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WO2025038906A1 - Cellules testiculaires artificielles et leur production - Google Patents

Cellules testiculaires artificielles et leur production Download PDF

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WO2025038906A1
WO2025038906A1 PCT/US2024/042621 US2024042621W WO2025038906A1 WO 2025038906 A1 WO2025038906 A1 WO 2025038906A1 US 2024042621 W US2024042621 W US 2024042621W WO 2025038906 A1 WO2025038906 A1 WO 2025038906A1
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cells
artificial
testis
stem cells
day
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Sue HAMMOUD
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University of Michigan System
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University of Michigan System
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0683—Cells of the male genital tract, e.g. prostate, epididymis; Non-germinal cells from testis, e.g. Leydig cells, Sertoli cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/52—Sperm; Prostate; Seminal fluid; Leydig cells of testes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/105—Insulin-like growth factors [IGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/31—Pituitary sex hormones, e.g. follicle-stimulating hormone [FSH], luteinising hormone [LH]; Chorionic gonadotropins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • C12N2501/392—Sexual steroids

Definitions

  • testis cells e.g., Sertoli and/or Leydig cells
  • Infertility is a rapidly rising crisis worldwide, and in roughly 50% of cases the cause is male factor infertility.
  • Traditional treatment options such as in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), require sperm, and leave these men with no therapies.
  • IVF in vitro fertilization
  • ICSI intracytoplasmic sperm injection
  • PGCLCs primordial germ cell-like cells
  • ESCs mouse and human embryonic stem cells
  • somatic cells from human fetal testis to aid the progression of in vitro derived human PGCLCs has ethical and technical limitations related to fetal tissue-based research. Therefore, to generate testis-like somatic cells without relying on fetal tissue is of clinical significance. This has previously been attempted by reprogramming human fibroblasts into Sertoli- or Leydig-like cells using a combination of well-chosen transcription factors. Despite expressing a handful of known markers for Sertoli and Leydig cells, the extent to which the in vitro derived cells resemble in vivo cells or recapitulate endogenous functions remain difficult to assess. Furthermore, given the requirement for transgenesis for efficient induction, and for the continuous expression of transcription factors for maintenance of Sertoli and Leydig cell fates, the clinical utility of such cells is unclear.
  • the present invention relates to in vitro methods for production of testis cells and related organoids.
  • the present invention provides in vitro methods for production of artificial testis cells from vertebrate pluripotent stem cells comprising: deriving genital ridge cells from pluripotent stem cells; and treating the genital ridge cells with a base medium comprising fibroblast growth factor 9 (FGF9), insulin and/or Insulin-like Growth Factor 1 (IGFl),and Epidermal Growth Factor (EGF) so that the genital ridge cells differentiate into testis cells.
  • the base medium further comprises Prostaglandin D2 (PGD2) and/or retinoic acid (RA).
  • the base medium further comprises Follicle Stimulating Hormone (FSH) and/or luteinizing hormone (LH) or Human Chorionic Gonadotropin (HCG).
  • FSH Follicle Stimulating Hormone
  • LH luteinizing hormone
  • HCG Human Chorionic Gonadotropin
  • base medium further comprises testosterone (T).
  • T testosterone
  • the base medium further comprises IWR1.
  • the step of deriving genital ridge cells further comprises: providing vertebrate pluripotent stem cells in a maintenance medium comprising a ROCK inhibitor; at day 0, removing the maintenance medium comprising a ROCK inhibitor and culturing the vertebrate pluripotent stem cells with the base medium comprising CHIR99021 so that the vertebrate pluripotent stem cells differentiate into presomitic mesoderm cells; on about day 4, removing the base medium comprising CHIR99021 and culturing the presomitic mesoderm cells in base medium comprising fibroblast growth factor 9 (FGF9) and heparin so that the presomitic mesoderm cells differentiate into intermediate mesoderm cells; and on about day 7 or 8, removing the medium comprising FGF9 and heparin and culturing the cells in a base medium comprising FGF9, insulin and/or IGF1, EGF, RA,PGD2, LH, FSH and/or T or a base medium comprising insulin and/r I
  • FGF9
  • the genital ridge cells are treated with the base medium comprising FGF9, insulin and/or IGF1, EGF, LH, FSH and/or T on about day 8 or 9 to provide gonadogenesis-induced cells.
  • the gonadogenesis-induced cells are cultured to induce the formation of organoids.
  • the genital ridge cells are dissociated into single cells on about day 8.
  • the methods further comprise, preferably on about day 12, removing organoids from the culture and culturing the organoids at an air-liquid interphase to allow maturation of testis organoids.
  • the testis organoids comprise one or more of artificial Sertoli cells, artificial Leydig cells, artificial myoid cells and artificial stromal cells.
  • the testis organoids comprise two or more of artificial Sertoli cells, artificial Leydig cells, artificial myoid cells and artificial stromal cells.
  • the testis organoids comprise three or more of artificial Sertoli cells, artificial Leydig cells, artificial myoid cells and artificial stromal cells.
  • the testis organoids comprise artificial Sertoli cells, artificial Leydig cells, artificial myoid cells and artificial stromal cells.
  • the organoids demonstrate upregulation or expression of one or more markers selected from the group consisting of LHX9, PDGRA, COUPTFII, TCF21, SOX9, GATA4, SF1, SMA, DHH, STAR and 3BHSD.
  • the vertebrate pluripotent stem cells are human stem cells.
  • the human stem cells are human embryonic stem cells.
  • the human stem cells are induced pluripotent stem cells.
  • the methods further comprise the step of isolating 1) the artificial testis cells from the organoids or 2) the testis organoid.
  • the artificial testis cells are Sertoli-like cells.
  • the artificial testis cells are Leydig-like cells.
  • the artificial testis cells are Myoid-like cells.
  • the artificial testis cells are stromal progenitor cells.
  • the methods further comprise transplanting the isolated artificial testis cells or artificial testis organoid into a mammal.
  • the methods further comprise contacting the artificial testis cells or artificial testis cell organoid with a test reagent and assaying the effect of the test reagent on the artificial testis cells or artificial testis cell organoid.
  • the methods further comprise obtaining stem cells or tissue comprising stem cells from a patient and culturing the stem cells or tissue comprising stem cells from the patient with the artificial testis cells or artificial testis cell organoid to provide differentiated patient stem cells.
  • the stem cells are primordial germ cells, pro-spermatogonia or spermatogonial stem cells.
  • the primordial germ cell like cells and pro-spermatogonia stem cells differentiate into spermatogonia.
  • the methods further comprise transferring the stem cells or differentiated spermatogonia back to a patient in need thereof.
  • the patient is diagnosed with nonobstructive azoospermia or severe oligospermia or has previously undergone a gonadotoxic treatment.
  • the stem cells or tissue comprising stem cells are obtained from the patient prior to a gonadotoxic treatment.
  • the stem cells or tissue comprising stem cells are obtained from the patient from NOA.
  • the methods further comprise about day 8 coculturing primordial germ cells with the genital ridge cells.
  • the primordial germ cells are derived from an embryo.
  • the primordial germ cells are primordial germ cell-like cells.
  • the primordial germ cells differentiate into spermatogonia.
  • the methods further comprise isolating the spermatogonia.
  • the present invention provides a cell culture comprising artificial testis cells produced by the foregoing methods.
  • the present invention provides isolated artificial testis cells produced by the foregoing methods, preferably isolated artificial Sertoli cells, isolated artificial Leydig cells, isolated artificial Myoid cells and/or isolated artificial stromal progenitor cells.
  • the present invention provides an artificial testis organoid produced by the foregoing methods.
  • the present invention provides spermatogonia produced by the foregoing methods.
  • the present invention provides differentiated patient stem cells produced by the foregoing methods.
  • the present invention provides in vitro methods for production of artificial Leydig-like cells from vertebrate pluripotent stem cells comprising: deriving genital ridge cells from pluripotent stem cells; and treating the genital ridge cells with a base medium comprising SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), bFGF2 (Basic fibroblast growth factor), DAPT and LiCh so that the genital ridge cells differentiate into Leydig-like cells.
  • SAG Smoothened Agonist
  • PDGF-AA Platinum-derived growth factor AA
  • PDGF-BB Platinum-derived growth factor BB
  • bFGF2 Basic fibroblast growth factor
  • the step of deriving genital ridge cells further comprises: providing vertebrate pluripotent stem cells in a maintenance medium comprising a ROCK inhibitor; at day 0, removing the maintenance medium comprising a ROCK inhibitor and culturing the vertebrate pluripotent stem cells with the base medium comprising CHIR99021 so that the vertebrate pluripotent stem cells differentiate into presomatic mesoderm cells; on about day 4, removing the base medium comprising CHIR99021 and culturing the presomatic mesoderm cells in base medium comprising fibroblast growth factor 9 (FGF9) and heparin so that the presomatic mesoderm cells differentiate into intermediate mesoderm cells; and on about day 7 or 8, removing the medium comprising FGF9 and heparin and culturing the cells in a base medium comprising FGF9, insulin and/or IGF1, EGF, RA,PGD2, LH, FSH and/or T or a base medium comprising insulin and/r I
  • FGF9
  • the genital ridge cells are treated with the base medium comprising SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), bFGF2 (Basic fibroblast growth factor), DAPT and LiCh on about day 8.
  • the genital ridge cells are dissociated into single cells.
  • the Leydig-like cells are produced in the culture by about day 16. In some preferred embodiments, the Leydig-like cells demonstrate upregulation or expression of one or more markers selected from the group consisting of STAR and 3BHSD.
  • the vertebrate pluripotent stem cells are human stem cells.
  • the human stem cells are human embryonic stem cells.
  • the human stem cells are induced pluripotent stem cells.
  • the methods further comprise the step of isolating the Leydig-like cells.
  • the methods further comprise the step of transplanting the isolated Leydig-like cells into a mammal.
  • the methods further comprise contacting the Leydig- like cells with a test reagent and assaying the effect of the test reagent on the cells.
  • the methods further comprise obtaining stem cells or tissue comprising stem cells from a patient and culturing the stem cells or tissue comprising stem cells from the patient with the Leydig-like cells. In some preferred embodiments, the methods further comprise transferring the stem cells or cells differentiated from the stem cells back to a patient in need thereof.
  • the present invention provides artificial Leydig cells produced by the foregoing methods.
  • the present invention provides in vitro method for production of artificial Myoid-like cells from vertebrate pluripotent stem cells comprising: deriving genital ridge cells from pluripotent stem cells; and treating the genital ridge cells with a base medium comprising SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), valproic acid, BMP2 (Bone Morphogenetic Protein 2), BMP4 (Bone Morphogenetic Protein 4), and Activin A so that the genital ridge cells differentiate into Myoid-like cells.
  • SAG Smoothened Agonist
  • PDGF-AA Platinum-derived growth factor AA
  • PDGF-BB Platinum-derived growth factor BB
  • valproic acid BMP2 (Bone Morphogenetic Protein 2)
  • BMP4 Bone Morphogenetic Protein 4
  • the step of deriving genital ridge cells further comprises: providing vertebrate pluripotent stem cells in a maintenance medium comprising a ROCK inhibitor; at day 0, removing the maintenance medium comprising a ROCK inhibitor and culturing the vertebrate pluripotent stem cells with the base medium comprising CHIR99021 so that the vertebrate pluripotent stem cells differentiate into presomatic mesoderm cells; on about day 4, removing the base medium comprising CHIR99021 and culturing the presomatic mesoderm cells in base medium comprising fibroblast growth factor 9 (FGF9) and heparin so that the presomatic mesoderm cells differentiate into intermediate mesoderm cells; and on about day 7 or 8, removing the medium comprising FGF9 and heparin and culturing the cells in a base medium comprising FGF9, insulin and/or IGF1, EGF, RA,PGD2, LH, FSH and/or T or a base medium comprising insulin and/r I
  • FGF9
  • the genital ridge cells are treated with the base medium comprising SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), valproic acid, BMP2 (Bone Morphogenetic Protein 2), BMP4 (Bone Morphogenetic Protein 4), and Activin A on about day 8.
  • the genital ridge cells are dissociated into single cells.
  • the Myoid-like cells are produced in the culture by about day 16. In some preferred embodiments, the Myoid-like cells demonstrate upregulation or expression of one or more Myoid cell markers.
  • the vertebrate pluripotent stem cells are human stem cells.
  • the human stem cells are human embryonic stem cells.
  • the human stem cells are induced pluripotent stem cells.
  • the methods further comprise the step of isolating the Myoid-like cells. In some preferred embodiments, the methods further comprise transplanting the isolated Myoid-like cells into a mammal.
  • the methods further comprise contacting the Myoidlike cells with a test reagent and assaying the effect of the test reagent on the cells.
  • the methods further comprise obtaining stem cells or tissue comprising stem cells from a patient and culturing the stem cells or tissue comprising stem cells from the patient with the Leydig-like cells.
  • the stem cells or cells differentiated from the stem cells back to a patient in need thereof.
  • the present invention provides artificial Myoid cells produced by the foregoing methods.
  • the present invention provides in vitro methods for production of artificial testis cells from mouse pluripotent stem cells comprising: deriving intermediate mesoderm cells from mouse pluripotent stem cells; and treating the intermediate mesoderm cells with a base medium comprising FGF9, insulin and/or IGF1, PGD2, RA, BMP4 (Bone Morphogenetic Protein 4), and EGF or a base medium comprising FGF9, insulin and/or IGF1, PGD2, RA, BMP4 (Bone Morphogenetic Protein 4), EGF, FSH and LH or Human Chorionic Gonadotropin (HCG) so that the intermediate ridge cells differentiate into testis cells.
  • a base medium comprising FGF9, insulin and/or IGF1, PGD2, RA, BMP4 (Bone Morphogenetic Protein 4), and EGF or a base medium comprising FGF9, insulin and/or IGF1, PGD2, RA, BMP4 (Bone Morphogenetic Protein 4), EGF, FSH and LH or Human Chori
  • the step of deriving intermediate mesoderm cells further comprises: providing mouse pluripotent stem cells; at day 0, culturing the mouse pluripotent stem cells with the base medium comprising Activin A (A A) and bFGF (Basic Fibroblast Growth Factor); and on about day 2, removing the base medium comprising AA and bFGF and culturing the cells in base medium comprising AA, RA, and BMP4.
  • a A Activin A
  • bFGF Basic Fibroblast Growth Factor
  • the cells are treated with the base medium comprising FGF9, insulin and/or IGF1, PGD2, RA, BMP4, and EGF on about day 6 or FGF9, insulin and/or IGF1, PGD2, RA, BMP4, EGF, FSH, and LH or HCG on about day 6 or 7.
  • the culture medium is replaced with a Leydig cell differentiation medium comprising a base medium supplemented with SAG (Smoothened Agonist), PDGF- AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), bFGF2 (Basic fibroblast growth factor), DAPT and LiCh.
  • the culture medium is replaced with a Myoid cell differentiation medium comprising a base medium supplemented with SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), valproic acid, BMP2 (Bone Morphogenetic Protein 2), BMP4 (Bone Morphogenetic Protein 4), and Activin A.
  • SAG Smoothened Agonist
  • PDGF-AA Platinum-derived growth factor AA
  • PDGF-BB Platinum-derived growth factor BB
  • valproic acid valproic acid
  • BMP2 Bone Morphogenetic Protein 2
  • BMP4 Bone Morphogenetic Protein 4
  • testis organoids are formed in the culture by about day 8.
  • the testis organoids comprise one or more of artificial Sertoli cells, artificial Leydig cells, artificial myoid cells and artificial stromal cells.
  • the testis organoids comprise two or more of artificial Sertoli cells, artificial Leydig cells, artificial myoid cells and artificial stromal cells.
  • the testis organoids comprise three or more of artificial Sertoli cells, artificial Leydig cells, artificial myoid cells and artificial stromal cells.
  • the testis organoids comprise artificial Sertoli cells, artificial Leydig cells, artificial myoid cells and artificial stromal cells.
  • the organoids demonstrate upregulation or expression of one or more markers selected from the group consisting of LHX9, PDGRA, COUPTFII, TCF21, SOX9, GATA4, SF1, SMA, DHH, STAR and 3BHSD.
  • the mouse stem cells are mouse embryonic stem cells. In some preferred embodiments, the mouse stem cells are induced pluripotent stem cells.
  • the methods further comprise the step of isolating 1) the artificial testis cells from the organoids or 2) the testis organoid.
  • the artificial testis cells are Sertoli-like cells.
  • the artificial testis cells are Leydig-like cells.
  • the artificial testis cells are Myoid-like cells.
  • the artificial testis cells are stromal progenitor cells.
  • the methods further comprise transplanting the isolated artificial testis cells or artificial testis organoid into a mammal.
  • the methods further comprise contacting the artificial testis cells or artificial testis cell organoid with a test reagent and assaying the effect of the test reagent on the artificial testis cells or artificial testis cell organoid.
  • the present invention provides testis organoid produced by the foregoing methods.
  • the present invention provides artificial Leydig cells produced by the foregoing methods.
  • the present invention provides artificial Sertoli cells produced by the foregoing methods.
  • the present invention provides artificial Myoid cells produced by the foregoing methods.
  • the present invention provides artificial stromal progenitor cells produced by the foregoing methods.
  • the present invention provides methods comprising: contacting artificial testis cells or organoids as described in any aspect above with a test reagent; and assaying the effect of the test reagent on the artificial testis cells or organoids.
  • the present invention provides methods comprising: transplanting the artificial testis cells or organoids according to any aspect above into a subject.
  • the present invention provides methods comprising: obtaining stem cells or tissue comprising stem cells from a patient; and co-culturing the stem cells or tissue comprising stem cells from the patient with artificial testis cells or organoids according to any aspect described above.
  • the stem cells are selected from the group consisting of primordial germ cell like cells (PGCLC), prospermatogonia, and spermatogonial stem/progenitor cells (SSC/SPCs).
  • PPCLC primordial germ cell like cells
  • SSC/SPCs spermatogonial stem/progenitor cells
  • the prospermatogonia stem cells differentiate into spermatogonia.
  • the methods further comprise transferring the spermatogonia back to a patient in need thereof.
  • the patient has previously undergone a gonadotoxic treatment and/or has nonobstructive azoospermia patient or severe oligospermy.
  • the gonadotoxic treatment is selected from the group consisting of chemotherapy and radiation.
  • the stem cells or tissue comprising stem cells are obtained from the patient prior to a gonadotoxic treatment.
  • the present invention provides methods of expanding patient derived or in vitro derived germ cells comprising: co-culturing patient or in vitro derived germ cells with artificial testis cells or organoids according to any aspect described above.
  • the primordial germ cells are primordial germ cell-like cells (PGCLCs), prospermatogonia or spermatogonia.
  • co- culturing results in licensure of the primordial germ cell like cells or pro- spermatogonia or pro-spermatogonia-like cells to form spermatogonia.
  • FIG. 1 Schematic depiction of mouse ESC differentiation protocol.
  • FIG. 2 Fluorescence micrographs showing improved expression of many gonadal (WT1, GATA4, SF1, CoupTFII) and Sertoli cell markers (SOX9, GAT A3) in the improved mouse protocol as compared to an older protocol.
  • FIG. 3 Fluorescence micrographs demonstrating generation of Leydig-like cells after 15 days in culture. Cells are co-stained by SF1 and 3BHSD.
  • FIG. 4A-B Graphs demonstrating improved expression of multiple testicular cell type markers.
  • B peritubular myoid cells.
  • FIG. 5. Schematic depiction of human ESC differentiation protocol.
  • FIG. 6. Schematic depiction of human iPSC differentiation protocol.
  • FIG. 7. Schematic depiction of protocol for combining testis like cells derived from human ESCs with primordial germ cells.
  • FIG. 8 Schematic depiction of protocol for deriving peritubular myoid cells from human ESCs.
  • FIG. 9 Schematic depiction of protocol for deriving Leydig cells from human ESCs.
  • FIG. 10 Differentiation efficiency of human somatic-like cells in the presence of hormone using the protocol described in PCT US2023/13608.
  • FIG. 11 Revised testis differentiation protocol of the present invention.
  • A Schematic and media composition.
  • B Various gonadal, Sertoli and Leydig cell markers.
  • D16 Soma+PGC -(-/-Hormone is the most efficient and refined condition.
  • FIG. 12 Schematic depiction of the revised mouse ESC differentiation protocol. Briefly Day 4 cells are dissociated and 25 K cells are placed in a 96 well u-bottom plate. The 3D aggregates are maintained for 3 days before being transplanted to ThinCert® and collected at Dayl7.
  • FIG. 13A-E Graphical data demonstrating that the differentiation schema depicted in FIG. 12 effectively induces testis progenitor markers. Additionally, it promotes the expression of markers for Sertoli cells, Leydig cells, and myoid cells, with a few endothelial cell markers also beginning to peak. Two different color represents two different medium composition to grow organoids on ThinCert® cell culture inserts.
  • testis progenitor markers include Tcf21, Pdgfr-alfa and Nr2f2 (A), Sertoli cell markers are Sox9, Wtl, Gata4 and Inhbb (B), Leydig cell markers Cypl lal, Cypl7al, Hsd3b6, Nr5al, Cyp21al and Cyl lbl (C), Myoid markers Cnnl, Sma-Alfa and Myhl l (D) and Endothelial cell markers Pecaml, Esam and Cdh5 (E). The fold change of gene expression was calculated in compression with day 0 undifferentiated ESCs. Gapdh is used as housekeeping gene to normalize Ct values in qPCR analysis.
  • FIG. 14A-D Fluorescence micrographs demonstrating complete reconstitution of the testis microenvironment and tubular structure in organoids made by the schema depicted in FIG. 12.
  • the Leydig cells present in the organoids are mature Leydig cells as they express (SF1, HSD3B and StAR).
  • FIG. 15. The in vitro derived organoids produce testosterone in response to LH or HCG induction.
  • FIG. 16. Schema for Mixing of Postnatal Day 2 germ cells with our in vitro derived somatic cells. Aggregation occurs at day 4, following the differentiation schema depicted in FIG. 12.
  • FIG. 17A-D Graphs showing germ cell counts and percentage at day 1 and day 3 organoids in 96 well plates. The total number of germ cell number is determined by counting the total number of DDX4 or DAZL+ cells at day 1 and 3-day (A).
  • B Percentage and total number of OCT4-GFP positive Pro-spermatogonia
  • C Percentage and total number of PLZF+ undifferentiated spermatogonia
  • FIG. 18 Experimental schema for in vitro testis organoid development from human stem cells.
  • FIG. 19 Graphical data showing that expression levels of Sertoli cell markers are improved by day 22 by increasing EGF1 concentration to 50ng/ml.
  • FIG. 20A-B Immunofluorescence micrographs of testis organoids collected after 22 days in culture.
  • WT1 a marker for interstitial cells of the testis - progenitors for Leydig and myoid cells.
  • B Formation of S0X9+ and Gata4+ tubules.
  • stem cell refers to cells that can self-renew and differentiate into multiple lineages.
  • a stem cell is a developmentally pluripotent or multipotent cell.
  • a stem cell can divide to produce two daughter stem cells, or one daughter stem cell and one progenitor (“transit”) cell, which then proliferates into the tissue's mature, fully formed cells.
  • Stem cells may be derived, for example, from embryonic sources (“embryonic stem cells”) or derived from adult sources.
  • embryonic sources embryonic sources
  • U.S. Pat. No. 5,843,780 to Thompson describes the production of stem cell lines from human embryos.
  • PCT publications WO 00/52145 and WO 01/00650 describe the use of cells from adult humans in a nuclear transfer procedure to produce stem cell lines.
  • the term “stem cell” as used herein thus encompasses embryonic stem cells, adult stem cells and induced pluripotent stem cells.
  • adult stem cells include, but are not limited to, hematopoietic stem cells, neural stem cells, mesenchymal stem cells, and bone marrow stromal cells. These stem cells have demonstrated the ability to differentiate into a variety of cell types including adipocytes, chondrocytes, osteocytes, myocytes, bone marrow stromal cells, and thymic stroma (mesenchymal stem cells); hepatocytes, vascular cells, and muscle cells (hematopoietic stem cells); myocytes, hepatocytes, and glial cells (bone marrow stromal cells) and, indeed, cells from all three germ layers (adult neural stem cells).
  • totipotent cell refers to a cell that is able to form a complete embryo (e.g., a blastocyst).
  • pluripotent cell or “pluripotent stem cell” refers to a cell that has complete differentiation versatility, e.g., the capacity to grow into any of the mammalian body's approximately 260 cell types.
  • a pluripotent cell can be self-renewing and can remain dormant or quiescent within a tissue. Unlike a totipotent cell (e.g., a fertilized, diploid egg cell), a pluripotent cell, even a pluripotent embryonic stem cell, cannot usually form a new blastocyst.
  • iPSCs induced pluripotent stem cells
  • somatic cell e.g., a differentiated somatic cell
  • iPS cells are capable of self-renewal and differentiation into mature cells.
  • multipotent cell refers to a cell that has the capacity to grow into a subset of the mammalian body's approximately 260 cell types. Unlike a pluripotent cell, a multipotent cell does not have the capacity to form all of the cell types.
  • progenitor cell refers to a cell that is committed to differentiate into a specific type of cell or to form a specific type of tissue.
  • ES cell embryonic stem cell
  • ESC embryonic stem cell
  • feeder cells refers to cells used as a growth support in some tissue culture systems. Feeder cells may be embryonic striatum cells or stromal cells.
  • chemically defined media refers to culture media of known or essentially- known chemical composition, both quantitatively and qualitatively. Chemically defined media is free of all animal products, including serum or serum-derived components (e.g., albumin).
  • serum-free media refers to culture media that is devoid of serum, but not necessarily of other undefined components.
  • Methods, kits, compositions, and systems are provided for culturing pluripotent stem cells to produce populations of cells comprising artificial testis cells, such as Sertoli and Leydig cells.
  • culture conditions are provided that result in the generation of artificial testis cells from a starting culture of human pluripotent stem cells.
  • Methods of using the cells, for example in various therapies, are also provided.
  • Somatic cells of the testis are central to testis tissue homeostasis and men’s reproductive and overall health.
  • the somatic cells provide a series of unknown growth factors and cytokines that are necessary for guiding germ cell development in vivo and required for the complete reconstitution of germline development for females in vitro or promoting the differentiation of male primordial germ cell like cells (PGCLC) to spermatogonia.
  • PPCLC primordial germ cell like cells
  • the present inventors provide improved methods for the generation of human artificial testis cells, such as Sertoli and/or Leydig cells) from stem cells (e.g., embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) using the differentiation schema described below.
  • stem cells e.g., embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) using the differentiation schema described below.
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • Suitable pluripotent stem cells include, but are not limited to, embryonic stem cells, adult stem cells, and induced pluripotent stem cells.
  • the pluripotent stem cells are vertebrate pluripotent stem cells.
  • the pluripotent stem cells are human embryonic stem cells (hESCs).
  • the pluripotent stem cells are mouse embryonic stem cells (mESCs).
  • the pluripotent stem cells are induced pluripotent stem cells (iPSCs).
  • the pluripotent stem cells may be genetically modified by methods known in the art so that they comprise and express one or more exogenous genes.
  • CDM chemically defined media
  • CDM may include maintenance or basal media containing salts, vitamins, glucose and amino acids.
  • a mTeSR medium such as mTeSRlTM from StemCell Technologies may be utilized.
  • the maintenance medium preferably comprises a ROCK inhibitor such as Y27632.
  • Maintenance medium for mouse stem cells may preferably be GMEM from ThermoFisher Scientific, preferably supplemented with LIF (Leukemia Inhibitory Factor) and optionally knockout serum.
  • the basal differentiation medium can be any of a number of commercially available media.
  • a combination of Dulbecco's Modified Eagle Medium and Hams F12 medium, sold as a combination (DMEM/F12; Invitrogen) may be utilized.
  • an APEL medium may be utilized, for example, STEMdiffTM APELTM medium from StemCell Technologies.
  • STEMdiff TM APEL TM Medium is a serum-free and animal component-free medium specifically developed to support hPSC differentiation. It was first described for the induction of hemato-endothelial cells, when supplemented with VEGF, BMP-4, SCF, and Activin A, but it has also been proven to be an effective basal medium for hPSC differentiation to other lineages, including cardiomyocytes.
  • an mTeSR medium may be utilized for maintenance of stem cells.
  • the present invention provides methods and reagents for producing artificial testis cells (e.g., Sertoli cells, Leydig cells, Myoid cells, and/or stromal cells) from pluripotent stems cells.
  • artificial testis cells e.g., Sertoli cells, Leydig cells, Myoid cells, and/or stromal cells
  • the present invention is not limited to the use of any particular pluripotent stem cells or chemically defined media.
  • the methods described herein for the production of artificial testis cells are described in relation to events occurring at various time points. It will be recognized that the methods may be varied by making alterations to the described time schedules. “Day 0” as used herein refers to the day and time that the pluripotent stem cells are removed from a maintenance medium and exposed to a differentiation medium. The differentiation timeline is then defined from the Day 0 starting point.
  • X days refers to the number of days from the Day 0 starting time point plus or minus 12 hours.
  • “on about Day 4” means 96 hours (i.e., 4 days) from the Day 0 starting point plus or minus 12 hours. If the Day 0 stating time was 9:00 AM, “about Day 4” then refers to 96 hours from that time point plus or minus 12 hours.
  • the first step in a method for producing human artificial testis cells according to the invention comprises providing pluripotent hESC or iPSCs as described above.
  • the pluripotent stem cells are provided in a stem cell maintenance medium.
  • the stem cell maintenance medium is a chemically defined medium such as an mTeSR medium.
  • the stem cell maintenance medium comprises a ROCK inhibitor.
  • the ROCK inhibitor is Y27632.
  • the second step of the method of the present invention comprises removing the pluripotent stem cells from the maintenance medium and culturing the pluripotent stem cells in a basal medium supplemented with agents suitable for directing the pluripotent stem cells to a presomatic mesoderm lineage.
  • the basal medium is a chemically defined medium.
  • the basal medium is an APEL medium such as STEMdiffTM APELTM medium from StemCell Technologies.
  • the basal medium is supplemented with from between 0.5 to 15 pM (e.g., 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0. 12.0, 13.0, and 14.0 pM and values and ranges therein) CHIR99021.
  • This step defines Day 0 of the process.
  • the base medium with supplements is preferably changed daily.
  • the third step of the method of the present invention comprises on about or at day 4 culturing the presomitic mesoderm cells produced in step 2 in a basal medium supplemented with agents for directing the presomitic mesoderm cells to form intermediate mesoderm.
  • the basal medium is a chemically defined medium.
  • the basal medium is an APEL medium such as STEMdiffTM APELTM medium from StemCell Technologies.
  • the basal medium is supplemented with from between 20 and 500 ng/ml (50, 100, 150, 200, 250, 300, 350, 400, 450 ng/ml and values and ranges therein) FGF 9.
  • the basal medium is further supplemented with from between 0.1 and 10 pg/ml (e.g., 0.4, 0.8, 1.0. 1.5, 2.0, 3.0. 5.0. 6.0, 7.0, 8.0, 9.0 pg/ml and values and ranges therein) heparin.
  • the base medium with supplements may preferably be changed every two days.
  • the fourth step of the method of the present invention comprises on about or at day 7 culturing the intermediate mesoderm cells produced in step 3 in a basal medium to direct the intermediate mesoderm cells to form genital ridge cells.
  • the basal medium is a chemically defined medium.
  • the basal medium is an APEL medium such as STEMdiffTM APELTM medium from StemCell Technologies.
  • the base medium may preferably be changed every two days.
  • the basal medium is supplemented with from between 5 and 100 nM (e.g., 10, 17, 20, 30, 40, 50, 60, 70, 80, 90 nM and values and ranges therein) IGF1.
  • the basal medium is further supplemented with from between 10 and 500 nM (e.g., 20, 50, 100, 200, 300, 400 nM and values and ranges therein) insulin.
  • the base medium may be further supplemented with from between 0.01 and 10 pM (e.g., 0.05, 0.1, 1.0, 5.0, 8.0 pM and values and ranges therein) retinoic acid (RA).
  • RA retinoic acid
  • the base medium may be further supplemented with from between 50 and 1000 ng/ml (e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900 ng/ml and values and ranges therein) PGD2.
  • the base medium may be further supplemented with from between 50 and 500 ng/ml (e.g., 100, 200, 300, 400 ng/ml and values and ranges therein) FGF9.
  • the base medium used in step 5 may be further be supplemented with from 5 to 100 ng/ml (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90 ng/ml and values and ranges therein) bone morphogenetic protein 4 (BMP4).
  • BMP4 bone morphogenetic protein 4
  • the base medium used in step 4 may be further be supplemented with from 5 to 200 ng/ml (e.g., 10, 20, 30, 50, 70, 100, 150 ng/ml and values and ranges therein) epidermal growth factor (EGF).
  • the base medium used in step 5 may be further be supplemented with from 0.5 to 10 pM (e.g., 1.0, 2.0, 3.0, 4.0 5.0, 6.0, 7.0, 8.0, 9.0 pM and values and ranges therein) IWR1.
  • the base medium used in step 4 is further supplemented with supplemented with from 5 to 100 ng/ml (10, 20, 30, 40, 50, 60, 70, 80, 90 ng/ml and values and ranges therein) luteinizing hormone (LH).
  • LH luteinizing hormone
  • human chorionic gonadotropin (HCG) is used instead of LH at an amount of 1 to 10 units (1, 2, 3, 5 or 10 units and ranges and values therein).
  • the cells are treated first with HCG at the recited range for until about day 15 and then with LH at the recited range after about day 15.
  • the base medium used in step 4 is further supplemented with from 5 to 300 ng/ml (10, 20, 30, 40, 50, 100, 150, 200, 250 ng/ml and values and ranges therein) follicle stimulating hormone (FSH).
  • FSH follicle stimulating hormone
  • a combination of insulin and IGF1 in the above ranges is utilized.
  • a combination of insulin, IGF1, and FGF9 in the above ranges is utilized.
  • a combination of insulin, IGF1, FGF9, RA, EGF, and PG Di in the above ranges is utilized.
  • the fifth step of the method of the present invention comprises on about or at day 8 culturing the genital ridge cells produced in step 4 in a basal medium supplemented with agents for directing the genital ridge cells to form artificial testis cells.
  • the cells are dissociated and plated into an Aggrewell® plate or a u-bottom plate. In some preferred embodiments, from 10000 to about 50000, and most preferably about 30000 dissociated cells are transferred into u-bottom plates.
  • the basal medium is a chemically defined medium.
  • the basal medium is an APEL medium such as STEMdiff® APEL® medium from StemCell Technologies.
  • the basal medium is supplemented with from between 5 and 100 nM (e.g., 10, 17, 20, 30, 40, 50, 60, 70, 80, 90 nM and values and ranges therein) IGF1. In some particularly preferred embodiments, the basal medium is further supplemented with from between 10 and 500 nM (e.g., 20, 50, 100, 200, 300, 400 nM and values and ranges therein) insulin. In some embodiments, the base medium may be further supplemented with from between 0.01 and 10 pM (e.g., 0.05, 0.1, 1.0, 5.0, 8.0 pM and values and ranges therein) retinoic acid (RA).
  • RA retinoic acid
  • the base medium may be further supplemented with from between 50 and 1000 ng/ml (e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900 ng/ml and values and ranges therein) PGD2. In some embodiments, the base medium may be further supplemented with from between 50 and 500 ng/ml (e.g., 100, 200, 300, 400 ng/ml and values and ranges therein) FGF9.
  • ng/ml e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900 ng/ml and values and ranges therein
  • FGF9 FGF9.
  • the base medium used in step 5 may be further be supplemented with from 5 to 100 ng/ml (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90 ng/ml and values and ranges therein) bone morphogenetic protein 4 (BMP4). In some further preferred embodiments, the base medium used in step 5 may be further be supplemented with from 1 to 200 ng/ml (e.g., 5, 10, 20, 30, 50, 70, 100, 150 ng/ml and values and ranges therein) epidermal growth factor (EGF).
  • EGF epidermal growth factor
  • the base medium used in step 5 may be further be supplemented with from 0.1 to 10 pM (e.g., 0.5, 1.0, 2.0, 3.0, 4.0 5.0, 6.0, 7.0, 8.0, 9.0 pM and values and ranges therein) IWR1.
  • a combination of insulin and IGF1 in the above ranges is utilized.
  • a combination of insulin, IGF1, and FGF9 in the above ranges is utilized.
  • a combination of insulin, IGF1, FGF9, RA and PGD2 in the above ranges is utilized.
  • a combination of insulin, IGF1, FGF9, EGF and IWR1 in the above ranges is utilized. In some preferred embodiments, a combination of insulin, IGF1, FGF9, EGF, RA, IWR1, and PGD2 in the above ranges is utilized.
  • the base medium with supplements may preferably be changed every two days.
  • the base medium used in step 5 is further supplemented with one or more hormones (FSH, LH or HCG).
  • the one or more hormones are added on or at about day 9.
  • the base medium (which may preferably comprise one or more of insulin, IGF1, FGF9, EGF, RA, IWR1 and PGD2 in the above ranges) is further supplemented with from 5 to 100 ng/ml (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90 ng/ml and values and ranges therein) luteinizing hormone (LH). ).
  • human chorionic gonadotropin is used instead of LH at an amount of 1 to 10 units (1, 2, 3, 5 or 10 units and ranges and values therein).
  • the base medium (which may preferably comprise one or more of insulin, IGF1, FGF9, EGF, RA, IWR1 and PGD2 in the above ranges) is further supplemented with from 20 to 300 ng/ml (e.g., 50, 100, 150, 200, 250 ng/ml and values and ranges therein) follicle stimulating hormone (FSH).
  • FSH follicle stimulating hormone
  • the base medium (which may preferably comprise one or more of insulin, IGF1, FGF9, BMP4, EGF, RA, IWR1, and PGD2 in the above ranges) is further supplemented with from between 0.01 and 10 pM (e.g., 0.05, 0.1, L0, 5.0, 8.0 pM and values and ranges therein) testosterone.
  • the base medium (which may preferably comprise one or more of insulin, IGF1, FGF9, EGF, RA, IWR1 and PGD2 in the above ranges) with LH, FSH and testosterone in the ranges described herein.
  • a sixth step the cultures of step 5 for hESC are maintained until about day 12 so that organoids are formed and the organoids are then transferred to a culture system for culture at an air liquid interface.
  • the base medium is preferably supplemented with the same factors as described for step 5.
  • the air- liquid interface culture system utilizes a ThinCert® cell culture insert in a Transwell® plate.
  • organoids comprising one or more of Sertoli cells, Leydig cells, Myoid cells and stromal cells are produced by about day 22. At this point, the organoids may be collected for use or dissociated for isolation of, for example, Leydig, Sertoli, Myoid or stromal cells.
  • the present invention provides methods for producing artificial Leydig cells.
  • the steps described above for deriving testis cells are followed up until on or about day 8 (i.e., steps 1 to 4).
  • the genital ridge cells are preferably dissociated and cultured in a basal medium supplemented with agents for directing the genital ridge cells to form artificial Leydig cells.
  • the basal media is supplemented with one or more of SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), bFGF2 (Basic fibroblast growth factor), DAPT and LiCh.
  • the basal media is supplemented with SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), bFGF2 (Basic fibroblast growth factor), DAPT and LiCh.
  • SAG is included in the basal medium at from 0.1 to 5.0 pM (e.g., 0.2, 0.5, 1.0, 2.0, 2.0, 4.0 pM and ranges and values therein).
  • PDGF-AA is included in the basal medium at from 1 to 50 ng/ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng/ml and ranges and values therein.
  • PDGF-BB is included in the basal medium at from 1 to 50 ng/ml (1, 5, 10, 15, 20, 30, 40 ng/ml and ranges and values therein.
  • bFGF is included in the basal medium at from 1 to 50 ng/ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng/ml and ranges and values therein.
  • DAPT is included in the basal medium at from 1.0 to 20.0 pM (e.g., 2.0, 5.0. 10.0, 15.0 pM and ranges and values therein).
  • LiCh is included in the basal medium at from 1.0 to 20.0 mM (e.g., 2.0, 5.0. 10.0, 15.0 mM and ranges and values therein).
  • Leydig cells characterized by the expression of Leydig cell markers as described in the Examples are produced and collected on or about on day 16.
  • the present invention provides methods for producing artificial Myoid cells.
  • the steps described above for deriving testis cells are followed up until on or about day 8 (i.e., steps 1 to 4).
  • the genital ridge cells are preferably dissociated and cultured in a basal medium supplemented with agents for directing the genital ridge cells to form artificial Myoid cells.
  • the basal medium is supplemented with one or more of SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), valproic acid, BMP2 (Bone Morphogenetic Protein 2), BMP4 (Bone Morphogenetic Protein 4), and Activin A so that the genital ridge cells differentiate into Myoid-like cells.
  • SAG Smoothened Agonist
  • PDGF-AA Platinum-derived growth factor AA
  • PDGF-BB Platinum-derived growth factor BB
  • valproic acid valproic acid
  • BMP2 Bone Morphogenetic Protein 2
  • BMP4 Bone Morphogenetic Protein 4
  • 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.
  • SAG is included in the basal medium at from 0.1 to 5.0 M (e.g., 0.2, 0.5, 1.0, 2.0, 2.0, 4.0 M and ranges and values therein).
  • PDGF-AA is included in the basal medium at from 1 to 50 ng/ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng/ml and ranges and values therein.
  • PDGF-BB is included in the basal medium at from 1 to 50 ng/ml (1, 5, 10, 15, 20, 30, 40 ng/ml and ranges and values therein.
  • BMP2 is included in the basal medium at from 1 to 50 ng/ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng/ml and ranges and values therein.
  • BMP4 is included in the basal medium at from 1 to 50 ng/ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng/ml and ranges and values therein.
  • Activin A is included in the basal medium at from 1 to 50 ng/ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng/ml and ranges and values therein.
  • valproic is included in the basal medium at from 1.0 to 50.0 nM (e.g., 2.0, 10.0, 20.0, 30.0, 40.0 nM and ranges and values therein).
  • Myoid cells most preferably peritubular myoid cells characterized by the expression of Myoid cell markers as described in the Examples are produced and collected on or about on day 16.
  • the present invention provides methods for expanding primordial germ cells, such as primordial germ cell-like cells (PGCLCs).
  • PPCLCs primordial germ cell-like cells
  • the steps described above for deriving testis cells are followed up until on or about day 8 (i.e., steps 1 to 4).
  • the genital ridge cells are dissociated and combined with primordial germs cells.
  • the basal medium is supplemented with the same agents in the same concentration ranges as described for production of the artificial testis cells and organoids described above.
  • the dissociated genital ridge cells are combined with the primordial germ cells in a ratio of about 9: 1.
  • the medium is replaced with base medium comprising insulin, FGF9, RA, PDG2, EGF, FSH, LH and testosterone in the same concentration ranges as described above for derivation of artificial testis cells and organoids.
  • IWR1 is not included in this culture medium.
  • the organoids form by about day 12 and on or about day 12 the organoids are preferably cultured at an air-liquid interface as described above. Organoids may preferably be collected on about day 16 for further use.
  • the present invention provides methods for producing artificial testis cells and organoids from pluripotent mouse cells, such as mESC.
  • the pluripotent stem cells are provided in a stem cell maintenance medium.
  • the stem cell maintenance medium is GMEM supplemented with LIF and serum.
  • the second step of the method of the present invention comprises removing the pluripotent stem cells from the maintenance medium and culturing the pluripotent stem cells in a basal medium supplemented with agents suitable for directing the pluripotent stem cells to epiblast. This is defined as Day 0.
  • the basal medium is DMEM/F12 and Neurobasal Medium (both from ThermoFisher Scientific) in a ratio of from 2:1 to 1 :2 and most preferably at a ratio of about 1 :1.
  • the basal medium is supplemented with N2 supplement, B-27 supplement, and Knockout Serum Replacer (KSR; all from ThermoFisher Scientific), Activin A and bFGF.
  • the supplemented basal medium is referred to as priming medium.
  • the basal medium is supplemented with from 1 to 10 pl/ml (e.g., 1.0. 2.0, 3.0, 4.0, 5.0, 6.0. 7.0, 8.0, 9.0, 10.0 pl/ml and ranges and values therein) N2 supplement.
  • the basal medium is supplemented with from 1 to 20 pl/ml (e.g., 1.0. 5.0, 10.0, 20.0 pl/ml and ranges and values therein) B-27 supplement.
  • the basal medium is supplemented with from 1 to 20 pl/ml (e.g., 1.0.
  • the basal medium is supplemented with from 1 to 20 ng/ml (e.g., 1.0. 5.0, 10.0, 20.0 ng/ml and ranges and values therein) Activin A.
  • the basal medium is supplemented with from 1 to 20 ng/ml (e.g., 1.0. 5.0, 10.0, 20.0 ng/ml and ranges and values therein) bFGF.
  • the third step of the method of the present invention comprises removing the epiblasts from the priming medium used in step 2 at about day 2 and culturing the epiblasts in a basal medium supplemented with agents suitable for directing the epiblast cells to form anterior intermediate mesoderm.
  • the basal medium is DMEM/F12.
  • the basal medium is supplemented with BMP4, Activin A and Retinoic Acid (RA).
  • the supplemented basal medium is referred to as differentiation medium.
  • the basal medium is supplemented with 1 to 20 ng/ml (e.g., 1.0. 5.0, 10.0, 20.0 ng/ml and ranges and values therein) Activin A.
  • the basal medium is supplemented with from 10 to 200 nM (e.g., 10.0, 50.0. 100.0, 200.0 nM) and ranges and values therein) RA. In some particularly preferred embodiments, the basal medium is supplemented with 1 to 20 ng/ml (1.0. 2.5, 5.0, 10.0, 20.0 ng/ml and ranges and values therein) BMP4.
  • the anterior intermediate mesoderm cells are dissociated on about day 4 and transferred into a u-bottom 96-well plate. In some embodiments, from 10,000 to 50,000, and most preferably about 25,000 dissociated cells are transferred to the plates. In an alternative embodiment, about 100000 to 500000, and most preferably about 300000 dissociated cells are plated into Aggrewell® plates.
  • the intermediate mesoderm cells on day 4 are dissociated and reaggregated in Aggrewell or u-bottom dishes and cultured in a progenitor medium comprising RA, IGF1, insulin, FGF9, PDG2, BMP4, EGF1 and/or y 27632.
  • the basal medium is supplemented with from between 5 and 100 nM (e.g., 10, 17, 20, 30, 40, 50, 60, 70, 80, 90 nM and values and ranges therein) IGF1.
  • the basal medium is further supplemented with from between 10 and 500 nM (e.g., 20, 50, 100, 200, 300, 400 nm and values and ranges therein) insulin.
  • the base medium may be further supplemented with from between 10.0 and 200 nM (e.g., 10.0, 20.0, 50.0, 100.0 and 200.0 nM and values and ranges therein) RA. In some embodiments, the base medium may be further supplemented with from between 50 and 1000 ng/ml (e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900 ng/ml and values and ranges therein) PGD2. In some embodiments, the base medium may be further supplemented with from between 5 and 40 ng/ml (10, 20, 30 ng/ml and values and ranges therein) FGF9.
  • 10.0 and 200 nM e.g., 10.0, 20.0, 50.0, 100.0 and 200.0 nM and values and ranges therein
  • RA e.g., 10.0, 20.0, 50.0, 100.0 and 200.0 nM and values and ranges therein
  • the base medium may be further supplemented with from between 50 and 1000
  • the base medium may be further supplemented with from between 1 and 10 pM (e.g., 1.0. 2.0, 3.0, 4.0, 5.0, 6.0. 7.0, 8.0, 9.0, 10.0 pM and ranges and values therein) Y-27632.
  • the base medium used in step 5 may be further be supplemented with from 5 to 100 ng/ml (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90 ng/ml and values and ranges therein) bone morphogenetic protein 4 (BMP4).
  • BMP4 bone morphogenetic protein 4
  • the base medium used in step 5 may be further be supplemented with from 1 to 200 ng/ml (e.g., 2, 5, 10, 20, 30, 50, 70, 100, 150 and values and ranges therein) epidermal growth factor (EGF).
  • the base medium used in step 4 is further supplemented with 5 to 100 ng/ml (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90 ng/ml and values and ranges therein) luteinizing hormone (LH).
  • human chorionic gonadotropin (HCG) is used instead of LH at an amount of 1 to 10 units (1, 2, 3, 5 or 10 units and ranges and values therein).
  • the base medium used in step 4 is further supplemented with from 5 to 300 ng/ml (e.g., 10, 20, 30, 40, 50, 100, 150, 200, 250 ng/ml and values and ranges therein) follicle stimulating hormone (FSH).
  • FSH follicle stimulating hormone
  • the cells are transferred to a ThinCert® cell culture inserts on about day 7 or an about day 3 days after culture in the Aggrewell® or u-bottom plates.
  • the base medium with supplements may preferably be changed every two days.
  • testis organoids are formed.
  • the progenitor medium is replaced after about 24 hours with Myoid differentiation medium to enrich production of myoid cells in the organoids.
  • the basal medium is supplemented with one or more of SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF- BB (Platelet-derived growth factor BB), valproic acid, BMP2 (Bone Morphogenetic Protein 2), BMP4 (Bone Morphogenetic Protein 4), and Activin A so that the genital ridge cells differentiate into Myoid-like cells.
  • 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.
  • SAG is included in the basal medium at from 0.1 to 5.0 pM (e.g., 0.2, 0.5, 1.0, 2.0, 2.0, 4.0 pM and ranges and values therein).
  • PDGF-AA is included in the basal medium at from 1 to 50 ng/ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng/ml and ranges and values therein.
  • PDGF-BB is included in the basal medium at from 1 to 50 ng/ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng/ml and ranges and values therein.
  • BMP2 is included in the basal medium at from 1 to 50 ng/ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng/ml and ranges and values therein.
  • BMP4 is included in the basal medium at from 1 to 50 ng/ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng/ml and ranges and values therein.
  • Activin A is included in the basal medium at from 1 to 50 ng/ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng/ml and ranges and values therein.
  • valproic is included in the basal medium at from 1.0 to 50.0 nM (e.g., 2.0, 10.0, 20.0, 30.0, 40.0 nM and ranges and values therein).
  • Myoid cells (most preferably peritubular myoid cells) characterized by the expression of Myoid cell markers as described in the Examples are produced and collected on or about on day 8.
  • the progenitor medium is replaced after about 24 hours with Leydig differentiation medium to enrich production of myoid cells in the organoids.
  • the basal media is supplemented with one or more of SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet-derived growth factor BB), bFGF2 (Basic fibroblast growth factor), DAPT and LiCh.
  • the basal media is supplemented with SAG (Smoothened Agonist), PDGF-AA (Platelet-derived growth factor AA), PDGF-BB (Platelet- derived growth factor BB), bFGF2 (Basic fibroblast growth factor), DAPT and LiCh.
  • SAG is included in the basal medium at from 0.1 to 5.0 pM (e.g., 0.2, 0.5, 1.0, 2.0, 2.0, 4.0 pM and ranges and values therein).
  • PDGF-AA is included in the basal medium at from 1 to 50 ng/ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng/ml and ranges and values therein.
  • PDGF-BB is included in the basal medium at from 1 to 50 ng/ml (e.g., 1 , 5, 10, 15, 20, 30, 40 ng/ml and ranges and values therein.
  • bFGF is included in the basal medium at from 1 to 50 ng/ml (e.g., 1, 5, 10, 15, 20, 30, 40 ng/ml and ranges and values therein.
  • DAPT is included in the basal medium at from 1.0 to 20.0 pM (e.g., 2.0, 5.0. 10.0, 15.0 pM and ranges and values therein).
  • LiCh is included in the basal medium at from 1.0 to 20.0 mM (e.g., 2.0, 5.0. 10.0, 15.0 mM and ranges and values therein).
  • Leydig cells characterized by the expression of Leydig cell markers as described in the Examples are produced and collected on or about on day 8.
  • the artificial testis cells express one or more of the following markers: LHX9, PDGRA, COUPTFII, TCF21, SOX9, GATA4, SF1, SMA, DHH, STAR and 3BHSD.
  • the artificial testis cells produced by the methods described above express at least one of the markers selected from the group consisting of EMX2, WT, SOX9, LHX9, DHH, STAR and 3BHSD.
  • the artificial testis cells express at least two of the markers selected from the group consisting of EMX2, WT, SOX9, LHX9, DHH, STAR and 3BHSD.
  • the artificial testis cells express at least three of the markers selected from the group consisting of EMX2, WT, SOX9, LHX9, DHH, STAR and 3BHSD. In some preferred embodiments, the artificial testis cells express the markers EMX2, WT, SOX9, LHX9, DHH, STAR and 3BHSD.
  • the cultured are maintained until organoids are formed.
  • the organoids preferably are three dimensional organoids having a roughly spherical shape.
  • the organoids are characterized by comprising tubule structures.
  • the organoids are characterized by comprising smooth muscle actin.
  • the artificial testis cells or organoids may be harvested or isolated from the cultures for further use.
  • methods, reagents, and kits described herein, as well as the artificial testis cells and organoids generated therewith find use in various research, diagnostic, clinical, and therapeutic applications.
  • artificial testis cells or organoids are used for direct transplantation into a subject.
  • the artificial testis cells or organoids are used for somatic cell replacement therapy in a subject in need thereof.
  • artificial testis cells generated by methods herein are useful for diagnostic, prognostic, and/or therapeutic uses.
  • the isolated artificial testis cells or organoids may be directly transplanted in a subject. If appropriate, cells are co-administered with one or more pharmaceutical agents or bioactives that facilitate the survival and function of the transplanted cells.
  • human organoids are transplanted into mice for additional differentiation and/or maturation of the cells in the organoids.
  • the organoids are preferably combined with in vivo or in vitro derived germ cells to achieve human germline stem cell expansion and further promote differentiation. It is contemplated that these methods will result in production of haploid round or elongating spermatids which find use in assisted reproductive technologies such as IVF/ICSI.
  • the testis cells or organoids of the invention may be used to coculture gamete stem cells such as primordial germ cell like cells, prospermatogonia or spermatogonial stem/progenitor cells (SSC/SPCs) from a patient.
  • gamete stem cells such as primordial germ cell like cells, prospermatogonia or spermatogonial stem/progenitor cells (SSC/SPCs) from a patient.
  • the cells are cultured so that the stem cells from the patient differentiate into spermatogonia.
  • the gamete stem cells or cells derived from the gamete stem cells such as spermatogonia are transplanted back into the patient or a patient in need thereof.
  • the patient suffers from non-obstructive azoospermia.
  • the patient has previously undergone a gonadotoxic treatment, including but not limited to chemotherapy and/or radiation.
  • the stem cells or tissue comprising stem cells are obtained from the patient prior to a gonado toxic treatment.
  • the testicular tissue or somatic cells of the subject prior to gonadotoxic treatment (chemotherapy and radiation), are preserved so that germline stem cells may be produced or isolated in the future.
  • the patient has been born with a genetic disorder that affects fertility or renders them infertile.
  • the germ line stem cells obtained from the tissue can be expanded using the in vitro derived cells of the present invention.
  • the methods described above further comprise obtaining fibroblast tissue to reprogram to induced pluripotent stem cells from a patient which a can be used to make autologous artificial testis cells.
  • these differentiated cells can be combined with germline stem cells: either primordial germ cell like cells (PGCLC), prospermatogonia (proSSC), or neonatal/adult spermatogonial stem/progenitor cells (SSC/SPCs).
  • PPCLC primordial germ cell like cells
  • proSSC vigogonia
  • SSC/SPCs neonatal/adult spermatogonial stem/progenitor cells
  • the stem cells can either proliferate or differentiate into spermatogonia or later germ cell stages.
  • the methods further comprise transferring the expanded stem cells or differentiated spermatogonia back to a patient in need thereof.
  • the artificial testis cells or organoids may be provided on a support material.
  • Support materials suitable for use for purposes of the present invention include tissue templates, conduits, barriers, and reservoirs useful for tissue repair.
  • synthetic and natural materials in the form of foams, sponges, gels, hydrogels, textiles, and nonwoven structures which have been used in vitro and in vivo to reconstruct or regenerate biological tissue, as well as to deliver chemotactic agents for inducing tissue growth, are suitable for use in practicing the methods of the present invention. See, for example, the materials disclosed in U.S. Pat. No. 5,770,417, U.S. Pat. No. 6,022,743, U.S. Pat. No. 5,567,612, U.S. Pat. No.
  • Cells generated with methods and reagents herein may be implanted as dispersed cells or formed into implantable clusters.
  • cells are provided in biocompatible degradable polymeric supports; porous, permeable, or semi-permeable non- degradable devices; or encapsulated (e.g., to protect implanted cells from host immune response, etc.).
  • Cells may be implanted into an appropriate site in a recipient. Suitable implantation sites may include, for example, the testis or subcutaneously.
  • cells or cell clusters are encapsulated for transplantation into a subject.
  • Encapsulation techniques are generally classified as microencapsulation, involving small spherical vehicles, and macroencapsulation, involving larger flat-sheet and hollow-fiber membranes (Uludag, H. et al. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64, herein incorporated by reference in its entirety).
  • Methods of preparing microcapsules include those disclosed by Lu M Z, et al. Biotechnol Bioeng. 2000, 70: 479-83; Chang T M and Prakash S, Mol Biotechnol. 2001, 17: 249-60; and Lu M Z, et al, J. Microencapsul.
  • microcapsules may be prepared by complexing modified collagen with a terpolymer shell of 2 -hydroxy ethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 pm.
  • HEMA 2 -hydroxy ethyl methylacrylate
  • MAA methacrylic acid
  • MMA methyl methacrylate
  • Such microcapsules can be further encapsulated with additional 2-5 pt
  • microcapsules are based on alginate, a marine polysaccharide (Sambanis, Diabetes Technol. Ther. 2003, 5: 665-8; herein incorporated by reference in its entirety) or its derivatives.
  • microcapsules can be prepared by the poly electrolyte complex ation between the poly anions sodium alginate and sodium cellulose sulphate with the polycation poly(methylene-co-guanidine) hydrochloride in the presence of calcium chloride.
  • cells generated using methods and reagents described herein are microencapsulated for transplantation into a subject (e.g., to prevent immune destruction of the cells).
  • Microencapsulation of cells e.g., pancreatic lineage cells, beta-like cells, etc.
  • provides local protection of implanted/ transplanted cells from immune attack e.g., along with or without the use of systemic immune suppressive drugs.
  • cells and/or cell clusters are microencapsulated in a polymeric, hydrogel, or other suitable material, including but not limited to: poly(orthoesters), poly (anhydrides), poly(phosphoesters), poly (phosphazenes), polysaccharides, polyesters, poly(lactic acid), poly (L-ly sine), poly(glycolic acid), poly(lactic-co-glycolic acid), poly(lactic acid-co-lysine), poly(lactic acid- graft-lysine), polyanhydrides, poly(fatty acid dimer), poly(fumaric acid), poly(sebacic acid), poly(carboxyphenoxy propane), poly(carboxyphenoxy hexane), poly (anhydride-co-imi des), poly(amides), poly(ortho esters), poly(iminocarbonates), poly (urethanes), poly(organophasphazenes), poly (phosphates), poly(ethylene vinyl acetate), poly (caprolactone), poly(carbon
  • cell are microencapsulated in an encapsulant comprising or consisting of alginate.
  • Cells may be embedded in a material or within a particle (e.g., nanoparticle, microparticle, etc.) or other structure (e.g., matrix, nanotube, vesicle, globule, etc.).
  • microencapsulating structures are modified with immune- modulating or immunosuppressive compounds to reduce or prevent immune response to encapsulated cells.
  • pancreatic lineage cells are encapsulated within an encapsulant material (e.g., alginate hydrogel) that has been modified by attachment of an immune-modulating agent (e.g., the immune modulating chemokine, CXCL12 (also known as SDF-1).
  • an immune modulating agent e.g., the immune modulating chemokine, CXCL12 (also known as SDF-1).
  • an immune modulating agent is a T-cell chemorepellent and/or a pro-survival factor.
  • cells generated using methods and reagents described herein are macroencapsulated for transplantation into a subject.
  • Macroencapsulation of cells for example, within a permeable or semipermeable chamber, provides local protection of implanted/transplanted cells from immune attack (e.g., along with or without the use of systemic immune suppressive drugs), prevents spread of cells to other tissues or areas of the body, and/or allows for efficient removal of cells.
  • Suitable devices for macroencapsulation include those described in, for example, U.S. Pat. No. 5,914,262; Uludag, et al, Advanced Drug Delivery Reviews, 2000, pp. 29-64, vol. 42, herein incorporated by reference in their entireties.
  • testis cells or organoids of the present invention may be used for hormone therapy.
  • the organoids are encapsulated and subcutaneously implanted in the subject.
  • testis cells or organoids of the present invention may be used for fertility restoration.
  • endogenous defective somatic cells in the testis are combined or replaced by the testis cells or organoids of the present invention.
  • the organoids are transplanted into a subject to allow spermatogenesis to occur at an ectopic location (e.g., a subcutaneous location) other than the testis.
  • populations of artificial testis cells and organoids may be used to prepare antibodies and cDNA libraries that are specific for the differentiated phenotype.
  • General techniques used in raising, purifying and modifying antibodies, and their use in immunoassays and immunoisolation methods are described in Handbook of Experimental Immunology (Weir & Blackwell, eds.); Current Protocols in Immunology (Coligan et al, eds.); and Methods of Immunological Analysis (Masseyeff et al, eds., Weinheim: VCH Verlags GmbH).
  • General techniques involved in preparation of mRNA and cDNA libraries are described in R A Methodologies: A Laboratory Guide for Isolation and Characterization (R. E.
  • the artificial testis cells and organoids generated by methods provided herein are used to screen for agents (e.g., small molecule drugs, peptides, polynucleotides, and the like) or environmental conditions (such as culture conditions or manipulation) that affect the cells.
  • agents e.g., small molecule drugs, peptides, polynucleotides, and the like
  • environmental conditions such as culture conditions or manipulation
  • Particular screening applications relate to the testing of pharmaceutical compounds in drug research and to agents for use in cryopreservation of gametes including sperm.
  • Assessment of the activity of candidate pharmaceutical compounds generally involves combining the cells with the candidate compound, determining any change in the morphology, marker phenotype, or metabolic activity of the cells that is attributable to the compound (compared with untreated cells or cells treated with an inert compound), and then correlating the effect of the compound with the observed change.
  • Any suitable assays for detecting changes associated with test agents may find use in such embodiments.
  • the screening may be done, for example, either because the compound is designed to have a pharmacological effect on testis cell types, because a compound designed to have effects elsewhere may have unintended side effects, or because the compound is part of a library screen for a desired effect.
  • Two or more drugs can be tested in combination (by combining with the cells either simultaneously or sequentially), to detect possible drug-drug interaction effects.
  • compounds are screened for cytotoxicity.
  • methods and systems are provided for assessing the safety and efficacy of drugs that act upon testis cells, or drugs that might be used for another purpose but may have unintended effects upon testis cells.
  • cells described herein find use in high throughput screening (HTS) applications.
  • HTS screening platform e.g., cells and plates
  • agents e.g., small molecule compounds, peptides, etc.
  • artificial testis cells or organoids generated using methods and reagents described herein are utilized for therapeutic delivery to a subject.
  • Cells may be placed directly in contact with subject tissue or may be otherwise sealed or encapsulated (e.g., to avoid direct contact). In embodiments in which cells are encapsulated, exchange of nutrients, gases, etc. between the encapsulated cells and the subject tissue is allowed. In some embodiments, cells are implanted/transplanted on a matrix or other delivery platform.
  • the methods and kits described herein are useful for identifying additional factors, reagents, and methods for the generation of artificial testis cells or other cell types.
  • the methods used herein may be used to screen factors, reagents and /or conditions for their effect of differentiation.
  • any screening performed in this or other embodiments discussed herein may be high-throughput screening.
  • mESC mouse Embryonic Stem Cells
  • growth medium A small amount of growth medium can be prepared and stored in 4°C for up to one month.
  • Growth medium composition for 100 ml - Glasgow's MEM (GMEM) 92 ml, 10 ml fetal bovine serum ES cell qualified, 1 ml of Nonessential amino acid, 1ml of sodium pyruvate, 1 ml of Penicillin-Streptomycin, 100 pl of 2-mercaptoethanol, 100 pl LIF.
  • GMEM Glasgow's MEM
  • basal priming media A small amount of basal priming media can be prepared and stored in 4°C for up to one month.
  • Priming medium composition for 100 ml medium add 46.9 ml of Neurobasal Medium, 46.9 ml of DMEM-F12 Medium, 500 pl of N2 supplement, 1 ml B27 supplement, 500 l of Glutamax, 100 pl 2-Mercaptoethanol, 1 ml Sodium pyruvate, 1 ml knockout serum replacer, 1 ml of non-essential amino acids, 1 ml of Penicillin-Streptomycin.
  • the additional growth factors, 10 ng/ml Activin A and 10 ng/ml bFGF are mixed in medium just before the addition of medium into the cells.
  • a small amount of basal AIM differentiation media can be prepared and stored in 4°C.
  • AIM differentiation medium composition for 100 ml of medium add 91.9 ml of DMEM- F12 medium, 1 ml of Sodium pyruvate, 4 ml of knockout serum replacer, 1 ml of non- essential amino acids, 100 pl of 2-Mercaptoethanol, 1 ml of Penicillin-Streptomycin. Addition of growth factors 10 ng/ml Activin A, 100 nM RA, 2.5 ng/ml BMP4 are mixed in medium right before the addition of medium into the cells.
  • the starting cells need to be in undifferentiated and growth phase.
  • Harvest cells from 80% confluent plate (at least one passage after thawing) a- Aspirate growth media and wash cells with 1 ml of PBS-/- of 6 well plate.
  • c- Take out cells from incubator and quench TrypLE express enzyme by adding 2 mL of growth medium.
  • c- Take out cells from incubator and quench TrypLE express enzyme by adding 2 mL of growth medium.
  • the cells samples were collected every day after the second day of differentiation for RNA isolation. qPCR was done for multiple AIM markers, gonadal markers, pluripotency marker Oct4, posterior intermediate mesoderm marker HoxDl 1. The data was confirmed by immunostaining of AIM as well as gonadal markers Wtl, gonadal marker Sox9. Differentiating peritubular Myoid cells from 4 day differentiated AIM cells.
  • Insulin Transferrin Selenium (100X) (ThermoFisher Scientific Cat # 41400045)
  • Progenitor Medium- Progenitor medium composition for 100 ml of medium add 91.9 ml of DMEM-F12 medium, 1 ml of Sodium pyruvate, 5 ml of knockout serum replacer, 1 ml of non-essential amino acids, 100 pl of 2-Mercaptoethanol, 1 ml of Penicillin-Streptomycin. Additional factors, 1% Matrigel, lOOnM Retinoic Acid, 17nM IGF1, lOOnM Insulin, 10 ng/ml FGF9, 500ng/ml PGD2, 20 ng/ml BMP4, 50 ng/ml EGF1, 5 M Y-27632 was mixed right before adding the media in the cells.
  • Myoid differentiation medium Myoid differentiation medium for 100 ml of medium: add 98.9 ml of DMEM-F12 medium, 100 pl of ITS, 1 ml BSA. Additional factors, SAG 0.5 pM, PDGFRAA 10 ng/ml, PDGFRBB 10 ng/ml, Valproic Acid 10 nm, BMP2 10 ng/ml, BMP4 10 ng/ml, Activin A 10 ng/ml was mixed right before adding the media in the cells.
  • BSA Bovine Serum Albumin
  • Progenitor Medium- Progenitor medium composition for 100 ml of medium add 91.9 ml of DMEM-F12 medium, 1 ml of Sodium pyruvate, 5 ml of knockout serum replacer, 1 ml of non-essential amino acids, 100 pl of 2-Mercaptoethanol, 1 ml of Penicillin-Streptomycin. Additional factors, 1% Matrigel, lOOnM Retinoic Acid, 17nM IGF1, lOOnM Insulin, 10 ng/ml FGF9, 500ng/ml PGD2, 20 ng/ml BMP4, 50 ng/ml EGF1, 5 M Y-27632 was mixed right before adding the media in the cells.
  • Leydig cell basal medium- Leydig cell basal medium composition for 100 ml of medium add 98.9 ml of DMEM-F12 medium, 100 pl of ITS, 1 ml BSA. Additional factors, SAG 0.5 pM, PDGFRAA 10 ng/ml, PDGFRBB 10 ng/ml, FGF2 10 ng/ml was mixed right before adding the media in the cells.
  • Leydig cell differentiation medium- Myoid differentiation medium for 100 ml of medium add 98.9 ml of DMEM-F12 medium, 100 pl of ITS, 1 ml BSA. Additional factors, SAG 0.5 pM, PDGFRAA 10 ng/ml, PDGFRBB 10 ng/ml, LiCh 5 mM, FGF2 10 ng/ml, DAPT 10 pM was mixed right before adding the media in the cells.
  • IGF1 (Sigma- Aldrich cat # 13769)
  • Insulin Sigma- Aldrich cat #19278
  • ROCK inhibitor Y-27632 (Enzo Life Sciences Cat # ALX-270-333)
  • basal organoid differentiation media can be prepared and stored in 4°C for up to one month.
  • Organoid differentiation medium composition for 100 ml of medium add 91.9 ml of DMEM-F12 medium, 1 ml of Sodium pyruvate, 5 ml of knockout serum replacer, 1 ml of non-essential amino acids, 100 pl of 2-Mercaptoethanol, 1 ml of Penicillin-Streptomycin. Additional factors, 1% Matrigel, lOOnM Retinoic Acid, 17nM IGF1, lOOnM Insulin, 10 ng/ml FGF9, 500ng/ml PGD2, 20 ng/ml BMP4, 50 ng/ml EGF1, 5 pM Y-27632 was mixed right before adding the media in the cells.
  • the Aggrewell® 400-24 plate or a 96 u- bottom well plates are prepared as follow.
  • a- Add 500 pl of anti- adherence rinsing solution per well of Aggrewell® 400-24 well plate and incubate for 30 minutes at room temperature.
  • 2- Organoid differentiation a- Aspirate the AIM differentiation media and wash the cells thrice with 2 ml of PBS-/- per well of six well plate from 4 day differentiated AIM cells. b- Add 500 pl of TrypLE Express Enzyme per well of the six well plate and incubate for 5 minutes in CO2 incubator. c- Take out cells from incubator and quench TrypLE express enzyme by adding 2 mL of growth medium per well of six well plate. d- Centrifuge the cells at 1000 RPM for 5 minutes, aspirate the liquid and dilute the palleted cells in organoid differentiation medium. e- About 300,000 cells was mixed in 500 pl of organoid differentiation media and added to preprepared Aggrewell® plate per well.
  • the organoids from the 96 well plates are collected or transferred at Day 7.
  • the 96 well plate is the better method! k- Data was confirmed by whole mount immunostaining of progenitor marker CoupTf2, Sertoli cell markers Gata4, Sox9 and Wtl, Leydig cell marker 3BHSD, STAR, Cypl7al.
  • FSH 17- Follicle Stimulating Hormone
  • LH 18- Luteinizing Hormone
  • FIG. 1 An overview of the differentiation protocol is provided in FIG. 1. This protocol provides improvements to the protocol provided in PCT US2023/13608, the contents of which are incorporated herein by reference in their entirety. Modification to the protocol include addition of BMP4 between Days 2-4 as well reducing FGF9 concentration in ALLGF and adding BMP4 and EGF1. These modifications resulted in the improvement the expression of many gonadal and Sertoli cell markers (See. FIG. 2).
  • Leydig cells are detected that are double positive for SF1 and 3BHSD. These Leydig cells enrich in clusters like what is observed in mouse and human testis (See FIG. 3).
  • Leydig cells were further maturation of Sertoli cells (Figure 4A) was detected as well as detection of peritubular myoid cell markers ( Figure 4B).
  • Leydig and Peritubular myoid cells Pure populations of Leydig and Peritubular myoid cells are obtained by following the protocols described above to day 8 and then isolating the cells and continuing the culture of the gonadal progenitor cells on Matrigel for an addition 3-8 using established Leydig and Myoid cell differentiation. Applying the existing Leydig and Myoid media to ESC is not sufficient to generate myoid and Leydig cells.
  • the following example provides a description of the reagents and protocols for producing artificial testis cells according to the present invention from human Embryonic Stem Cells (hESC) or iPSCs.
  • hESC human Embryonic Stem Cells
  • iPSCs iPSCs
  • FIG. 5 A schematic of this protocol is provided as FIG. 5.
  • STEMdiff APEL Medium Stem Cell Technologies, cat. no. 05270 or 05275
  • DPBS Dulbecco's phosphate-buffered saline
  • CHIR99021 (R&D, cat. no. 9902): Stock Solution (10 mM) — Centrifuge the tube briefly before opening. Reconstitute 10 mg of CHIR99021 into 2.149 ml of DMSO to make 10 mM stock. Prepare 20 pl aliquots and label tubes as “Ch” store at -20°C. The thawed tube can be kept at 4°C for 24hr.
  • Y27632 ROCK inhibitor (Enzo, Cat. no. ALX-270-333): Stock Solution (5mM) — Dissolve Img in 625 pl sterile TC water Prepare 20 pl aliquots and label tubes as “Y”. Store at -20°C at stem cell core. Dilute in lOmL culture medium for final lOpM. Discard the tube after use. 15. Heparin (Sigma Aldrich, cat. no. H4784-250 mg): Stock solution (1 mg ml 1) — Reconstitute to 1 mg ml- ' in ultrapure water and filter through polyether sulfone (PES) 0.22 pm filter (syringe-driven filter unit). Prepare 40, u I aliquots and store at 4°C for up to 12 months.
  • PES polyether sulfone
  • FGF9 (R&D, cat. no. 273-F9-025): Stock Solution FGF9 (100 pg/ml) — Centrifuge the tube briefly before opening. Reconstitute to 100 pg/ml in filtered DPBS containing 0.1% (wt/vol) Bovine serum albumin. Prepare 20 pl aliquots and store them at -80°C for up to 6 months. Thawed FGF9 aliquot can be stored at 4°C for up to 2 weeks.
  • RA (Sigma, cat. no. R2625-5 mg): Stock Solution (0.5Mm) — Dissolve Img of RA dissolve in 3 ml of the DMSO to make 1.11 mM Stock Solution. Dilute the stock solution to 0.5 mM, prepare 50pl or lOOpl aliquots and this working solution can be stored at -20°C. Use 0.5 mM of stock solution to the cell culture media according to the experimental desired concentrations. Discard the tube after use.
  • Insulin Sigma, cat. no. 192778: Stock Solution (1.7mM) from Vendor.
  • IGF1-50 pg (Sigma, Cat. no. 13769): Stock solution (lOMm) — 50 pg of IGF1 is dissolved in 658 pl of 0.2% acetic acid to a stock concentration of 10 mM. Prepare lOpl aliquots and store at -80°C. Dilute the stock solution to a concentration of 100 pM in 0.2% acetic acid and add to the cell culture media according to the experimental desired concentrations. The thawed tube can be stored at 4°C for up to 1 month.
  • PGD2 (Cayman, cat. no.12010): Stock Solution (2mg/ml) — Dissolve PGD2 in 200pl PBS pH 7.4 to a stock concentration of 2mg/ml, prepare 5pl aliquots and store at -80°C. Discard the tube after use.
  • Matrigel (Coming, cat. no. 354234, Lot no. 9133008): Stock Solution (2mg/ml or Img/ml) — Dilute the Matrigel to desired concentration 2mg/ml or 1 mg/ml in DMEM/F12 media and prepare aliquots and store at -20°C until use.
  • Testosterone (Sigma, cat no. T1500-5g): Stock Solution(5mM)-6g of Testosterone was dissolved in 1ml of DMSO then serial diluted to make concentration 5mM.
  • Follicle stimulating hormone (Sigma: cat no. F4021-25ug)- Stock Solution (100 pg/ml)- Dissolve 25ug of FSH in 250ul of ultrapure water and prepare aliquots and store at -20°C until use.
  • Luteinizing Hormone (Sigma: cat no. L6420-10ug): Stock Solution (100 pg/ml)- Dissolve 25ug of LH in lOOul of ultrapure water and prepare aliquots and store at -20°C until use.
  • EGF Epidermal Growth Factor from murine submaxillary gland, EGF (Sigma, cat no. E4127-.1 mg): Stock Solution(100ug/ml)-0.1mg of EGF was dissolved in 10ml of 0.1%BSA/PBS in 10ml then aliquoted in 1 ml and store at -20°C until use.
  • IWR1 (Sigma: cat no. 681669-lOmg)- Dissolve 10 mg/ml in DMSO and prepare aliquots and store at -20°C until use.
  • FIG. 6 A schematic of this protocol is provided as FIG. 6.
  • STEMdiff APEL Medium Stem Cell Technologies, cat. no. 05270 or 05275
  • CHIR99021 (R&D, cat. no. 9902): Stock Solution (10 mM) — Centrifuge the tube briefly before opening. Reconstitute 10 mg of CHIR99021 into 2.149 ml of DMSO to make 10 mM stock. Prepare 20 pl aliquots and label tubes as “Ch” store at -20°C. The thawed tube can be kept at 4°C for 24hr.
  • Y27632 ROCK inhibitor (Enzo, Cat. no. ALX-270-333): Stock Solution (5mM) — Dissolve Img in 625 pl sterile TC water Prepare 20 pl aliquots and label tubes as “Y”. Store at -20°C at stem cell core. Dilute in lOmL culture medium for final lOpM. Discard the tube after use.
  • Heparin Sigma Aldrich, cat. no. H4784-250 mg
  • Stock solution (1 mg ml“l) — Reconstitute to 1 mg ml- ' in ultrapure water and filter through polyether sulfone (PES) 0.22 pm filter (syringe-driven filter unit).
  • PES polyether sulfone
  • FGF9 (R&D, cat. no. 273-F9-025): Stock Solution FGF9 (100 pg/ml) — Centrifuge the tube briefly before opening. Reconstitute to 100 pg/ml in filtered DPBS containing 0.1% (wt/vol) Bovine serum albumin. Prepare 20 pl aliquots and store them at -80°C for up to 6 months. Thawed FGF9 aliquot can be stored at 4°C for up to 2 weeks.
  • RA (Sigma, cat. no. R2625-5 mg): Stock Solution (0.5Mm) — Dissolve Img of RA dissolve in 3 ml of the DMSO to make 1.11 mM Stock Solution. Dilute the stock solution to 0.5 mM, prepare 50pl or lOOpl aliquots and this working solution can be stored at -20°C. Use 0.5 mM of stock solution to the cell culture media according to the experimental desired concentrations. Discard the tube after use.
  • Insulin Sigma, cat. no. 192778: Stock Solution (1.7mM) from Vendor.
  • IGF1-50 pg (Sigma, Cat. no. 13769): Stock solution (lOMm) — 50 pg of IGF1 is dissolved in 658 pl of 0.2% acetic acid to a stock concentration of 10 mM. Prepare lOpl aliquots and store at -80°C. Dilute the stock solution to a concentration of 100 pM in 0.2% acetic acid and add to the cell culture media according to the experimental desired concentrations. The thawed tube can be stored at 4°C for up to 1 month.
  • PGD2 (Cayman, cat. no. 12010): Stock Solution (2mg/ml) — Dissolve PGD2 in 200pl PBS pH 7.4 to a stock concentration of 2mg/ml, prepare 5p 1 aliquots and store at -80°C. Discard the tube after use.
  • Matrigel (Coming, cat. no. 354234, Lot no. 9133008): Stock Solution (2mg/ml or Img/ml) — Dilute the Matrigel to desired concentration 2mg/ml or 1 mg/ml in DMEM/F12 media and prepare aliquots and store at -20°C until use.
  • Testosterone (Sigma, cat no. T1500-5g): Stock Solution(5mM)-6g of Testosterone was dissolved in 1ml of DMSO then serial diluted to make concentration 5mM.
  • Follicle stimulating hormone (Sigma: cat no. F4021-25ug)- Stock Solution (100 pg/ml)- Dissolve 25ug of FSH in 250ul of ultrapure water and prepare aliquots and store at -20°C until use.
  • Luteinizing Hormone Sigma: cat no. L6420-10ug
  • Stock Solution 100 pg/ml
  • Dissolve 25ug of LH in lOOul of ultrapure water and prepare aliquots and store at -20°C until use.
  • EGF Epidermal Growth Factor from murine submaxillary gland, EGF (Sigma, cat no. E4127-.1 mg): Stock Solution(100ug/ml)-0.1mg of EGF was dissolved in 10ml of 0.1%BSA/PBS in 10ml then aliquoted in 1 ml and store at -20°C until use.
  • IWR1 Sigma: cat no. 681669-lOmg
  • Stock Solution ()- Dissolve 10 mg/ml in DMSO and prepare aliquots and store at -20°C until use.
  • Freezing container Naalgene, Mr. Frosty
  • Inverted contrasting tissue culture microscope KL1500CD, Leica
  • Dav 18 Change the media for every two days with growth factors.
  • FIG. 7 A schematic of this protocol is provided as FIG. 7.
  • STEMdiff APEL Medium Stem Cell Technologies, cat. no. 05270 or 05275
  • DPBS Dulbecco's phosphate-buffered saline
  • CHIR99021 (R&D, cat. no. 9902): Stock Solution (10 mM) — Centrifuge the tube briefly before opening. Reconstitute 10 mg of CHIR99021 into 2.149 ml of DMSO to make 10 mM stock. Prepare 20 pl aliquots and label tubes as “Ch” store at -20°C. The thawed tube can be kept at 4°C for 24hr.
  • Y27632 ROCK inhibitor (Enzo, Cat. no. ALX-270-333): Stock Solution (5mM) — Dissolve Img in 625 pl sterile TC water Prepare 20 pl aliquots and label tubes as “Y”. Store at -20°C at stem cell core. Dilute in lOmL culture medium for final lOpM. Discard the tube after use.
  • Heparin Sigma Aldrich, cat. no. H4784-250 mg
  • Stock solution (1 mg ml“l) — Reconstitute to 1 mg ml- ' in ultrapure water and filter through polyether sulfone (PES) 0.22 pm filter (syringe-driven filter unit).
  • PES polyether sulfone
  • FGF9 (R&D, cat. no. 273-F9-025): Stock Solution FGF9 (100 pg/ml) — Centrifuge the tube briefly before opening. Reconstitute to 100 pg/ml in filtered DPBS containing 0.1% (wt/vol) Bovine serum albumin. Prepare 20 pl aliquots and store them at -80°C for up to 6 months. Thawed FGF9 aliquot can be stored at 4°C for up to 2 weeks.
  • RA (Sigma, cat. no. R2625-5 mg): Stock Solution (0.5Mm) — Dissolve Img of RA dissolve in 3 ml of the DMSO to make 1.1 1 mM Stock Solution. Dilute the stock solution to 0.5 mM, prepare 50pl or lOOpl aliquots and this working solution can be stored at -20°C. Use 0.5 mM of stock solution to the cell culture media according to the experimental desired concentrations. Discard the tube after use.
  • Insulin Sigma, cat. no. 192778: Stock Solution (1.7mM) from Vendor.
  • IGF1-50 pg (Sigma, Cat. no. 13769): Stock solution (lOMm) — 50 pg of IGF1 is dissolved in 658 pl of 0.2% acetic acid to a stock concentration of 10 mM. Prepare lOpl aliquots and store at -80°C. Dilute the stock solution to a concentration of 100 pM in 0.2% acetic acid and add to the cell culture media according to the experimental desired concentrations. The thawed tube can be stored at 4°C for up to 1 month.
  • PGD2 (Cayman, cat. no.12010): Stock Solution (2mg/ml) — Dissolve PGD2 in 200pl PBS pH 7.4 to a stock concentration of 2mg/ml, prepare 5pl aliquots and store at -80°C. Discard the tube after use.
  • Matrigel (Coming, cat. no. 354234, Lot no. 9133008): Stock Solution (2mg/ml or Img/ml) — Dilute the Matrigel to desired concentration 2mg/ml or 1 mg/ml in DMEM/F12 media and prepare aliquots and store at -20°C until use.
  • Testosterone (Sigma, cat no. T1500-5g): Stock Solution(5mM)-6g of Testosterone was dissolved in 1ml of DMSO then serial diluted to make concentration 5mM.
  • Follicle stimulating hormone (Sigma: cat no. F4021-25ug)- Stock Solution (100 pg/ml)- Dissolve 25ug of FSH in 250ul of ultrapure water and prepare aliquots and store at -20°C until use.
  • Luteinizing Hormone Sigma: cat no. L6420-10ug
  • Stock Solution 100 pg/ml
  • Dissolve 25ug of LH in lOOul of ultrapure water and prepare aliquots and store at -20°C until use.
  • EGF Epidermal Growth Factor from murine submaxillary gland, EGF (Sigma, cat no. E4127-.1 mg): Stock Solution(100ug/ml)-0.1mg of EGF was dissolved in 10ml of 0.1%BSA/PBS in 10ml then aliquoted in 1 ml and store at -20°C until use.
  • IWR1 Sigma: cat no. 681669-lOmg
  • Stock Solution ()- Dissolve 10 mg/ml in DMSO and prepare aliquots and store at -20°C until use.
  • Next morning cells can be used to start differentiation.
  • Dav 11 Collect organoids from required number of wells on day 11 and transfer the Organoids into ThinCert® plates.
  • Lower chamber receives at least 700ul of APEL ⁇ 0.1 pM RA + 100 nM Insulin + 17 nM IGF1 + 500 ng/ml PGD2 + 200 ng/ml FGF9 I pM Testosterone ⁇ lOOng/ml Follicle stimulating hormone ⁇ 200ng/ml Luteinizing Hormonei +50ng/ml EGF.
  • testis somatic like cells derived from iPSC with primordial germ cell like cells to promote PGCLC maturation.
  • STEMdiff APEL Medium Stem Cell Technologies, cat. no. 05270 or 05275
  • DPBS Dulbecco's phosphate-buffered saline
  • CHIR99021 (R&D, cat. no. 9902): Stock Solution (10 mM) — Centrifuge the tube briefly before opening. Reconstitute 10 mg of CHIR99021 into 2.149 ml of DMSO to make 10 mM stock. Prepare 20 pl aliquots and label tubes as “Ch” store at -20°C. The thawed tube can be kept at 4°C for 24hr.
  • Y27632 ROCK inhibitor (Enzo, Cat. no. ALX-270-333): Stock Solution (5mM) — Dissolve Img in 625 pl sterile TC water Prepare 20 pl aliquots and label tubes as “Y”. Store at -20°C at stem cell core. Dilute in lOmL culture medium for final lOpM. Discard the tube after use.
  • Heparin Sigma Aldrich, cat. no. H4784-250 mg
  • Stock solution (1 mg ml _ l) — Reconstitute to 1 mg ml- ' in ultrapure water and filter through polyether sulfone (PES) 0.22 pm filter (syringe-driven filter unit).
  • PES polyether sulfone
  • FGF9 (R&D, cat. no. 273-F9-025): Stock Solution FGF9 (100 pg/ml) — Centrifuge the tube briefly before opening. Reconstitute to 100 pg/ml in filtered DPBS containing 0.1% (wt/vol) Bovine serum albumin. Prepare 20 pl aliquots and store them at -80°C for up to 6 months. Thawed FGF9 aliquot can be stored at 4°C for up to 2 weeks.
  • RA (Sigma, cat. no. R2625-5 mg): Stock Solution (0.5Mm) — Dissolve Img of RA dissolve in 3 ml of the DMSO to make 1.1 1 mM Stock Solution. Dilute the stock solution to 0.5 mM, prepare 50pl or lOOpl aliquots and this working solution can be stored at -20°C. Use 0.5 mM of stock solution to the cell culture media according to the experimental desired concentrations. Discard the tube after use.
  • Insulin Sigma, cat. no. 192778: Stock Solution (1.7mM) from Vendor.
  • IGF1-50 pg (Sigma, Cat. no. 13769): Stock solution (lOMm) — 50 pg of IGF1 is dissolved in 658 pl of 0.2% acetic acid to a stock concentration of 10 mM. Prepare lOpl aliquots and store at -80°C. Dilute the stock solution to a concentration of 100 pM in 0.2% acetic acid and add to the cell culture media according to the experimental desired concentrations. The thawed tube can be stored at 4°C for up to 1 month.
  • PGD2 (Cayman, cat. no.12010): Stock Solution (2mg/ml) — Dissolve PGD2 in 200pl PBS pH 7.4 to a stock concentration of 2mg/ml, prepare 5pl aliquots and store at -80°C. Discard the tube after use.
  • Matrigel (Coming, cat. no. 354234, Lot no. 9133008): Stock Solution (2mg/ml or Img/ml) — Dilute the Matrigel to desired concentration 2mg/ml or 1 mg/ml in DMEM/F12 media and prepare aliquots and store at -20°C until use.
  • Testosterone (Sigma, cat no. T1500-5g): Stock Solution(5mM)-6g of Testosterone was dissolved in 1ml of DMSO then serial diluted to make concentration 5mM.
  • Follicle stimulating hormone (Sigma: cat no. F4021-25ug)- Stock Solution (100 pg/ml)- Dissolve 25ug of FSH in 250ul of ultrapure water and prepare aliquots and store at -20°C until use.
  • Luteinizing Hormone Sigma: cat no. L6420-10ug
  • Stock Solution 100 pg/ml
  • Dissolve 25ug of LH in lOOul of ultrapure water and prepare aliquots and store at -20°C until use.
  • EGF Epidermal Growth Factor from murine submaxillary gland, EGF (Sigma, cat no. E4127-.1 mg): Stock Solution(100ug/ml)-0.1mg of EGF was dissolved in 10ml of 0.1%BSA/PBS in 10ml then aliquoted in 1 ml and store at -20°C until use.
  • IWR1 Sigma: cat no. 681669-lOmg
  • Stock Solution ()- Dissolve 10 mg/ml in DMSO and prepare aliquots and store at -20°C until use.
  • FIG. 8 A schematic of this protocol is provided as FIG. 8.
  • STEMdiff APEL Medium Stem Cell Technologies, cat. no. 05270 or 05275
  • DMSO Sigma Aldrich, cat. no. D5879
  • DPBS Dulbecco's phosphate-buffered saline
  • CHIR99021 (R&D, cat. no. 9902): Stock Solution (10 mM) — Centrifuge the tube briefly before opening. Reconstitute 10 mg of CHIR99021 into 2.149 ml of DMSO to make 10 mM stock. Prepare 20 pl aliquots and label tubes as “Ch” store at -20°C. The thawed tube can be kept at 4°C for 24hr.
  • Y27632 ROCK inhibitor (Enzo, Cat. no. ALX-270-333): Stock Solution (5mM) — Dissolve Img in 625 pl sterile TC water Prepare 20 pl aliquots and label tubes as “Y”. Store at -20°C at stem cell core. Dilute in lOmL culture medium for final lOpM. Discard the tube after use.
  • Heparin Sigma Aldrich, cat. no. H4784-250 mg
  • Stock solution (1 mg ml _ l) — Reconstitute to 1 mg ml-' in ultrapure water and filter through polyether sulfone (PES) 0.22 pm filter (syringe-driven filter unit).
  • PES polyether sulfone
  • FGF9 (R&D, cat. no. 273-F9-025): Stock Solution FGF9 (100 pg/ml) — Centrifuge the tube briefly before opening. Reconstitute to 100 pg/ml in filtered DPBS containing 0.1% (wt/vol) Bovine serum albumin. Prepare 20 pl aliquots and store them at -80°C for up to 6 months. Thawed FGF9 aliquot can be stored at 4°C for up to 2 weeks.
  • RA (Sigma, cat. no. R2625-5 mg): Stock Solution (0.5Mm) — Dissolve Img of RA dissolve in 3 ml of the DMSO to make 1.11 mM Stock Solution. Dilute the stock solution to 0.5 mM, prepare 50pl or lOOpl aliquots and this working solution can be stored at -20°C. Use 0.5 mM of stock solution to the cell culture media according to the experimental desired concentrations. Discard the tube after use.
  • Insulin Sigma, cat. no. 19278: Stock Solution (1.7mM) from Vendor. 19.
  • IGF1-50 pg (Sigma, Cat. no. 13769): Stock solution (lOMm) — 50 pg of IGF1 is dissolved in 658 pl of 0.2% acetic acid to a stock concentration of 10 mM. Prepare lOpl aliquots and store at -80°C. Dilute the stock solution to a concentration of 100 pM in 0.2% acetic acid and add to the cell culture media according to the experimental desired concentrations. The thawed tube can be stored at 4°C for up to 1 month.
  • PGD2 (Cayman, cat. no.12010): Stock Solution (2mg/ml) — Dissolve PGD2 in 200pl PBS pH 7.4 to a stock concentration of 2mg/ml, prepare 5 pl aliquots and store at -80°C. Discard the tube after use.
  • Matrigel (Corning, cat. no. 354234, Lot no. 9133008): Stock Solution (2mg/ml or Img/ml) — Dilute the Matrigel to desired concentration 2mg/ml or 1 mg/ml in DMEM/F12 media and prepare aliquots and store at -20°C until use.
  • SAG (Millipore, cat.no.566661): Stock Solution(0.5mM). Prepare 5ul of aliquots and store at -20 °C.
  • PDGFAA (Sigma, cat.no. SRP3228-10ug): Stock Solution(10ug/ml)- Dissolve lOug in 1ml of 0.1%BSA. Prepare 50ul of aliquots and store at -20°C.
  • PDGFBB (Sigma, cat.no. SRP3229-10ug): Stock Solution(10ug/ml)-Dissolve lOug in 1ml of 0.1%BSA. Prepare 50ul of aliquots and store at -20°C.
  • Valproic acid (Sigma, cat.no. P4543-10G): Stock Solution (ImM)-Dissolve 1.6619mg in 1ml of Sterile H20.Prepare 50ul of aliquots and store at-80°C.
  • BMP2 (R&D, cat.no. 355-BM-050): Stock Solution(50ug/ml)-Dissolve 50ug in 1ml Sterile 4mM HCL containing 0.1%BSA and Store 50ul aliquots at 80°C.
  • BMP4 (R&D: cat no. 314-BP-500): Stock Solution (200 pg/ml)- Dissolve 200 pg/mL in sterile 4 mM HC1 containing 0.1% BSA and store 50ul aliquots at -80°C until use.
  • Freezing container (Nalgene, Mr. Frosty)

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Abstract

La présente invention concerne des procédés in vitro pour la production de cellules testiculaire (par exemple, des cellules Sertoli et/ou Leydig) et d'organoïdes associés. Les cellules testiculaires et les organoïdes de type testiculaire peuvent être utilisés à des fins thérapeutiques, y compris pour permettre la production de spermatogonies à partir de cellules souches de pro-spermatogonies.
PCT/US2024/042621 2023-08-17 2024-08-16 Cellules testiculaires artificielles et leur production Pending WO2025038906A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230058240A1 (en) * 2014-05-23 2023-02-23 Craig S. Atwood Methods of generating hormone-producing organoids and reversing hypogonadism
WO2023163983A1 (fr) * 2022-02-22 2023-08-31 The Regents Of The University Of Michigan Cellules de sertoli artificielles et leur méthode de production

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230058240A1 (en) * 2014-05-23 2023-02-23 Craig S. Atwood Methods of generating hormone-producing organoids and reversing hypogonadism
WO2023163983A1 (fr) * 2022-02-22 2023-08-31 The Regents Of The University Of Michigan Cellules de sertoli artificielles et leur méthode de production

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"Master's Thesis, University of Helsinki", 1 January 2020, article LINTALA ANNIKA: "The effect of bFGF, FGF9 and PGD2 on differentiation of human induced pluripotent stem cells into testicular Sertoli-like cells", XP093283091 *
YANG YAN, LI QUAN, HUANG RUFEI, XIA HUAN, TANG YAN, MAI WANWEN, LIANG JINLIAN, MA SIYING, CHEN DERONG, FENG YUQING, LEI YALING, ZH: "Small‐Molecule‐Driven Direct Reprogramming of Fibroblasts into Functional Sertoli‐Like Cells as a Model for Male Reproductive Toxicology", ADVANCED BIOLOGY, vol. 6, no. 5, 1 May 2022 (2022-05-01), XP093283092, ISSN: 2701-0198, DOI: 10.1002/adbi.202101184 *

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