 μl of the dispensed solution at -80°C.

[0643] 28. Activin A (R&D, cat.no.338-AC-050 / CF). Stock solution (50 ug / ml) - Dissolve 50 ug in 1 ml of sterile 4 mM HCl, and store 50 ug of dispensed solution at -80°C.

[0644] (material) 1. Nunc® 4 Welldish for IVF (Fisher, cat.no.144444) 2. Nunc® Cell Culture-Treated Multi-Dish - 24 Wells (Fisher, Cat. No. 142475) 3. Ultra-low adsorption U-shaped bottom microplate (Corning, cat. no. 12-456-721) 4. Greiner Bio-One ThinCert® Tissue Culture Insert (Greiner Bio-One, cat.no.10443845) 5. Costar 24-well plate (Corning, Cat. No. 3526) 6. Costar 6-well plate (Corning, Cat. No. 3506) 7. Coverslip 12mm (Hampton Research, Cat. No. HR3-277) 8. Benchtop centrifuge (Eppendrof, 5417c) 9. Biological safety cabinet 10. CO2 Incubator (Heraeus) 11. Conical tube 15ml (Falcon, cat.no.352096) 12. Conical tube 50ml (Falcon, cat.no.352070) 13. Freezer container (Nalgene, Mr. Frosty) 14. Inverted phase-contrast tissue culture microscope (KL1500CD, Leica) 15. Laser confocal microscope (Nikon Eclipse Ti2) 16. Pipette Boy (Drummond) 17. Pipette (Gilson) 18. Automatic cell counter (BioRad, TC20) 19. Serum pipettes (Fisher, Cat. No. 13-678-11D, 13-676-10J, 13-676-10R) 20. Stericup (registered trademark) 0.22 μm filter unit (Millipore, 03290) 21. Sterile filter pipette tips (Genesee Scientific, 24-815, 24-804, 24-830, 26-401) 22. Sterile microcentrifuge tube (Fisher, Cat. No. 05-408-120) 23. Aggrewell® 800 24-well, 5-pack (Stem cell technologies, Cat. No. 34815) (Prepare cells according to the instructions for ESC or iPSC.) (Day 7 (Intermediate Mesoderm)) Remove FGF9 and heparin, and add the following: APEL ±0.1 μM, RA ±100 nM, insulin ±17 nM, IGF 1 ±500 ng / ml, PGD 2 ±200 ng / ml, FGF 9.

[0645] (Day 8) • Dissociate the wells containing differentiated cells into single cells.

[0646] Wash the cells in each well of the 24-well plate with 0.5 ml of DPBS buffer.

[0647] Add 0.5 ml of 1x Accutase, incubate in an incubator for 3-5 minutes, and then add 1 ml of DMEM / F12 medium to stop the reaction.

[0648] • Centrifuge at 1000 rpm for 3 minutes.

[0649] • Aspirate the culture medium.

[0650] Resuspend the cells in APEL medium containing the corresponding growth factor and 10 μm of (Y27632) Rock inhibitor, and count the number of cells.

[0651] • Add 3 ml (5 × 10) per well to APEL medium containing the corresponding growth factor. 5 Add (1) cells.

[0652] The culture medium is replaced every other day with the following: APEL ±0.5 μM SAG ±10 nM valproic acid ±10 ng / ml activin A ±10 ng / ml BMP2 ±10 ng / ml BMP4 ±10 ng / ml PDGFAA ±10 ng / ml PDGFBB ±0.1% BSA.

[0653] (Day 12) Replace the culture medium with one containing growth factors every two days.

[0654] APEL ±0.5 μM, SAG ±10 nM, valproic acid ±10 ng / ml, activin A ±10 ng / ml, BMP2 ±10 ng / ml, BMP4 ±10 ng / ml, PDGFAA ±10 ng / ml, PDGFBB ±0.1% BSA.

[0655] (Day 14) Replace the culture medium with one containing growth factors every two days.

[0656] APEL ±0.5 μM, SAG ±10 nM, valproic acid ±10 ng / ml, activin A ±10 ng / ml, BMP2 ±10 ng / ml, BMP4 ±10 ng / ml, PDGFAA ±10 ng / ml, PDGFBB ±0.1% BSA.

[0657] (Day 16) • Recover cells for RNA production.

[0658] Wash the cells with 0.5 ml of DPBS.

[0659] Add 0.5 ml of 1x Accutase, incubate in an incubator for 3-5 minutes, and then add 1 ml of DMEM / F12 medium to stop the reaction.

[0660] • Centrifuge at 10,000g for 2 minutes.

[0661] • Aspirate the culture medium.

[0662] • Flash-freeze with liquid nitrogen and store at -80°C until use (Leydig-like cells).

[0663] • Fix some wells of cells covered with coverslips in 4% PFA for 10 minutes, then wash three times with DPBS. Store in DBPS buffer at 4°C until staining.

[0664] (Single-layer coverslip staining protocol) Fix the cells with 4% PFA for 20 minutes.

[0665] Wash three times in PBS- / - for 5 minutes each, then store in PBS- / - at 4°C until staining.

[0666] • Rinse quickly three times with PBS- / - before staining.

[0667] Blocking buffer PBS+ / +, 0.1% Triton-x 100, 10% donkey serum or goat serum, and 10% Na-Azide (8.85 ml PBS+ / +, 50 μl Triton-X 100 + 1 ml donkey or goat serum + 100 μl Na-Azide).

[0668] Add 200 μl of blocking buffer to a 24-well plate and incubate for 1 hour in a lidded, humidified chamber at room temperature.

[0669] Add 200 μl of primary antibody to a 24-well plate and incubate overnight in a lidded, humidified chamber at 4°C.

[0670] • Dilute the blocking buffer to 50% as the primary antibody buffer (PBS+ / +, 0.25% Triton-X, 2.5% donkey or goat serum + 50 μl of NA-Azide).

[0671] The following day, wash the slides three times each with PBS- / - and 0.1% Triton-X for 10 minutes each.

[0672] Add 200 μl of secondary Ab along with DAPI and allow to stand for 1.5 hours in a lidded humidified chamber at room temperature.

[0673] (8.85 ml of PBS+ / +, 1 ml of donkey or goat serum + 100 ml of Na-Azide) Wash with PBS- / - three times for 10 minutes each.

[0674] Mount the image onto a coverslip using Vectashield or Prolong gold, dry it, and store it at 4°C until imaging.

[0675] [Differentiation into presomatic mesoderm, intermediate mesoderm, and Leydig-like cells.] A schematic diagram of this protocol is shown in Figure 9.

[0676] (reagent) Culture medium and supplements 1. STEMdiff APEL medium (Stem Cell Technologies, cat.no.05270 or no.05275) 2.DMEM / F-12(ThermoFisher scientific cat.no.11320-082) 3.DMSO (Sigma Aldrich, cat.no.D5879) 4. Dulbecco's phosphate-buffered saline (DPBS) (ThermoFisher Scientific, Cat. no. 14190-144) 5. Accutase (Cat.no.07920) 6.0.5M EDTA stock solution (Lonza 51201) 7.Quick-RNA Miniprep Kit (Zymo Research, Cat.No.R1055) 8. Anti-adhesion cleaning solution (Stem cell Technologies Cat.No.07010) 9. Primary antibody solution 1 (EMD Millipore, Cat.No.KP31812-200ml) 10.Secondary antibody solution 2 (EMD Millipore, Cat.No.KP31855-200ml) 11.0.5M EDTA stock (Lonza 51201) 12. Cleaning solution 13. CHIR99021 (R&D, cat.no.9902): Stock solution (10mM) Gently centrifuge the tube before opening. Reconstitute 10mg of CHIR99021 in 2.149ml of DMSO to make a 10mM stock solution. Dispense into 20μL aliquots, label with "Ch", and store at -20°C. Thawed tubes can be stored at 4°C for 24 hours.

[0677] 14. Y27632 ROCK inhibitor (Enzo, Cat. no. ALX-270-333): Dissolve 1 mg of stock solution (5 mM) in 625 μL of sterile TC water. Dispense into 20 μL aliquots and label with "Y". Store in a stem cell core at -20°C. Dilute with 10 mL of culture medium to a final concentration of 10 μM. Discard the tubing after use.

[0678] 15. Heparin (Sigma Aldrich, cat.no.: H4784-250mg): Stock solution (1mg ml) -1 ) 1 mg ml in ultrapure water -1 Reconstitute the solution and filter it through a 0.22 μm polyethersulfone (PES) filter (syringe-driven filter unit). Dispense into 40 μL portions and store at 4°C for up to 12 months.

[0679] 16. FGF9 (R&D, cat.no.273-F9-025): Stock solution FGF9 (100 μg / ml). Gently centrifuge before opening the tube. Reconstitute into 100 μg / ml filtered DPBS containing 0.1% (wt / vol) bovine serum albumin. Dispense into 20 μl portions and store at -80°C for up to 6 months. Store the stock at -80°C. Thawed FGF9 aliquots can be stored at 4°C for up to 2 weeks.

[0680] 17. RA (Sigma, cat.no. R2625-5mg): Stock solution (0.5mm) Dissolve 1mg of RA in 3ml of DMSO to prepare a 1.11mM stock solution. Dilute the stock solution to 0.5mM and prepare 50μl or 100μl dispensing solutions. This working solution can be stored at -20°C. Use the 0.5mM stock solution in the cell culture medium at the concentration required for the experiment. Discard the tube after use.

[0681] 18. Insulin (Sigma, cat.no.I9278): Stock solution (1.7mM) obtained from the vendor.

[0682] 19. IGF1-50μg (Sigma, Cat.no.I3769): Stock solution (10 mM) Dissolve 50 μg of IGF1 in 658 μl of 0.2% acetic acid to make a 10 mM stock solution. Dispense into 10 μl aliquots and store at -80°C. Dilute the stock solution to 100 μM with 0.2% acetic acid and add to the cell culture medium according to the concentration required for the experiment. Thawed tubes can be stored at 4°C for up to 1 month.

[0683] 20. PGD2 (Cayman, cat.no.12010): Stock solution (2 mg / ml) Dissolve PGD2 in 200 μl of PBS (pH 7.4) to make a stock concentration of 2 mg / ml. Dispense into 5 μl aliquots and store at -80°C. Discard the tubing after use.

[0684] 21. Matrigel (Corning, cat.no.354234, Lot no.9133008): Stock solution (2 mg / ml or 1 mg / ml) Dilute Matrigel with DMEM / F12 medium to the desired concentration (2 mg / ml or 1 mg / ml), dispense, and store at -20°C until use.

[0685] 22. SAG (Millipore, cat.no.566661): Stock solution (0.5 mM). Prepare 5 µl of dispensed solution and store at -20°C.

[0686] 23. PDGFAA (Sigma, cat.no. SRP3228-10ug): Stock solution (10ug / ml) - Dissolve 10ug in 1ml of 0.1% BSA. Prepare 50ul of dispensed solution and store at -20°C.

[0687] 24. PDGFBB (Sigma, cat.no. SRP3229-10ug): Stock solution (10ug / ml) - Dissolve 10ug in 1ml of 0.1% BSA. Prepare 50ul of dispensed solution and store at -20°C.

[0688] 25. LiCl2 (Sigma, cat.no. L7026-100ml): Stock solution (8M) provided by the supplier. Store at room temperature.

[0689] 26. Basic FGF2 (Proteintech, cat.no. HZ-1285): Stock solution (10 ug / ml) - Dissolve 10 ug in 1 ml of 0.2% BSA DPBS, and store 50 ug of the dispensed solution at -20°C.

[0690] 27. DAPT (Stem Cell Technologies, cat.no.72082). Dissolve 5 mg of DAPT in 1.16 ml of DMSO in a 10 mM stock solution. Prepare 50 µl of the solution and store at -80°C.

[0691] (material) 1. Nunc® 4 Welldish for IVF (Fisher, cat.no.144444) 2. Nunc® Cell Culture-Treated Multi-Dish - 24 Wells (Fisher, Cat. No. 142475) 3. Ultra-low adsorption U-shaped bottom microplate (Corning, cat. no. 12-456-721) 4. Greiner Bio-One ThinCert® Tissue Culture Insert (Greiner Bio-One, cat.no.10443845) 5. Costar 24-well plate (Corning, Cat. No. 3526) 6. Costar 6-well plate (Corning, Cat. No. 3506) 7. Coverslip 12mm (Hampton Research, Cat. No. HR3-277) 8. Benchtop centrifuge (Eppendrof, 5417c) 9. Biological safety cabinet 10. CO2 Incubator (Heraeus) 11. Conical tube 15ml (Falcon, cat.no.352096) 12. Conical tube 50ml (Falcon, cat.no.352070) 13. Freezer container (Nalgene, Mr. Frosty) 14. Inverted phase-contrast tissue culture microscope (KL1500CD, Leica) 15. Laser confocal microscope (Nikon Eclipse Ti2) 16. Pipette Boy (Drummond) 17. Pipette (Gilson) 18. Automatic cell counter (BioRad, TC20) 19. Serum pipettes (Fisher, Cat. No. 13-678-11D, 13-676-10J, 13-676-10R) 20. Stericup (registered trademark) 0.22 μm filter unit (Millipore, 03290) 21. Sterile filter pipette tips (Genesee Scientific, 24-815, 24-804, 24-830, 26-401) 22. Sterile microcentrifuge tube (Fisher, Cat. No. 05-408-120) 23. Aggrewell® 800 24-well, 5-pack (Stem cell technologies, Cat. No. 34815) (The same protocol as for ESC or iPSC will be followed until day 6.) (Day 7 (Intermediate Mesoderm)) Remove the FGF9 medium and heparin, and add FGF9 with APEL ±0.1 μM, RA ±100 nM, insulin ±17 nM, IGF1 ±500 ng / ml, and PGD2 ±200 ng / ml.

[0692] (Day 8) (Induction of differentiated cells into Leydig cells) (Preparation of cells for plating) • Dissociate gonadal progenitor cells induced in vitro.

[0693] Wash the cells in each well of the 24-well plate with 0.5 ml of DPBS buffer.

[0694] Add 0.5 ml of 1x Accutase, incubate in an incubator for 3-5 minutes, and then add 1 ml of DMEM / F12 medium to stop the reaction.

[0695] • Centrifuge at 1000 rpm for 3 minutes.

[0696] • Aspirate the culture medium.

[0697] Resuspend the cells in APEL medium containing the corresponding growth factor and 10 μm of (Y27632) Rock inhibitor, and count the number of cells.

[0698] • Add 3 ml (5 × 10) per well to APEL medium containing the corresponding growth factor. 5 Add (1) cells.

[0699] Replace the culture medium every other day with the following: APEL ±0.5 μM, SAG ±5 mM, LiCl2 ±5 μM, DAPT ±10 ng / ml, bFGf2 ±10 ng / ml, PDGFAA ±10 ng / ml, PDGFBB ±0.1% BSA.

[0700] (Day 12) Replace the culture medium with one containing growth factors every two days.

[0701] APEL ±0.5 μM, SAG ±5 mM, LiCl2 ±5 μM, DAPT ±10 ng / ml, bFGf2 ±10 ng / ml, PDGFAA ±10 ng / ml, PDGFBB ±0.1% BSA.

[0702] (Day 14) Replace the culture medium with one containing growth factors every two days.

[0703] APEL ±0.5 μM, SAG ±5 mM, LiCl2 ±5 μM, DAPT ±10 ng / ml, bFGf2 ±10 ng / ml, PDGFAA ±10 ng / ml, PDGFBB ±0.1% BSA.

[0704] (Day 16) • Recover cells for RNA production.

[0705] Wash the cells in each well of the 24-well plate with 0.5 ml of DPBS buffer.

[0706] Add 0.5 ml of 1x Accutase, incubate in an incubator for 3-5 minutes, and then add 1 ml of DMEM / F12 medium to stop the reaction.

[0707] • Centrifuge at 10,000g for 2 minutes.

[0708] • Aspirate the culture medium.

[0709] • Flash-freeze with liquid nitrogen and store at -80°C until use (Leydig-like cells).

[0710] • Fix some wells of cells covered with coverslips in 4% PFA for 10 minutes, then wash three times with DPBS. Store in DBPS buffer at 4°C until staining.

[0711] (Single-layer coverslip staining protocol) Fix the cells with 4% PFA for 20 minutes.

[0712] Wash three times in PBS- / - for 5 minutes each, then store in PBS- / - at 4°C until staining.

[0713] • Rinse quickly three times with PBS- / - before staining.

[0714] Blocking buffer PBS+ / +, 0.1% Triton-x 100, 10% donkey serum or goat serum, and 10% Na-Azide (8.85 ml PBS+ / +, 50 μl Triton-X 100 + 1 ml donkey or goat serum + 100 μl Na-Azide).

[0715] Add 200 μl of blocking buffer to a 24-well plate and incubate for 1 hour in a lidded, humidified chamber at room temperature.

[0716] Add 200 μl of primary antibody to a 24-well plate and incubate overnight in a lidded, humidified chamber at 4°C.

[0717] • Dilute the blocking buffer to 50% as the primary antibody buffer (PBS+ / +, 0.25% Triton-X, 2.5% donkey or goat serum + 50 μl of NA-Azide).

[0718] The following day, wash the slides three times each with PBS- / - and 0.1% Triton-X for 10 minutes each.

[0719] Add 200 μl of secondary Ab along with DAPI and allow to stand for 1.5 hours in a lidded humidified chamber at room temperature.

[0720] (8.85 ml of PBS+ / +, 1 ml of donkey or goat serum + 100 ml of Na-Azide) Wash with PBS- / - three times for 10 minutes each.

[0721] Mount the image onto a coverslip using Vectashield or Prolong gold, dry it, and store it at 4°C until imaging.

[0722] [Results and Discussion] Differentiation protocols for effectively enriching Sertoli-like cells and testicular stromal cells have been previously described (see PCT US2023 / 13608, which is incorporated herein by reference in its entirety). These previously described protocols have been modified in this embodiment to improve organoid differentiation. The original protocol was completed on day 13, at which point moderate expression levels of key Sertoli cell markers such as Gata4, Nr5a1, Sox9, and Fshr were observed. In particular, comprehensive single-cell RNA sequencing analysis and comparative studies of in vitro and in vivo derived cells demonstrated a correlation between in vitro derived cells and early gonadal and pre-Sertoli cell progenitor cells, as described in PCT US2023 / 13608.

[0723] The differentiation process was further optimized on day 8 of the differentiation timeline by introducing IWR1 and EGF1 into the previously established ALL GF cocktail. To maintain organoid proliferation in the absence of primordial germ cell-like cells (PGCLCs), cells are maintained in ALLGF+EGF+IWR1+hormone medium. Notably, upon introduction of germ cells, IWR1 supplementation is removed due to observed effects on germ cell maturation and differentiation. An additional modification involves transferring organoids to a Transwell® plate system on day 12. This change allows for organoid culture at the gas-liquid interface, enabling long-term culture that more accurately mimics the in vivo environment. The introduction of this novel approach provides an excellent model for studying and understanding the complexity of intercellular interactions and developmental processes within the testis.

[0724] In addition to the incorporation of IWR1 and EGF1, the transition to Transwell® plate culture significantly increased the expression of markers that were not previously present. In particular, these modifications promoted the induction of major markers in the gonadal cell lineage, with LHX9 being upregulated as an example. Similarly, Sertoli cell-specific markers such as DHH were strongly induced along with the Leydig cell markers STAR and 3BHSD (see comparison of D13 in Figure 10 and D16+ / - hormones in Figure 11).

[0725] Furthermore, introducing primordial germ cell-like cells (PGCLCs) into testicular organoids significantly improved the overall efficiency of gonadal progenitor cell differentiation, resulting in a remarkable enrichment of over 1,000 times. Notably, Sertoli cell markers showed induction of over 10 times, encompassing the entire spectrum of identified markers. In addition, improvements to the protocol have also led to successful induction of Leydig cells.

[0726] These adaptations to previously described differentiation protocols, combined with the introduction of in vitro-derived germ cell-like cells, collectively resulted in enhanced determination of all gonadal cell fate. This improved protocol not only amplifies differentiation efficiency and increases the proportion of cells derived from gonadal cell lineages, but also induces the expression of previously unobserved markers. Of particular note is the emergence of SRY, a key gene involved in testicular sex determination (see Figure 11).

[0727] In summary, these modifications and advancements in differentiation protocols, coupled 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 highlight significant progress in our ability to replicate and investigate the complex processes governing gonadal cell fate determination.

[0728] [Somatic cells derived in vitro may support the transfer of PGCLCs to spermatogonial cells.] Maintaining PGCLCs and differentiating them into spermatogonial cells is a crucial step in the clinical application of in vitro gamete formation. The data presented herein demonstrate that in vitro-derived cells have the ability to effectively nurture human PGCLCs, which leads to decreased expression of PGCLC-related markers such as Sox17, TFAP2C, and PRDM1. This decrease in marker expression leads to the induction of genes characteristic of spermatogonial cells (including DDX4, PLZF, DNMT3, and various other related markers).

[0729] These findings demonstrate the ability of PGCLCs to guide their developmental trajectory toward spermatogonial cell lines, highlighting the importance of this groundbreaking process. This achievement not only contributes to a deeper understanding of the complex mechanisms governing germ cell differentiation but also significantly broadens the prospects for clinical applications in the field of in vitro gamete formation.

[0730] [Example 3] This embodiment provides an alternative protocol for producing testicular-like organoids from mouse pluripotent stem cells. A schematic diagram is shown in Figure 12.

[0731] In the protocol described in Example 1 above, intermediate mesoderm cells were dissociated, and 300,000 cells were mixed with 500 μl of organoid differentiation medium and added to a non-adherent Aggrewell® plate. In this protocol, intermediate mesoderm cells (cells on day 4) were dissociated, and 25,000 cells were seeded in 100 μl of organoid aggregation medium (DMEM / F12, 5% knockout serum substitute, 1X pyruvate, 1X non-essential amino acids, 1X β-mercapto(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 2 units / ml hCG), 20 ng / ml follicle-stimulating hormone) in the wells of a U-bottom non-adherent 96-well plate. After adding 100 μl of suspension, the 96-well plate is centrifuged at 500 g and cultured in a 5% CO2 incubator. On the day after agglutination (day 5), an additional 100 μl of the original organoid agglutination medium is added to each well. On day 2 of agglutination, 100 μl of the old medium is removed and 100 μl of organoid agglutination medium is added. On day 7, the organoids are transplanted into ThinCert® Transwell® dishes. The organoids are placed in the upper compartment, and the lower compartment is filled with 500 μl of organoid differentiation medium (DMEM / F12, 5% knockout serum substitute, 1X pyruvate, 1X non-essential amino acids, 1X β-mercapto (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 2 units / ml hCG), 20 ng / ml follicle-stimulating hormone). Half of the medium is replaced every other day for the next 10 days. On day 10, the organoids are imaged and retrieved from the ThinCert® cell culture insert. The retrieved samples are used for qPCR, sequencing, and immunostaining, and the medium is sent to a mass spectrometry facility for testosterone measurement.

[0732] Using this differentiation scheme, as shown in the data in Figure 13, mature tubular structures are formed, containing Sertoli cells, spontaneously identified Leydig cells, peritubular muscle-like cells, and a small number of endothelial cells.

[0733] Next, to confirm the differentiation results, organoids cultured under two conditions (LH or HCG) were sectioned, and protein expression and organization were examined by immunohistochemistry (see Figure 14). Figure 14 includes only the data for HCG treatment, but similar results were obtained with LH treatment.

[0734] To determine whether the Leydig cells in culture were functional, culture medium was collected from either HCG-treated or LH-treated organoids, and testosterone concentrations were measured by mass spectrometry. As a control, basal medium that had never been exposed to organoids was used for background correction. The results are shown in Figure 15. Although testosterone concentrations decreased with HCG treatment, these organoids self-organized more rapidly in culture and formed a more extensive tubular network compared to LH-treated organoids. A preferred approach is to treat the organoids with HCG for 10 days, followed by LH treatment for another 10 days.

[0735] Finally, we tested whether the organoids could support germ cell proliferation. A schematic diagram of the experiment is shown in Figure 16. As the data in Figure 17 shows, these organoids were found to support germ cell proliferation and differentiation. In the protocol of Example 1, in vivo-derived PND2 germ cells were undifferentiated and lacked Stra8 expression. In this example, the proportion of OCT4-positive cells, a marker for primordial germ cells or prespermatogonia, was reduced in the organoids. Simultaneously, the transition of these cells to undifferentiated spermatogonial cells (PLZF-positive) and / or differentiating spermatogonial cells (Stra8-positive) was observed.

[0736] [Example 4] This embodiment provides an alternative protocol for producing testicular organoids from human stem cells. A schematic diagram is shown in Figure 18.

[0737] 10,000 UCLA6 hESCs were plated onto Geltrex-coated 24-well plates in mTESR+ medium containing 10 μM Y27632. The following morning, the medium was replaced with APEL2 (StemCell Technologies) containing 3 μM CHIR99021 (day 0), and the cells were cultured in this medium until day 4. From day 4 to day 7, the cells were cultured in APEL2 + 200 ng / ml FGF9 and 1 μg / ml heparin. On day 7, the medium was replaced with APEL2 + 0.1 μM 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 μM testosterone. After 24 hours, the cells were dispersed using acutase, and 30,000 cells per well were re-aggregated in the medium from day 7 + 10 μM Y27632 in a 96-well ultra-low adsorption U-bottom plate. After another 24 hours, fresh medium (the same as the medium from day 8, but without Y27632) was added. After 3 days, 5-6 organoids were transferred to a Thincert® cell culture insert in a 24-well plate. The organoids were allowed to mature for a further 11 days before being harvested for analysis. This protocol omits the aggregation step in Aggrewell® plates. Instead, cells are seeded on day 8 into 96-well U-bottom plates. On day 22, more pronounced expression of several previously undetectable Sertoli cell markers (e.g., AMH, SRY, and NR5A1) is detected, and improved tubular organization is observed. See Figures 19 and 20.

[0738] [References] 1. Jemal, A. et al. Cancer statistics, 2004. CA Cancer J Clin 54, 8-29, doi:10.3322 / canjclin.54.1.8 (2004). 2.Gratwohl, A. et al. Hematopoietic stem cell transplantation: a global perspective. JAMA 303, 1617-1624, doi:10.1001 / jama.2010.491 (2010). 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). 4.Lambertini, M. et al. Cancer and fertility preservation: international recommendations from an expert meeting. BMC Med 14, 1, doi:10.1186 / s12916-015-0545-7 (2016). 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). 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). 7.Hayashi, K. et al. Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice. Science 338, 971-975, doi:10.1126 / science.1226889 (2012). 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). 9.Yamashiro, C. et al. Generation of human oogonia from induced pluripotent stem cells in vitro. Science 362, 356-360, doi:10.1126 / science.aat1674 (2018). 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). 11.Hwang, Y. S. et al. Reconstitution of prospermatogonial specification in vitro from human induced pluripotent stem cells. Nat Commun 11, 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). 13.Stevant, I. 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). 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). 15.Luz Garcia-Alonso et al. Single-cell roadmap of human gonadal development. Nature, doi:doi: 10.1038 / s41586-022-04918-4.(2022). 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). 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).

[0739] All publications, patents, patent applications, and accession numbers listed in the above specification are incorporated herein by reference in their entirety. Although the present invention has been described in relation to specific embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications and variations of the compositions and methods described in the present invention will be apparent to those skilled in the art and are intended to fall within the scope of the following claims. [Brief explanation of the drawing]

[0740] [Figure 1] Schematic diagram of the mouse ESC differentiation protocol. [Figure 2] Fluorescence micrographs showing improved expression of numerous gonadal markers (WT1, GATA4, SF1, CoupTFII) and Sertoli cell markers (SOX9, GATA3) in the improved mouse protocol compared to the conventional protocol. [Figure 3] Fluorescence micrograph showing the generation of Leydig-like cells after 15 days of culture. The cells are co-stained with SF1 and 3BHSD. [Figure 4A-B]Graph showing increased expression of multiple testicular cell type markers. (A) Sertoli cell markers (blue = old, yellow = new). (B) Peritusparenchymal myoform cells. [Figure 5] Schematic diagram of the human ESC differentiation protocol. [Figure 6] Schematic diagram of the human iPSC differentiation protocol. [Figure 7] A schematic diagram of a protocol combining human ESC-derived testis-like cells and primordial germ cells. [Figure 8] Schematic diagram of a protocol for inducing peritubular muscle-like cells from human ESCs. [Figure 9] A schematic diagram of the protocol for inducing Leydig cells from human ESCs. [Figure 10] Differentiation efficiency of human somatic cell-like cells in the presence of hormones, using the protocol described in PCT US2023 / 13608. A) Modified differentiation scheme. B) qPCR expression of gonads, Sertoli, Leydig, and off-target markers. [Figure 11] The modified testicular differentiation protocol of the present invention. (A) Schematic diagram and culture medium composition. (B) Various gonad, Sertoli, and Leydig cell markers. D16 Soma+PGC+ / - hormone is the most efficient and refined condition. [Figure 12] Schematic diagram of the modified mouse ESC differentiation protocol. Briefly, cells were dissociated on day 4 and plated with 25K cells onto a 96-well U-bottom plate. After maintaining the 3D aggregates for 3 days, they were transplanted into ThinCert® cells and harvested on day 17. [Figure 13A-E]Graph data showing that the differentiation scheme in Figure 12 effectively induces testicular progenitor cell markers. Furthermore, it promotes marker expression in Sertoli cells, Leydig cells, and myosoidal cells, and some endothelial cell markers also begin to peak. The two different colors represent two different culture medium compositions for culturing organoids on ThinCert® cell culture inserts. Note: Testicular progenitor cell markers include Tcf21, Pdgfr-alfa, and Nr2f2 (A); Sertoli cell markers include Sox9, Wt1, Gata4, and Inhbb (B); Leydig cell markers include Cyp11a1, Cyp17a1, Hsd3b6, Nr5a1, Cyp21a1, and Cy11b1 (C); myoiform cell markers include Cnn1, Sma-Alfa, and Myh11 (D); and endothelial cell markers include Pecam1, Esam, and Cdh5 (E). The multiplicative changes in gene expression were calculated relative to pre-differentiation ESCs (day 0). Gapdh was used as a housekeeping gene for normalizing Ct values ​​in qPCR analysis. [Figure 14A-D] Fluorescence micrographs showing complete reconstruction of the testicular microenvironment and tubular structure in organoids created by the scheme shown in Figure 12. A) Co-staining of Leydig cells (SF1), peritubular myoid cells (SMA), and Sertoli cells (SOX9). B) Sertoli cells (GATA4+SOX9) form the blood-testis barrier (ZO-1). C) Our in vitro-derived Sertoli cells (SOX9+) produce anti-Müllerian hormone (AMH). D) Leydig cells present in the organoids are mature Leydig cells expressing SF1, HSD3B, and StAR. [Figure 15] In vitro-induced organoids produce testosterone in response to LH or HCG induction. [Figure 16] A schematic diagram showing the mixing of germ cells on day 2 postnatal with somatic cells induced in vitro by our method. Aggregation occurs on day 4, according to the differentiation scheme shown in Figure 12. [Figure 17A-D]Graphs showing the germ cell count and percentage of organoids in a 96-well plate on days 1 and 3. The total germ cell count is determined by counting the total number of DDX4 or DAZL-positive cells on days 1 and 3 (A). Percentage and total number of OCT4-GFP-positive prespermatogonial cells (B), percentage and total number of PLZF-positive undifferentiated spermatogonial cells (C), and percentage and total number of differentiating Stra8-positive spermatogonial cells. [Figure 18] An experimental scheme for in vitro testicular organoid generation from human stem cells. [Figure 19] Graph data showing that increasing the EGF1 concentration to 50 ng / ml improves the expression level of Sertoli cell markers by day 22. [Figure 20A-B] Immunofluorescence micrograph of testicular organoids collected after 22 days of culture. (A) WT1: Marker for testicular stromal cells (Leydig cells and myosoidal cell progenitor cells). (B) Formation of SOX9-positive and Gata4-positive tubules.

Claims

1. An in vitro method for producing artificial testicular cells from pluripotent stem cells of vertebrates: A process for inducing germline cells from pluripotent stem cells; and, A step of differentiating the germline cells into testicular cells by treating them with a basal medium containing fibroblast growth factor 9 (FGF9), insulin and / or insulin-like growth factor 1 (IGF1), and epidermal growth factor (EGF), A method that includes this.

2. The basal medium further comprises prostaglandin D2 (PGD2) and / or retinoic acid (RA), The method according to claim 1.

3. The basal medium further comprises follicle-stimulating hormone (FSH) and / or luteinizing hormone (LH), or human chorionic gonadotropin (HCG). The method according to any one of claims 1 to 2.

4. The aforementioned basal culture medium further contains testosterone (T), The method according to any one of claims 1 to 3.

5. The aforementioned basal culture medium further comprises IWR1. The method according to any one of claims 1 to 4.

6. The process of inducing germline cells is: A step of providing pluripotent stem cells of vertebrates in a maintenance medium containing a ROCK inhibitor; On day 0, the steps include removing the maintenance medium containing the ROCK inhibitor, and culturing the pluripotent stem cells of the vertebrate in the basal medium containing CHIR99021 to differentiate the pluripotent stem cells of the vertebrate into unsegmented mesoderm cells; On approximately the fourth day, the process involves removing the basal medium containing CHIR99021, and culturing the unsegmented mesoderm cells in a basal medium containing fibroblast growth factor 9 (FGF9) and heparin to differentiate the unsegmented mesoderm cells into intermediate mesoderm cells; and, On approximately the 7th or 8th day, the steps include removing the medium containing FGF9 and heparin, and culturing the cells in a basal medium containing FGF9, insulin and / or IGF1, EGF, RA, PGD2, LH or hCG, FSH, and / or T, or in a basal medium containing insulin and / or IGF1, FGF9, RA, and PGD2 to differentiate the cells into germline cells. The method according to claim 1, further comprising:

7. The germline cells are treated on approximately day 8 or 9 with the basal medium containing FGF9, insulin and / or IGF1, EGF, LH or hCG, FSH, and / or T to provide gonadal formation-inducing cells. The method according to any one of claims 6.

8. The aforementioned gonadal formation-inducing cells are cultured to induce organoid formation. The method according to claim 7.

9. The aforementioned germinal prominence cells are dissociated into single cells on approximately the 8th day. The method according to any one of claims 1 to 8.

10. On the 12th day, The process of extracting organoids from the culture, and A step of culturing the organoids at the gas-liquid interface to mature the testicular organoids, The method according to any one of claims 1 to 9, further comprising:

11. The testicular organoid comprises one or more artificial Sertoli cells, artificial Leydig cells, artificial myosophical cells, and artificial stromal cells. The method according to claim 10.

12. The testicular organoid comprises two or more artificial Sertoli cells, artificial Leydig cells, artificial myosophical cells, and artificial stromal cells. The method according to claim 10.

13. The testicular organoid comprises three or more artificial Sertoli cells, artificial Leydig cells, artificial myosophical cells, and artificial stromal cells. The method according to claim 10.

14. The testicular organoid comprises artificial Sertoli cells, artificial Leydig cells, artificial myosophical cells, and artificial stromal cells. The method according to claim 10.

15. The organoid exhibits 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. The method according to any one of claims 10 to 14.

16. The pluripotent stem cells of the aforementioned vertebrate are human stem cells. The method according to any one of claims 1 to 12.

17. The aforementioned human stem cells are human embryonic stem cells. The method according to claim 16.

18. The aforementioned human stem cells are induced pluripotent stem cells. The method according to claim 16.

19. 1) A step of isolating the artificial testicular cells from the organoid, or 2) A step of isolating the testicular organoid, The method according to any one of claims 1 to 18, further comprising:

20. The artificial testicular cells are Sertoli-like cells. The method according to claim 19.

21. The artificial testicular cells are Leydig-like cells. The method according to claim 19.

22. The artificial testicular cells are myoform cells. The method according to claim 19.

23. The artificial testicular cells are interstitial progenitor cells. The method according to claim 19.

24. The process further includes transplanting the isolated artificial testicular cells or artificial testicular organoids into a mammal. The method according to any one of claims 19 to 23.

25. The steps include: bringing the artificial testicular cells or artificial testicular cell organoids into contact with a test reagent; A step of evaluating the effect of the test reagent on the artificial testicular cells or artificial testicular cell organoids, The method according to any one of claims 1 to 23, further comprising:

26. A process for obtaining stem cells or tissue containing stem cells from a patient, and A step of providing differentiated patient stem cells by culturing the patient-derived stem cells or tissue containing stem cells with artificial testicular cells or artificial testicular cell organoids. The method according to any one of claims 1 to 23, further comprising:

27. The aforementioned stem cells are primordial germ cells, prespermatogonia, or spermatogonial stem cells. The method according to claim 26.

28. The aforementioned primordial germ cell-like cells and prespermatogonial stem cells differentiate into spermatogonial cells. The method according to claim 27.

29. The process further includes returning the aforementioned stem cells or differentiated spermatogonial cells to a patient who needs them. The method according to any one of claims 26 to 28.

30. The patient has previously received gonadal toxicity treatment and / or has non-obstructive azoospermia or severe oligozoospermia, The method according to any one of claims 26 to 28.

31. The aforementioned gonadal toxic treatment is selected from the group consisting of chemotherapy and radiotherapy. The method according to claim 30.

32. The aforementioned stem cells or tissue containing stem cells are obtained from the patient prior to gonadal toxicity treatment. The method according to any one of claims 26 to 31.

33. The process further includes a step of co-culturing primordial germ cells with the aforementioned germline cells on approximately the 8th day. The method according to any one of claims 1 to 18.

34. The aforementioned primordial germ cells originate from the embryo. The method according to claim 33.

35. The aforementioned primordial germ cells are primordial germ cell-like cells. The method according to any one of claims 33 to 34.

36. The aforementioned primordial germ cells differentiate into spermatogonial cells. The method according to any one of claims 33 to 35.

37. The process further includes isolating the spermatogonial cells, The method according to claim 36.

38. A cell culture comprising artificial testicular cells produced by any one of claims 1 to 23 and 33 to 37.

39. Isolated artificial testicular cells produced by the method described in any one of claims 19 to 23.

40. Artificial Leydig cells produced by the method described in claim 19.

41. Artificial Sertoli cells produced by the method described in claim 19.

42. Artificial muscle-like cells produced by the method described in claim 19.

43. Artificial stromal progenitor cells produced by the method described in claim 19.

44. An artificial testicular organoid produced by the method described in claim 19.

45. spermatogonial cells produced by the method described in any one of claims 33 to 37.

46. Differentiated patient stem cells produced by the method described in any one of claims 26-28 and 30-32.

47. An in vitro method for producing artificial Leydig-like cells from vertebrate pluripotent stem cells: A process for inducing germline cells from pluripotent stem cells; and, The aforementioned germline cells are treated 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 LiCl ２ A step of treating the germline cells with a basal medium containing the above to differentiate them into Leydig-like cells, A method that includes this.

48. The process of inducing germline cells is: A step of providing pluripotent stem cells of vertebrates in a maintenance medium containing a ROCK inhibitor; On day 0, the steps include removing the maintenance medium containing the ROCK inhibitor, and culturing the pluripotent stem cells of the vertebrate in the basal medium containing CHIR99021 to differentiate the pluripotent stem cells of the vertebrate into presomatic mesoderm cells; On approximately the fourth day, the steps include: removing the basal medium containing CHIR99021; and culturing the presegmented mesoderm cells in a basal medium containing fibroblast growth factor 9 (FGF9) and heparin to differentiate the presegmented mesoderm cells into intermediate mesoderm cells; and On approximately the 7th or 8th day, the steps include removing the medium containing FGF9 and heparin, and culturing the cells in a basal medium containing FGF9, insulin and / or IGF1, EGF, RA, PDG2, LH, FSH, and / or T, or in a basal medium containing insulin and / or IGF1, FGF9, RA, and PGD2 to differentiate the cells into germline cells. The method according to claim 47, further comprising:

49. The aforementioned germinal prominence cells, around day 8, produce SAG (Smoothened agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), bFGF2 (Basic fibroblast growth factor), DAPT, and LiCl. ２ The basal medium containing the above is used to treat the following: The method according to any one of claims 47 to 48.

50. The aforementioned germline cells are dissociated into single cells. The method according to claim 49.

51. The Leydig-like cells are produced in the culture by approximately day 16. The method according to any one of claims 47 to 50.

52. The Leydig-like cells exhibit upregulation or expression of one or more markers selected from the group consisting of STAR and 3BHSD. The method according to any one of claims 47 to 51.

53. The pluripotent stem cells of the aforementioned vertebrate are human stem cells. The method according to any one of claims 47 to 52.

54. The aforementioned human stem cells are human embryonic stem cells. The method according to claim 53.

55. The aforementioned human stem cells are induced pluripotent stem cells. The method according to claim 53.

56. The process further includes isolating the Leydig-like cells, The method according to any one of claims 47 to 55.

57. The process further includes transplanting the isolated Leydig-like cells into a mammal. The method according to claim 56.

58. The steps include bringing the Leydig-like cells into contact with a test reagent, and A step of evaluating the effect of the test reagent on the cells, The method according to any one of claims 47 to 56, further comprising:

59. A process for obtaining stem cells or tissue containing stem cells from a patient, and A step of culturing the patient-derived stem cells or tissue containing stem cells with the Leydig-like cells, The method according to any one of claims 47 to 56, further comprising:

60. The process further includes returning the aforementioned stem cells or cells differentiated from the aforementioned stem cells to a patient in need. The method according to claim 59.

61. Artificial Leydig cells produced by the method described in any one of claims 47 to 56.

62. An in vitro method for producing artificial muscle-like cells from pluripotent stem cells of vertebrates: A process for inducing germline cells from pluripotent stem cells; and, A step of differentiating the germline cells into myosocyte-like cells by treating them with a basal medium containing 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. A method that includes this.

63. The process of inducing germline cells is: A step of providing pluripotent stem cells of vertebrates in a maintenance medium containing a ROCK inhibitor; On day 0, the steps include removing the maintenance medium containing the ROCK inhibitor, and culturing the pluripotent stem cells of the vertebrate in the basal medium containing CHIR99021 to differentiate the pluripotent stem cells of the vertebrate into presomatic mesoderm cells; On approximately the fourth day, the steps include: removing the basal medium containing CHIR99021; and culturing the presegmented mesoderm cells in a basal medium containing fibroblast growth factor 9 (FGF9) and heparin to differentiate the presegmented mesoderm cells into intermediate mesoderm cells; and On approximately the 7th or 8th day, the steps include removing the medium containing FGF9 and heparin, and culturing the cells in a basal medium containing FGF9, insulin and / or IGF1, EGF, RA, PDG2, LH, FSH, and / or T, or in a basal medium containing insulin and / or IGF1, FGF9, RA, and PGD2 to differentiate the cells into germline cells. The method according to claim 62, further comprising:

64. The germinal protuberance cells are treated on approximately day 8 with the basal medium containing 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. The method according to any one of claims 62 to 63.

65. The aforementioned germline cells are dissociated into single cells. The method according to claim 64.

66. The muscle-like cells mentioned above are produced in the culture by approximately day 16. The method according to any one of claims 62 to 65.

67. The aforementioned myosophical cell-like cells exhibit upregulation or expression of one or more myosophical cell markers. The method according to any one of claims 62 to 66.

68. The pluripotent stem cells of the aforementioned vertebrate are human stem cells. The method according to any one of claims 62 to 67.

69. The aforementioned human stem cells are human embryonic stem cells. The method according to claim 68.

70. The aforementioned human stem cells are induced pluripotent stem cells. The method according to claim 68.

71. The process further includes isolating the muscle-like cell-like cells, The method according to any one of claims 62 to 70.

72. The process further includes transplanting the isolated myosophical cell-like cells into a mammal. The method according to claim 71.

73. The steps include bringing the myosoidal cell-like cells into contact with a test reagent, and A step of evaluating the effect of the test reagent on the cells, The method according to any one of claims 62 to 71, further comprising:

74. A process for obtaining stem cells or tissue containing stem cells from a patient, and A step of culturing the patient-derived stem cells or tissue containing stem cells with the Leydig-like cells, The method according to any one of claims 62 to 71, further comprising:

75. The process further includes returning the aforementioned stem cells or cells differentiated from the aforementioned stem cells to a patient in need. The method according to claim 72.

76. Artificial muscle-like cells produced by the method described in any one of claims 62 to 71.

77. An in vitro method for producing artificial testicular cells from mouse pluripotent stem cells: A process for inducing intermediate mesoderm cells from mouse pluripotent stem cells; and, A step of differentiating the intermediate mesoderm cells into testicular cells by treating them with a basal medium containing FGF9, insulin and / or IGF1, PGD2, RA, BMP4 (bone morphogenetic protein 4), and EGF, or with a basal medium containing FGF9, insulin and / or IGF1, PGD2, RA, BMP4 (bone morphogenetic protein 4), EGF, FSH, and LH or human chorionic gonadotropin (hCG). A method that includes this.

78. The process of inducing intermediate mesoderm cells is: A process for providing mouse pluripotent stem cells; On day 0, the pluripotent stem cells of the mouse are cultured in the basal medium containing activin A (AA) and bFGF (basic fibroblast growth factor); and, On approximately the second day, the process involves removing the basal medium containing AA and bFGF, and culturing the cells in a basal medium containing AA, RA, and BMP4. The method according to claim 77, further comprising:

79. The aforementioned cells, On approximately the 6th day, the cells are treated with the basal medium containing FGF9, insulin and / or IGF1, PGD2, RA, BMP4, and EGF, or On approximately day 6 or 7, the cells are treated with the basal medium containing FGF9, insulin and / or IGF1, PGD2, RA, BMP4, EGF, FSH, and LH or hCG. The method according to any one of claims 77 to 78.

80. The culture medium, on approximately day 7, contains SAG (Smoothened agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), bFGF2 (Basic fibroblast growth factor), DAPT, and LiCl. ２ The basal medium is replaced with Leydig cell differentiation medium containing supplemented basal medium. The method according to any one of claims 77 to 79.

81. The culture medium is replaced on approximately day 7 with a myosocyte differentiation medium containing a basal 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. The method according to any one of claims 77 to 79.

82. Testicular organoids are formed in the culture by approximately the 8th day. The method according to any one of claims 77 to 81.

83. The testicular organoid comprises one or more artificial Sertoli cells, artificial Leydig cells, artificial myosophical cells, and artificial stromal cells. The method according to claim 82.

84. The testicular organoid comprises two or more artificial Sertoli cells, artificial Leydig cells, artificial myosophical cells, and artificial stromal cells. The method according to claim 82.

85. The testicular organoid comprises three or more artificial Sertoli cells, artificial Leydig cells, artificial myosophical cells, and artificial stromal cells. The method according to claim 82.

86. The testicular organoid comprises artificial Sertoli cells, artificial Leydig cells, artificial myosophical cells, and artificial stromal cells. The method according to claim 82.

87. The organoid exhibits 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. The method according to any one of claims 82 to 86.

88. The aforementioned mouse stem cells are mouse embryonic stem cells. The method according to any one of claims 77 to 87.

89. The aforementioned mouse stem cells are induced pluripotent stem cells. The method according to any one of claims 77 to 87.

90. 1) A step of isolating the artificial testicular cells from the organoid, or 2) A step of isolating the testicular organoid, The method according to any one of claims 77 to 87, further comprising:

91. The artificial testicular cells are Sertoli-like cells. The method according to claim 90.

92. The artificial testicular cells are Leydig-like cells. The method according to claim 90.

93. The artificial testicular cells are myoform cells. The method according to claim 90.

94. The artificial testicular cells are interstitial progenitor cells. The method according to claim 90.

95. The process further includes transplanting the isolated artificial testicular cells or artificial testicular organoids into a mammal. The method according to any one of claims 90 to 92.

96. The steps include: bringing the artificial testicular cells or artificial testicular cell organoids into contact with a test reagent; A step of evaluating the effect of the test reagent on the artificial testicular cells or artificial testicular cell organoids, The method according to any one of claims 77 to 94, further comprising:

97. A testicular organoid produced by the method described in any one of claims 77 to 94.

98. Artificial Leydig cells produced by the method described in any one of claims 77 to 94.

99. Artificial Sertoli cells produced by the method described in any one of claims 77 to 94.

100. Artificial muscle-like cells produced by the method described in any one of claims 77 to 94.

101. Artificial stromal progenitor cells produced by the method described in any one of claims 77 to 94.

102. A step of contacting artificial testicular cells or organoids according to any one of claims 38 to 46, 61, 76, and 97 to 101 with a test reagent; and, A step of evaluating the effect of the test reagent on the artificial testicular cells or organoids, A method that includes this.

103. A method comprising the step of transplanting artificial testicular cells or organoids according to any one of claims 38 to 46, 61, 76, and 97 to 101.

104. The process of obtaining stem cells or tissue containing stem cells from a patient; and, A step of co-culturing the patient-derived stem cells or tissue containing stem cells with artificial testicular cells or organoids according to any one of claims 38 to 46, 61, 76, and 97 to 101. A method that includes this.

105. The aforementioned stem cells are selected from the group consisting of primordial germ cell-like cells (PGCLCs), prespermatogonia, and spermatogonial stem / progenitor cells (SSCs / SPCs). The method according to claim 104.

106. The aforementioned pre-spermatogonial stem cells differentiate into spermatogonial cells. The method according to claim 105.

107. The process further includes returning the spermatogonial cells to a patient who needs them. The method according to any one of claims 104 to 106.

108. The aforementioned patient has previously received gonadal toxicity treatment and / or has non-obstructive azoospermia or severe oligozoospermia, The method according to any one of claims 104 to 107.

109. The aforementioned gonadal toxic treatment is selected from the group consisting of chemotherapy and radiotherapy. The method according to claim 108.

110. The aforementioned stem cells or tissue containing stem cells are obtained from the patient prior to gonadal toxicity treatment. The method according to any one of claims 104 to 109.

111. A method for growing patient-derived or in vitro-induced germ cells: A step of co-culturing germ cells derived from the patient or induced in vitro with artificial testicular cells or organoids according to any one of claims 38-46, 61, 76, and 97-101. A method that includes this.

112. The germ cells derived from the aforementioned patient, or induced in vitro, are primordial germ cell-like cells (PGCLCs), prespermatogonia, or prespermatogonia-like cells. The method according to claim 111.

113. Co-culturing brings forth the ability of the aforementioned primordial germ cell-like cells (PGCLCs), prespermatogonia, and prespermatogonia-like cells to form spermatogonies. The method according to any one of claims 111 to 112.

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