Artificial Sertoli cells and methods for producing the same - Patents.com

Abstract

The present invention relates to a method for the in vitro production of Sertoli cells and related organoids. Sertoli cells and testis-like organoids can be used for therapeutic purposes, including promoting the production of spermatogonia from pre-spermatogonial stem cells.

Description

Detailed Description of the Invention

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims priority to U.S. Provisional Patent Application No. 63 / 312,528, filed February 22, 2022, the contents of which are incorporated by reference in their entirety herein.

[0002] [Federal Research and Development Statement] This invention was made with Government support under award HD091949 from the National Institutes of Health. The Government has certain rights in this invention.

[0003] [Field of the Invention] The present invention relates to a method for the in vitro production of Sertoli cells and associated organoids.

[0004] [Background of the invention] Infertility is a rapidly growing crisis worldwide. 1% of couples of reproductive age (20-50 years) worldwide suffer from infertility, with male infertility accounting for approximately 50% of the causes. In the United States, 300,000 men have nonobstructive azoospermia (i.e., no germ cells or mature sperm cells), most of which are genetically unexplained. Conventional treatments such as in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) require sperm, and these men have no cure. In addition to adult infertility, approximately 10,000 prepubertal boys develop cancer each year in the United States, requiring gonadotoxic treatments such as chemotherapy and radiation therapy (1). In addition, approximately 1,000 pediatric patients with hematologic and immune deficiencies and autoimmune diseases undergo myeloablative conditioning prior to bone marrow transplantation, which is also gonadotoxic (2). These treatments, which involve alkylating chemotherapy agents, total body irradiation (3), and gonadal radiation (4), damage both somatic and germ cell populations in the testes, placing patients at significant risk of infertility. However, with medical advances, 85% of these children are cured and will desire to have their own children when they reach adulthood (5). As gametes are required for any reproductive therapy, developing methods to reconstitute germ cell development in vitro or in vivo for these patients remains a clinical challenge and will remain a focus of the reproductive biology community for decades to come. Successful generation of patient-derived testicular cells in vitro would provide novel cell-based therapies for genetic and ectopic infertility (treatment-induced infertility). Such treatments could also be applied to restore endocrine function in aging men, hypogonadal men, and patients with gender identity disorder. Furthermore, if in vitro induced cells or cell populations (sometimes called organoids) could recapitulate the natural spermatogenic process, they could be used as a novel experimental system to screen new classes of male contraceptives.

[0005] Currently, the race for cell-based therapies is centered on the development of germ cell-like precursors known as primordial germ cell-like cells (PGCLCs). PGCLCs have been derived in vitro from mouse and human embryonic stem cells (ESCs), but they cannot be maintained in vitro (6). Only when transplanted into neonatal mouse testes can these mouse PGCLCs develop into sperm, which can then be used to generate live pups. Furthermore, reconstitution of female and male mouse PGCLCs with fetal gonadal tissue in vitro successfully reconstituted oogenesis and spermatogenesis programs, although the frequency of live pups obtained from in vitro-induced germ cells was roughly around 1–3% (6–9). These preliminary findings are exciting and highlight the importance of somatic cells to execute the gametogenesis program. Similar experiments with mouse embryonic gonadal tissue from three experimental groups using human or rhesus PGCLCs showed that when female and male human hPGCLCs or male rhesus PGCLCs were combined with fetal mouse ovarian or testicular tissue, respectively, the PGCLCs initiated differentiation but failed to initiate meiosis (9-11). These observations indicate that the sources of somatic and germ cells must be compatible (i.e., from the same species) to initiate meiosis.

[0006] Utilizing somatic cells from human fetal testis to support the progression of in vitro induced human PGCLCs has ethical and technical constraints associated with fetal tissue-based research. Therefore, generating testicular somatic cells without relying on fetal tissue is of clinical importance. Previously, attempts have been made to reprogram human fibroblasts into Sertoli-like or Leydig-like cells using a combination of select transcription factors. Despite expressing some known markers of Sertoli and Leydig cells, it remains difficult to assess the extent to which in vitro induced cells resemble their in vivo counterparts or recapitulate endogenous functions. Furthermore, the clinical utility of such cells is unclear, given that efficient induction requires gene transfer and continuous expression of transcription factors is required to maintain the Sertoli and Leydig cell fate.

[0007] [Summary of the Invention] The present invention relates to an in vitro method for producing Sertoli cells and associated organoids.

[0008] In some preferred embodiments, the present invention provides an in vitro method for producing Sertoli cells from vertebrate pluripotent stem cells, comprising: inducing genital ridge cells from pluripotent stem cells; treating the genital ridge cells with a basal medium containing fibroblast growth factor 9 (FGF9), insulin and / or IGF1, thereby causing the genital ridge cells to differentiate into Sertoli cells.

[0009] In some preferred embodiments, the step of inducing gonadal ridge cells further comprises: providing vertebrate pluripotent stem cells in a maintenance medium comprising a ROCK inhibitor; on day 0, removing the maintenance medium comprising a ROCK inhibitor and culturing the vertebrate pluripotent stem cells in the basal medium comprising CHIR99021, thereby allowing the vertebrate pluripotent stem cells to differentiate into promesodermal cells; on about day 4, removing the basal medium comprising CHIR99021 and culturing the promesodermal cells in basal medium comprising fibroblast growth factor 9 (FGF9) and heparin, thereby allowing the promesodermal cells to differentiate into intermediate mesodermal cells; on about day 7, removing the medium comprising FGF9 and heparin and culturing the intermediate mesodermal cells in basal medium, thereby allowing the intermediate mesodermal cells to differentiate into gonadal ridge cells.

[0010] In some preferred embodiments, the step of treating the genital ridge cells with a culture medium containing insulin and / or IGF1 to cause the genital ridge cells to differentiate into Sertoli cells further comprises, on about day 10, removing the basal medium and culturing the genital ridge cells in a basal medium containing insulin-like growth factor 1 (IGF1) and insulin to cause the genital ridge cells to differentiate into artificial Sertoli cells.

[0011] In some preferred embodiments, the step of treating the genital ridge cells with a basal medium containing fibroblast growth factor 9 (FGF9), insulin, and / or IGF1 to cause the genital ridge cells to differentiate into Sertoli cells further comprises treating the genital ridge cells with epidermal growth factor (EGF), bone morphogenetic protein 4 (BMP4), IWR1, or a combination thereof. In some preferred embodiments, the step of treating the genital ridge cells with a basal medium containing fibroblast growth factor 9 (FGF9), insulin, and / or IGF1 to cause the genital ridge cells to differentiate into Sertoli cells further comprises treating the genital ridge cells with follicle stimulating hormone (FSH) and / or luteinizing hormone and / or testosterone, or a combination thereof.

[0012] In some preferred embodiments, the vertebrate pluripotent stem cells are human embryonic stem cells (hESCs).

[0013] In some preferred embodiments, the present invention provides an in vitro method for producing Sertoli cells from vertebrate pluripotent stem cells, comprising inducing pre-intermediate mesoderm cells from pluripotent stem cells; treating said pre-intermediate mesoderm cells with a basal medium comprising insulin and / or IGF1, thereby causing said pre-intermediate mesoderm cells to differentiate into Sertoli cells.

[0014] In some preferred embodiments, the step of inducing pre-intermediate mesoderm cells further comprises the steps of: providing vertebrate pluripotent stem cells in a maintenance medium comprising a ROCK inhibitor; on day 0, removing the maintenance medium comprising a ROCK inhibitor and culturing the vertebrate pluripotent stem cells in the basal medium comprising CHIR99021, thereby allowing the vertebrate pluripotent stem cells to differentiate into pre-mesoderm cells; and on about day 4, removing the basal medium comprising CHIR99021 and culturing the pre-mesoderm cells in basal medium comprising fibroblast growth factor 9 (FGF9) and heparin, thereby allowing the pre-mesoderm cells to differentiate into intermediate mesoderm cells.

[0015] In some preferred embodiments, the step of treating the intermediate mesoderm cells with a culture medium containing insulin and / or IGF1, thereby causing the intermediate mesoderm cells to differentiate into Sertoli cells, further comprises, on about day 7, removing the medium containing FGF9 and heparin, and culturing the intermediate mesoderm cells in a basal medium containing insulin-like growth factor 1 (IGF1) and insulin, thereby causing the intermediate mesoderm cells to differentiate into artificial Sertoli cells.

[0016] In some preferred embodiments, the step of treating the intermediate mesoderm cells further comprises treating the intermediate mesoderm cells with FGF9, epidermal growth factor (EGF), bone morphogenetic protein 4 (BMP4), IWR1, or a combination thereof. In some preferred embodiments, FGF9, epidermal growth factor (EGF), and bone morphogenetic protein 4 (BMP4) are utilized. In some preferred embodiments, FGF9, epidermal growth factor (EGF), bone morphogenetic protein 4 (BMP4), and IWR1 are utilized. In some preferred embodiments, the step of treating the intermediate mesoderm cells further comprises treating the gonadal ridge cells with follicle stimulating hormone (FSH) and / or luteinizing hormone (LH) and / or testosterone, or a combination thereof. In some preferred embodiments, one or more hormones are added at about day 9.

[0017] In some preferred embodiments, the vertebrate pluripotent stem cells are human embryonic stem cells (hESCs).

[0018] In some preferred embodiments, the step of inducing pre-intermediate mesoderm cells further comprises the steps of: providing vertebrate pluripotent stem cells in a maintenance medium comprising LIF; on day 0, removing the maintenance medium comprising LIF and culturing the vertebrate pluripotent stem cells in the basal medium comprising activin A and bFGF, thereby allowing the vertebrate pluripotent stem cells to differentiate into epidermal cells; and on about day 2, removing the basal medium comprising activin A and bFGF and culturing the epidermal cells in a basal medium comprising activin A and RA, thereby allowing the epidermal cells to differentiate into intermediate mesoderm cells.

[0019] In some preferred embodiments, the step of treating the intermediate mesoderm cells with a culture medium containing insulin and / or IGF1, thereby causing the intermediate mesoderm cells to differentiate into Sertoli cells, further comprises removing the medium containing activin A and RA on about day 4, and culturing the intermediate mesoderm cells in a basal medium containing insulin-like growth factor 1 (IGF1) and insulin, thereby causing the intermediate mesoderm cells to differentiate into artificial Sertoli cells. In some preferred embodiments, the vertebrate pluripotent stem cells are mouse embryonic stem cells (mESCs).

[0020] In some preferred embodiments, the artificial Sertoli cells produced by any of the above methods express at least one marker selected from the group consisting of EMX2, WT, SOX9, and LHX9. In some preferred embodiments, the artificial Sertoli cells express at least two markers selected from the group consisting of EMX2, WT, SOX9, and LHX9. In some preferred embodiments, the artificial Sertoli cells express at least three markers selected from the group consisting of EMX2, WT, SOX9, and LHX9. In some preferred embodiments, the artificial Sertoli cells express the markers EMX2, WT, SOX9, and LHX9.

[0021] In some preferred embodiments, the basal medium utilized in the above-mentioned methods is APEL2 medium. In some preferred embodiments, the maintenance medium is mTESR medium. In some preferred embodiments, the basal medium is DMEM medium.

[0022] In some preferred embodiments, in any of the aforementioned methods, the basal medium further comprising insulin-like growth factor 1 (IGF1) and insulin comprises one or more of retinoic acid, PDG2, and FGF9, preferably a combination thereof.

[0023] In some preferred embodiments, the vertebrate pluripotent stem cells utilized in any of the aforementioned methods comprise an exogenous gene.

[0024] In some preferred embodiments, the above method further comprises culturing the artificial Sertoli cell under conditions such that the artificial Sertoli cell forms artificial Sertoli cell organoid.In some preferred embodiments, the cell is first dissociated.In some preferred embodiments, the dissociated cell is further cultured in Aggrewell plate.

[0025] In some preferred embodiments, the artificial Sertoli cell organoids are characterized by having tubule structures. In some preferred embodiments, the artificial Sertoli cell organoids are further characterized by comprising smooth muscle actin.

[0026] In some preferred embodiments, the above-mentioned method further comprises isolating the artificial Sertoli cell or artificial Sertoli cell organoid.

[0027] In some preferred embodiments, the above method further comprises transplanting the isolated artificial Sertoli cell or artificial Sertoli cell organoid into a mammal.

[0028] In some preferred embodiments, the method further comprises contacting the artificial Sertoli cells or artificial Sertoli cell organoids with a test reagent and evaluating the effect of the test reagent on the artificial Sertoli cells or artificial Sertoli cell organoids.

[0029] In some preferred embodiments, the patient may preserve testicular tissue or somatic cells prior to undergoing gonadotoxic treatment (chemotherapy or radiation therapy) to allow for future production or isolation of germline stem cells. In some preferred embodiments, the germline stem cells obtained from tissue can be expanded using the in vitro derived cells of the invention. In some preferred embodiments, the above method further comprises obtaining fibroblast tissue and reprogramming it into induced pluripotent stem cells from the patient that can be used to generate autologous artificial Sertoli cells. In some preferred embodiments, these differentiated cells can be combined with germline stem cells: either primordial germ cell-like cells (PGCLCs), precursor spermatogonia (proSSCs), or newborn / adult spermatogonia stem / progenitor cells (SSCs / SPCs). In some preferred embodiments, the stem cells can be expanded or differentiated into spermatogonia or further germ cell stages. In some preferred embodiments, the method further comprises returning the expanded stem cells or differentiated spermatogonia to a patient in need thereof.

[0030] In some preferred embodiments, the invention provides cell cultures comprising artificial Sertoli cells produced by any of the methods described above.

[0031] In some preferred embodiments, the present invention provides an isolated artificial Sertoli cell produced by any of the aforementioned methods.

[0032] In some preferred embodiments, the present invention provides artificial Sertoli cell organoids produced by any of the above methods.

[0033] In some preferred embodiments, the present invention provides a method comprising the steps of: providing an artificial Sertoli cell or organoid as described above; contacting the artificial Sertoli cell or organoid with a test reagent; and evaluating the effect of the test reagent on the artificial Sertoli cell or organoid.

[0034] In some preferred embodiments, the present invention provides a method comprising: providing an artificial Sertoli cell or organoid as described above; and transplanting the artificial Sertoli cell or organoid into a subject.

[0035] In some preferred embodiments, the present invention provides methods comprising the steps of: Providing artificial Sertoli cells or organoids as described above; harvesting stem cells or tissues containing stem cells from a patient; and culturing the stem cells or tissues containing stem cells from the patient as the artificial Sertoli cells. In some preferred embodiments, the stem cells or tissues containing stem cells are harvested and stored from the patient before gonadotoxic treatment. In some preferred embodiments, the germline stem cells obtained from the tissue can be expanded using the in vitro derived cells of the present invention. In some preferred embodiments, the above-mentioned method further comprises harvesting fibroblast tissues from the patient for reprogramming into induced pluripotent stem cells, which can be used to generate autologous artificial Sertoli cells. In some preferred embodiments, these differentiated cells can be combined with germline stem cells: either primordial germ cell-like cells (PGCLCs), precursor spermatogonia (proSSCs), or newborn / adult spermatogonia stem / progenitor cells (SSCs / SPCs). In some preferred embodiments, the stem cells can be expanded or differentiated into spermatogonia or later germ cell stages. In some preferred embodiments, the methods further comprise returning the expanded stem cells or differentiated spermatogonia to a patient in need thereof.

[0036] In some preferred embodiments, the present invention provides a kit comprising a plurality of containers, at least one container comprising a basal medium comprising fibroblast growth factor 9 (FGF9) and heparin, and at least one container housing a basal medium comprising insulin-like growth factor 1 (IGF1) and insulin. In some preferred embodiments, the kit further comprises at least one container housing an inhibitor of ROCK I and / or ROCK II in a maintenance medium. In some preferred embodiments, the kit further comprises at least one container comprising CHIR99021. In some preferred embodiments, the kit further comprises at least one container comprising BMP4. In some preferred embodiments, the kit further comprises at least one container comprising EGF. In some preferred embodiments, the kit further comprises at least one container comprising IWR1. In some preferred embodiments, the kit further comprises at least one container comprising FSH. In some preferred embodiments, the kit further comprises at least one container comprising LH. In some preferred embodiments, the kit further comprises at least one container having testosterone. In some preferred embodiments, the kit further comprises instructions for carrying out the method as described above.

[0037] [Brief explanation of the figure] Figure 1. Schematic diagram showing the developmental process of testicular somatic cells and key markers identifying key stages.

[0038] Figure 2. In vitro generation of testicular cells and the resulting organoids. (A) Schematic of the experimental method used to generate the organoids. (B-D) Data from RT-qPCR analysis of different markers along the differentiation time course.

[0039] Figure 3. Micrographs showing the expression dynamics of pluripotency and gonadal markers during our mouse ESC differentiation trajectory.

[0040] Figure 4. In vitro derived somatic cells resemble embryonic gonadal cells in vivo. A) Single cell RNA-seq time course analysis of in vitro differentiation (days 0, 2, 4, 8) identified 8 clusters visualized in UMAP space. Note: Shown is one experiment, but 9 successful in vitro differentiations. B) Cells harvested on days 0, 2, 4, and 8 contribute to 8 clusters, showing emergence of new cell types at later days. (C). Expression profile of selected specific and non-specific (D) gonadal differentiation markers. (E) Correlation of our scRNAseq daily centroids with published in vivo daily cell centroids. (F) Daily cell counts per cluster in our data (left) and correlation of our 8 cluster centroids with 4 in vivo cell types (right).

[0041] Figure 5. Schematic diagram showing the strategy to combine in vitro derived somatic cells with in vivo derived Oct4-egfp precursor spermatogonia.

[0042] Figure 6. Micrographs showing that our in vitro induced somatic cells incorporate progenitor spermatogonia and promote their differentiation in vitro. In vitro induced somatic-like cells are mixed with purified Oct4+ prospermatogonia harvested from the testes of PND1-2 Oct4-egfptg / + mice. During the experiment, a subset of Oct4-egfp+ prospermatogonia maintains expression of a known undifferentiated spermatogonia marker (known as PLZF), but after 4 days of co-culture, a subset of cells transitions to an OCT4-dim or OCT4-low state and acquires strong expression of Stra8.

[0043] Figure 7. Improved somatic cell differentiation protocol. A) Schematic of somatic cell generation including the most promising four-drug combination. B) Expression of testicular somatic cell markers in all three cocktails described in 7A. C) Renal differentiation markers. D) Granulosa cell markers.

[0044] Figure 8. Photomicrographs of representative staining patterns of gonadal markers obtained from ALL-GF organoids compared to other culture conditions.

[0045] Figure 9. Graph of data showing that testicular cells derived from cocktail #3 improve stem cell expansion on days 3 and 5 of in vitro co-culture. Germ cell numbers are determined by quantifying the number of EGFP+ germline stem cells. The starting numbers of somatic and germ cells are the same for all combinations.

[0046] Figure 10. Generation of human testicular somatic-like cells. A) Optimized differentiation scheme used for H1 and U6 hESC lines. B) Graph showing tracking of relative RNA expression of several cell type specific markers over the differentiation trajectory.

[0047] Figure 11. Micrographs showing that our differentiation protocol induces expression of key protein markers across the differentiation trajectory. Results are similar for H1 and U6 lines.

[0048] Figure 12. Data showing that scRNAseq analysis identified 13 clusters, a subset of which emerged at later stages of culture and resembled known differentiated cells observed in vivo in human gonadal tissue.

[0049] Figure 13. Improved differentiation efficiency of human somatic-like cells in the presence of hormones. A) Modified differentiation scheme. B) qPCR expression of gonadal, Sertoli, Leydig, and off-target markers.

[0050] Figure 14. Representative images of gonadal markers at day 22 from both H1 ESC lines.

[0051] Figure 15. Photomicrograph showing in vitro derived organoids consisting of germ and somatic cells after 5 days of mixed culture.

[0052] Figure 16. Graphs showing data related to validation of IPSC differentiation schema and gonadal cell generation.

[0053] [Definition] The term "stem cell" ("SC") as used herein refers to a cell capable of self-renewal and differentiation into multiple lineages. Stem cells are developmentally pluripotent or multipotent cells. Stem cells can divide to produce two daughter stem cells, or one daughter stem cell and one progenitor cell ("transit"), which then grow into mature, fully formed cells of the tissue. Stem cells can be, for example, from embryos ("embryonic stem cells") or from adults. For example, U.S. Patent No. 5,843,780 to Thompson describes the production of stem cell lines from human embryos. PCT Publications WO00 / 52145 and WO01 / 00650 describe the use of cells from human adults in nuclear transfer methods to produce stem cell lines.

[0054] Examples of adult stem cells include, but are not limited to, hematopoietic stem cells, neural stem cells, mesenchymal stem cells, and bone marrow stromal cells. These stem cells have demonstrated the ability to differentiate into various cell types, such as adipocytes, chondrocytes, bone cells, muscle cells, bone marrow stromal cells, and thymic stromal cells (mesenchymal stem cells); hepatocytes, vascular cells, and muscle cells (hematopoietic stem cells); muscle cells, hepatocytes, and glial cells (bone marrow stromal cells), and even cells derived from all three germ cell layers (adult neural stem cells).

[0055] As used herein, the term "totipotent cell" refers to a cell that is capable of forming a complete embryo (eg, a blastocyst).

[0056] As used herein, the term "pluripotent cell" or "pluripotent stem cell" refers to a cell that has complete differentiation potential, e.g., a cell that has the ability to develop into any of the approximately 260 types of cells present in the mammalian body. Pluripotent cells can self-replicate and remain dormant or quiescent in tissues. Unlike totipotent cells (e.g., fertilized diploid egg cells), pluripotent cells, even pluripotent embryonic stem cells, usually cannot form new blastocysts.

[0057] As used herein, the term "induced pluripotent stem cells" ("iPS cells") refers to stem cells derived from somatic cells, e.g., differentiated somatic cells, and that have greater potential than the somatic cells. iPS cells are capable of self-renewal and differentiation into mature cells.

[0058] As used herein, the term "multiple potential cells" refers to cells that have the ability to develop into a subset of the approximately 260 types of cells present in the mammalian body. Unlike pluripotent cells, multiple potential cells do not have the ability to form all types of cells.

[0059] As used herein, the term "progenitor cell" refers to a cell that is committed to differentiate into a particular type of cell or to form a particular type of tissue.

[0060] As used herein, the term "embryonic stem cell" ("ES cell" or "ESC") refers to a pluripotent cell that is derived from the inner cell mass of a blastocyst (e.g., a 4- to 5-day old human embryo) and has the ability to produce many or all of the types of cells present in a mature animal.

[0061] The term "feeder cells" as used herein refers to cells used as a growth support in some tissue culture systems. Feeder cells may be embryonic striatal cells or interstitial cells. The term "chemically defined medium" as used herein refers to a culture medium of known or essentially known chemical composition, both quantitatively and qualitatively. Chemically defined medium is free of all animal products, including serum or serum-derived components (e.g., albumin).

[0062] As used herein, the term "serum-free medium" refers to a culture medium that does not contain serum, but does not necessarily contain other undefined components.

[0063] [Detailed Description of the Invention] Methods, kits, compositions, and systems are provided for culturing pluripotent stem cells to produce cell populations that include artificial Sertoli cells. In particular, culture conditions are provided for generating artificial Sertoli cells from starting cultures of human pluripotent stem cells.

[0064] These methods overcome the limitations noted in the Background of the Invention and exploit the genetic, evolutionary, and molecular biological insights gained from the scRNAseq data we have collected across developmental stages and multiple species to develop a novel, highly efficient, directed somatic cell differentiation protocol. We tuned many parameters of the protocol in mouse ESCs and two human ESC lines and analyzed the scRNAseq data to confirm progression through expected cell states along the developmental trajectory. Benchmarking and classification of in vitro induced cell states relies on the abundance of in vivo markers described above during gonadal differentiation (see Figure 1).

[0065] Testicular somatic cells are key to testicular tissue homeostasis and male reproductive and systemic health. Somatic cells provide a set of unknown growth factors and cytokines required to induce germ cell development in vivo, fully reconstitute female germ cell development in vitro, and promote differentiation of male primordial germ cell-like cells (PGCLCs) into spermatogonia.

[0066] When using mice, it is possible to co-culture in vitro induced PGCLCs with fetal somatic cells or seed embryonic gonads isolated from littermates, but this is costly in non-human primates and ethically unthinkable in humans. Therefore, improving our understanding of somatic cell specification programs and generating alternative somatic cell sources is essential for reconstituting somatic cells in a dish, using inherited / spontaneous gene mutations, ectopic drugs from cancer therapy, or replacing damaged cells in vivo to rejuvenate aged gonads. To address this gap, we utilized single-cell RNAseq data to generate human artificial Sertoli cells from ESCs using the differentiation schema described below.

[0067] [Pluripotent stem cells] The method system, system and kit of the present invention finds use in various pluripotent cells.Suitable pluripotent stem cells include, but are not limited to, embryonic stem cells, adult stem cells, and induced pluripotent stem cells.In some preferred embodiments, the pluripotent stem cells are vertebrate pluripotent stem cells.In some particularly preferred embodiments, the pluripotent stem cells are human embryonic stem cells (hESCs).In other particularly preferred embodiments, the pluripotent stem cells are mouse embryonic stem cells (mESCs).

[0068] In some preferred embodiments, the pluripotent stem cells may be genetically modified by methods known in the art to contain and express one or more exogenous genes.

[0069] [Basal medium] A concern in culturing human ES cells is to remove as many undefined and animal-derived components as possible from the culture conditions for ES cells. Standardizing culture conditions can minimize normal variations in biological materials to which the cells are exposed. Furthermore, avoiding the use of materials, cells, exudates, or components of animal origin can avoid the possibility of cross-species viral transmission through the culture system. Thus, utilizing a chemically defined medium (CDM) that avoids the use of animal products provides a baseline culture condition to which differentiation factors can be added that have predictable effects.

[0070] CDM (e.g., for hESCs) includes a maintenance or basal medium that includes salts, vitamins, glucose, and amino acids. Maintenance of human stem cells prior to differentiation protocols may utilize mTeSR medium, such as mTeSR1™ from StemCell Technologies. In some embodiments, the maintenance medium preferably includes a ROCK inhibitor, such as Y27632. Maintenance medium for mouse stem cells may preferably be GMEM from ThermoFisher Scientific, supplemented with LIF (Leukemia Inhibitory Factor) and optionally knockout serum. The basal differentiation medium may be any of a number of commercially available media. In some preferred embodiments, a combination of Dulbecco's Modified Eagle Medium and Ham's F12 medium (DMEM / F12; Invitrogen) may be utilized. In other preferred embodiments, APEL medium, such as STEMdiff™ APEL™ medium from StemCell Technologies, may be utilized. STEMdiff™ APEL™ medium is a serum-free and animal component-free medium specifically developed to support the differentiation of hPSCs. This medium was originally described to induce blood endothelial cells when supplemented with VEGF, BMP-4, SCF, and activin A, but has also proven to be an effective basal medium for hPSC differentiation into other lineages, including cardiomyocytes. In another preferred embodiment, mTeSR medium may be utilized for stem cell maintenance.

[0071] [Differentiation into artificial Sertoli cells] The present invention provides methods and reagents for producing artificial Sertoli cells from pluripotent stem cells. The present invention is not limited to the use of a particular pluripotent stem cell or chemically defined medium. The methods described herein for producing artificial Sertoli cells are described in relation to events occurring at various time points. It will be recognized that the methods can be varied by modifying the time schedule described. As used herein, "day 0" refers to the day and time that the pluripotent stem cells are removed from the maintenance medium and exposed to the differentiation medium. The differentiation timeline is thus defined starting from day 0. When the term "about" X days is used, this refers to the number of days from the start of day 0 plus or minus 12 hours. For example, "about day 4" means 96 hours (i.e., 4 days) plus or minus 12 hours from the start of day 0. If the start time of day 0 was 9:00 a.m., then "around day 4" would therefore refer to 96 hours plus or minus 12 hours from that time point.

[0072] The first step of the method for producing human artificial Sertoli cells according to the present invention comprises providing pluripotent hESCs as described above. In some preferred embodiments, the pluripotent stem cells are provided in stem cell maintenance medium. In some preferred embodiments, the stem cell maintenance medium is a chemically defined medium, such as mTeSR medium. In some preferred embodiments, the stem cell maintenance medium comprises a ROCK inhibitor. In some preferred embodiments, the ROCK inhibitor is Y27632.

[0073] The second step of the method of the invention involves removing the pluripotent stem cells from the maintenance medium and culturing the pluripotent stem cells in a basal medium supplemented with an agent suitable for inducing the pluripotent stem cells to a promesodermal lineage. In some preferred embodiments, the basal medium is a chemically defined medium. In particularly preferred embodiments, the basal medium is APEL medium, such as StemCell Technologies' STEMdiff™ APEL™ medium. In some particularly preferred embodiments, the basal medium is supplemented with 0.5-15 μM (1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, and 14.0 μM and values ​​and ranges thereof) of CHIR99021. This step defines the process as day 0. The basal medium containing the supplement is preferably replaced daily.

[0074] The third step of the method of the invention comprises culturing the premesodermal cells produced in the second step on or around day 4 in a basal medium supplemented with an agent for inducing the premesodermal cells to intermediate mesoderm. In some preferred embodiments, the basal medium is a chemically defined medium. In some particularly preferred embodiments, the basal medium is APEL medium, such as StemCell Technologies' STEMdiff™ APEL™ medium. In some particularly preferred embodiments, the basal medium is supplemented with 20-500 ng / ml (50, 100, 150, 200, 250, 300, 350, 400, 450 ng / ml and values ​​and ranges thereof) of FGF9. In some particularly preferred embodiments, the basal medium is further supplemented with 0.1-10 μg / ml (0.4, 0.8, 1.0, 1.5, 2.0, 3.0, 5.0, 6.0, 7.0, 8.0, 9.0 μg / ml and values ​​and ranges thereof) of heparin. The basal medium containing the supplements may be changed preferably every two days.

[0075] The fourth step of the method of the present invention comprises culturing the intermediate mesoderm cells produced in the third step in a basal medium on or around day 7, and inducing the intermediate mesoderm cells to form gonadal ridge cells. In some preferred embodiments, the basal medium is a chemically defined medium. In some particularly preferred embodiments, the basal medium is an APEL medium, such as StemCell Technologies' STEMdiff™ APEL™ medium. In some particularly preferred embodiments, the basal medium is not supplemented with additional differentiation agents during this step. The basal medium may be preferably changed every 2 days.

[0076] Optionally, step 4 may be omitted and the intermediate mesoderm cells cultured in the medium described in step 5 on or around day 7.

[0077] The fifth step of the method of the invention involves culturing the genital ridge cells produced in the fourth step on or around day 10 (about day 7 if the fourth step is omitted, see FIG. 16A) in a basal medium supplemented with an agent for inducing the genital ridge cells to form artificial Sertoli cells and other gonadal progenitor cells. In some preferred embodiments, the basal medium is a chemically defined medium. In some particularly preferred embodiments, the basal medium is an APEL medium, such as StemCell Technologies' STEMdiff™ APEL™ medium. In some particularly preferred embodiments, the basal medium is supplemented with 5-100 nM (10, 17, 20, 30, 40, 50, 60, 70, 80, 90 nM and values ​​and ranges thereof) of IGF1. In some particularly preferred embodiments, the basal medium is further supplemented with 10-500 nM (20, 50, 100, 200, 300, 400 nM and values ​​and ranges thereof) of insulin. It is contemplated that the inclusion of IGF1 and insulin in the basal medium is sufficient to promote differentiation of genital ridge cells into artificial Sertoli cells.

[0078] However, in other additional embodiments, the basal medium may be supplemented with one or more other agents. In some embodiments, the basal medium may be further supplemented with retinoic acid (RA) at 0.01-10 μM (0.05, 0.1, 1.0, 5.0, 8.0 μM and values ​​and ranges thereof). In some embodiments, the basal medium may be further supplemented with PGD2 at 50-1000 ng / ml (100, 200, 300, 400, 500, 600, 700, 800, 900 and values ​​and ranges thereof). In some embodiments, the basal medium may be further supplemented with FGF9 at 50-500 ng / ml (100, 200, 300, 400 and values ​​and ranges thereof). In some further preferred embodiments, the basal medium used in step 5 may be further supplemented with 5-100 mg / ml (10, 20, 30, 40, 50, 60, 70, 80, 90 and values ​​and ranges thereof) of bone morphogenetic protein 4 (BMP4). In some further preferred embodiments, the basal medium used in step 5 may be further supplemented with 10-200 mg / ml (30, 50, 70, 100, 150 and values ​​and ranges thereof) of epidermal growth factor (EGF). In some further preferred embodiments, the basal medium used in step 5 may be further supplemented with 0.5-10 μM (1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 μM and values ​​and ranges thereof) of IWR1. In some preferred embodiments, a combination of insulin and IGF1 in the above ranges is utilized. In some preferred embodiments, a combination of insulin, IGF1, and FGF9 in the above ranges is utilized. In some preferred embodiments, the combination of insulin, IGF1, FGF9, RA and PGD2 in the above range is utilized. In some preferred embodiments, the combination of insulin, IGF1, FGF9, BMP4, EGF and / or IWR1 in the above range is utilized. In some preferred embodiments, the combination of insulin, IGF1, FGF9, BMP4, EGF, RA, IWR1 and PGD2 in the above range is utilized. The basal medium containing the supplements can be replaced preferably every 2 days.

[0079] In some further preferred embodiments, the basal medium used in step 5 is further supplemented with one or more hormones. In some further preferred embodiments, the one or more hormones are added on about day 9. In some preferred embodiments, the basal medium (preferably may contain one or more of insulin, IGF1, FGF9, BMP4, EGF, RA, IWR1 and PGD2 in the ranges above) is further supplemented with 5-100 mg / ml (10, 20, 30, 40, 50, 60, 70, 80, 90 and values ​​and ranges thereof) of luteinizing hormone (LH). In some preferred embodiments, the basal medium (preferably may contain one or more of insulin, IGF1, FGF9, BMP4, EGF, RA, IWR1 and PGD2 in the ranges above) is further supplemented with 20-300 mg / ml (50, 100, 150, 200, 250 and values ​​and ranges thereof) of follicle stimulating hormone (FSH). In some preferred embodiments, the basal medium (which may preferably include one or more of insulin, IGF1, FGF9, BMP4, EGF, RA, IWR1, and PGD2 in the ranges described above) is further supplemented with testosterone at 0.01 to 10 μM (0.05, 0.1, 1.0, 5.0, 8.0 μM and values ​​and ranges thereof). In some preferred embodiments, the basal medium (which may preferably include one or more of insulin, IGF1, FGF9, BMP4, EGF, RA, IWR1, and PGD2 in the ranges described above) includes LH, FSH, and testosterone in the ranges described herein.

[0080] In some preferred embodiments, the fifth step of culturing hESCs is maintained until artificial Sertoli cells and / or organoids are derived or until about day 13, most preferably until about day 18-22. In some preferred embodiments, the sixth step of the method of the invention comprises dissociating the differentiated cells. In some preferred embodiments, the differentiated cells are harvested for dissociation around day 10-20, preferably around day 15-18, most preferably around day 18. In some preferred embodiments, the cells are plated in Aggrewell (Aggrewell 400, 24-well plate (Stemcell Technologies Cat # 34421)) and allowed to form organoids. In some preferred embodiments, the organoids are harvested around day 20-24, most preferably around day 20-22.

[0081] The first step of the method for producing mouse artificial Sertoli cell according to the present invention comprises providing pluripotent mESC as described above.In some preferred embodiments, the pluripotent stem cell is provided in stem cell maintenance medium.In some preferred embodiments, the stem cell maintenance medium is GMEM supplemented with LIF and serum.

[0082] The second step of the method of the present invention involves removing the pluripotent stem cells from the maintenance medium and culturing the pluripotent stem cells in a basal medium supplemented with an agent suitable for inducing the pluripotent stem cells to epiderm. This step is defined as day 0. In some preferred embodiments, the basal medium uses DMEM / F12 and Neurobasal Medium (both from ThermoFisher Scientific) in a ratio of 2:1 to 1:2, most preferably in a ratio of about 1:1. In some preferred embodiments, the basal medium is supplemented with N2 supplement, B-27 supplement, and KSR (Knockout Serum Replacer; all from ThermoFisher Scientific), Activin A and bFGF. The supplemented basal medium is called priming medium. In some particularly preferred embodiments, the basal medium is supplemented with N2 supplement at 1-10 μl / ml (1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 μl / ml and ranges and values ​​thereof). In some particularly preferred embodiments, the basal medium is supplemented with B-27 supplement at 1-20 μl / ml (1.0, 5.0, 10.0, 20.0 μl / ml and ranges and values ​​thereof). In some particularly preferred embodiments, the basal medium is supplemented with KSR at 1-20 μl / ml (1.0, 5.0, 10.0, 20.0 μl / ml and ranges and values ​​thereof). In some particularly preferred embodiments, the basal medium is supplemented with Activin A at 1-20 ng / ml (1.0, 5.0, 10.0, 20.0 ng / ml and ranges and values ​​thereof). In some particularly preferred embodiments, the basal medium is supplemented with 1-20 ng / ml (1.0, 5.0, 10.0, 20.0 ng / ml and ranges and values ​​therein) of bFGF.

[0083] The third step of the method of the present invention involves removing the epiblast on about day 2 from the priming medium used in the second step and culturing it in a basal medium supplemented with an agent suitable for inducing the epiblast cells to form anterior intermediate mesoderm. In some preferred embodiments, the basal medium is DMEM / F12. In some preferred embodiments, the basal medium is supplemented with KSR, activin A and retinoic acid (RA). The supplemented basal medium is called differentiation medium. In some particularly preferred embodiments, the basal medium is supplemented with KSR at 1-20 μl / ml (1.0, 4.0, 5.0, 10.0, 20.0 μl / ml and ranges and values ​​thereof). In some particularly preferred embodiments, the basal medium is supplemented with activin A at 1-20 ng / ml (1.0, 5.0, 10.0, 20.0 ng / ml and ranges and values ​​thereof). In some particularly preferred embodiments, the basal medium is supplemented with 10-200 nM (10.0, 50.0, 100.0, 200.0 nM and ranges and values ​​therein) of RA.

[0084] The fourth step of the method of the present invention comprises removing the pre-intermediate mesoderm cells from the differentiation medium used in the third step on about day 4 and culturing them in a basal medium supplemented with an agent suitable for inducing the pre-intermediate mesoderm cells to form testicular organoids comprising Sertoli cells and / or artificial Sertoli cells. In some preferred embodiments, the basal medium is DMEM / F12. In some preferred embodiments, the basal medium is supplemented with KSR, FGF9, IGF1, insulin, PDG2 and RA. The supplemented medium may be referred to as an aggregation medium. In some particularly preferred embodiments, the basal medium is supplemented with 1-20 μl / ml (1.0, 4.0, 5.0, 10.0, 20.0 μl / ml and ranges and values ​​thereof) of KSR. In some particularly preferred embodiments, the basal medium is supplemented with 5-100 nM (10, 17, 20, 30, 40, 50, 60, 70, 80, 90 nM and values ​​and ranges thereof) of IGF1. In a particularly preferred embodiment, the basal medium is further supplemented with 10-500 nM (20, 50, 100, 200, 300, 400 nM and values ​​and ranges thereof) of insulin. It is believed that the inclusion of IGF1 and insulin in the basal medium is sufficient to promote the differentiation of anterior intermediate mesoderm cells and form artificial Sertoli cells and organoids containing the cells. However, in other further embodiments, the basal medium may be supplemented with one or more other agents. In some embodiments, the basal medium may be further supplemented with 10.0-200 nM (10.0, 20.0, 50.0, 100.0 and 200.0 nM and values ​​and ranges thereof) of RA. In some embodiments, the basal medium may be further supplemented with 50-1000 ng / ml (100, 200, 300, 400, 500, 600, 700, 800, 900 ng / ml and values ​​and ranges thereof) of PGD2. In some embodiments, the basal medium may be further supplemented with 50-500 ng / ml (100, 200, 300, 400 and values ​​and ranges thereof) of FGF9. In some preferred embodiments, the basal medium may be supplemented with 1-10 μM (1, 0, 2, 0, 3, 0, 4, 0, 5, 0, 6, 0, 7, 0, 8.0, 9.0, 10.0 μM and ranges and values ​​thereof) of Y-27632.In some further preferred embodiments, the basal medium used in the fifth step may be further supplemented with 5-100 mg / ml (10, 20, 30, 40, 50, 60, 70, 80, 90 and ranges and values ​​thereof) of bone morphogenetic protein 4 (BMP4). In some further preferred embodiments, the basal medium used in the fifth step may be further supplemented with 10-200 mg / ml (30, 50, 70, 100, 150 and values ​​and ranges thereof) of epidermal growth factor (EGF). In some further preferred embodiments, the basal medium used in the fifth step may be further supplemented with 0.5-10 μM (0.5, 1, 2, 4, 5, 7, 10 and values ​​and ranges thereof) of IWR1. The basal medium containing the supplements may be replaced, preferably every two days.

[0085] In some preferred embodiments, the fourth step culture of mESCs is maintained until artificial Sertoli cells and / or organoids are derived or until about day 8. In some preferred embodiments, the method of the present invention includes dissociating the differentiated mESC cells after the fourth step. In some preferred embodiments, the cells are plated in Aggrewell (Aggrewell 400, 24-well plate (Stemcell Technologies Cat # 34421)) and allowed to form organoids. In some preferred embodiments, the organoids are harvested around day 6-10, most preferably around day 8.

[0086] In some preferred embodiments, the artificial Sertoli cells produced by the above method express at least one marker selected from the group consisting of EMX2, WT, SOX9, and LHX9. In some preferred embodiments, the artificial Sertoli cells express at least two markers selected from the group consisting of EMX2, WT, SOX9, and LHX9. In some preferred embodiments, the artificial Sertoli cells express at least three markers selected from the group consisting of EMX2, WT, SOX9, and LHX9. In some preferred embodiments, the artificial Sertoli cells express the markers EMX2, WT, SOX9, and LHX9.

[0087] In some preferred embodiments, artificial Sertoli cells are maintained in culture until they form organoids.Artificial Sertoli cell organoids are preferably three-dimensional organoids with roughly spherical shapes.In some preferred embodiments, organoids are characterized by comprising tubular structures.In some preferred embodiments, organoids are characterized by comprising smooth muscle actin.

[0088] In some preferred embodiments, the artificial Sertoli cells or organoids may be harvested or isolated from the culture for further use.

[0089] [Use of artificial Sertoli cells and organoids] In some preferred embodiments, the methods, reagents and kits described herein and the artificial Sertoli cells and organoids produced thereby find use in various research, diagnostic, clinical and therapeutic applications.In some embodiments, artificial Sertoli cells or organoids are used for direct transplantation into subjects (such as for treating infertility).In some embodiments, the artificial Sertoli cells produced by the methods herein are useful for diagnostic, prognostic and / or therapeutic applications.

[0090] In some embodiments, isolated artificial Sertoli cells or organoids can be directly transplanted into subjects.If appropriate, cells are co-administered with one or more pharmaceuticals or bioactive agents that promote the survival and function of transplanted cells.

[0091] In some embodiments, human organoids are transplanted into mice for differentiation and / or maturation of cells in organoids.In other embodiments, organoids may be combined with germ cells, preferably in vivo or in vitro induced, to achieve proliferation of human germ stem cells and further promote differentiation.It is expected that these methods will produce haploid round or elongated sperm, which will find auxiliary use in reproductive medicine techniques such as IVF / ICSI.

[0092] In some embodiments, the Sertoli cells or organoids of the present invention can be used in co-culture with gamete stem cells, such as primordial germ cell-like cells, precursor spermatogonia, or spermatogonial stem / progenitor cells (SSC / SPC) from a patient. In some preferred embodiments, the cells are cultured so that the stem cells from the patient differentiate into spermatogonia. In some preferred embodiments, the gamete stem cells, or cells derived from gamete stem cells, such as spermatogonia, are transplanted into a patient or a patient in need thereof. In some preferred embodiments, the patient has previously undergone gonadotoxic treatment, including but not limited to chemotherapy and / or radiation therapy. In some preferred embodiments, stem cells or tissues containing stem cells are obtained from a patient prior to gonadotoxic treatment. In some preferred embodiments, prior to gonadotoxic treatment (chemotherapy and radiation therapy), the subject's testicular tissue or somatic cells are stored so that germline stem cells can be produced or isolated in the future. In some preferred embodiments, the germline stem cells obtained from the tissue can be expanded using the in vitro derived cells of the present invention. In some preferred embodiments, the above-mentioned method further comprises harvesting fibroblast tissue for reprogramming into patient-derived induced pluripotent stem cells, which can be used to generate autologous artificial Sertoli cells. In some preferred embodiments, these differentiated cells can be combined with germline stem cells: either primordial germ cell-like cells (PGCLCs), precursor spermatogonia (proSSCs), or newborn / adult spermatogonia stem / progenitor cells (SSCs / SPCs). In some preferred embodiments, the stem cells can be expanded or differentiated into spermatogonia or later germ cell stages. In some preferred embodiments, the method further comprises returning the expanded stem cells or differentiated spermatogonia to a patient in need thereof.

[0093] In some embodiments, the artificial Sertoli cells or organoids may be provided on a support material. Supports suitable for use for the purpose of the present invention include tissue templates, conduits, partitions, and reservoirs that are useful for tissue repair. In particular, synthetic and natural materials in the form of foams, sponges, gels, hydrogels, woven fabrics, and non-woven structures have been used in vitro and in vivo to reconstruct or regenerate biological tissues, and to deliver chemotactic agents to induce tissue growth, and these materials are suitable for use in the implementation of the method of the present invention. See, for example, the materials described in U.S. Pat. Nos. 5,770,417, 6,022,743, 5,567,612, 5,759,830, 6,626,950, 6,534,084, 6,306,424, 6,365,149, 6,599,323, 6,656,488, U.S. Patent Application Publication No. 2004 / 0062753 A1, U.S. Pat. No. 4,557,264, and 6,333,029.

[0094] The cells produced by using the methods and reagents described herein may be transplanted as dispersed cells or formed into transplantable clusters. In some embodiments, the cells are provided in a biocompatible degradable polymeric support; a porous, permeable, or semi-permeable non-degradable device; or encapsulated (e.g., to protect the transplanted cells from host immune response, etc.). The cells can be transplanted into a suitable site of the recipient. Suitable transplantation sites include, for example, the testis or subcutaneously.

[0095] In some embodiments, cells or cell clusters are encapsulated for transplantation into a subject.Encapsulation techniques are generally classified into microencapsulation, which includes small spherical vesicles, and macroencapsulation, which includes larger flat membranes and hollow fiber membranes (Uludag, H. et al.Adv Drug Deliv Rev. 2000; 42: 29-64, which is incorporated herein by reference in its entirety).Preparation methods for microcapsules include those described in: Lu MZ et al., Biotechnol Bioeng.2000, 70: 479-83; Chang TM and Prakash S, Mol Biotechnol.2001, 17: 249-60; and Lu MZ et al., J. Microencapsul.2000, 17: 245-51, which is incorporated herein by reference in its entirety. For example, microcapsules can be prepared by complexing modified collagen with a terpolymer shell of 2-hydroxyethylmethylacrylate (HEMA), methacrylic acid (MAA) and methylmethacrylate (MMA), resulting in capsules with a thickness of 2-5 μm. Such microcapsules can be further encapsulated with a terpolymer shell of 2-5 μm to provide a negatively charged smooth surface to minimize plasma protein adsorption (Chia, SM et al. Multi-layered microcapsules for cell encapsulation Biomaterials. 2002 23: 849-56; incorporated herein by reference in its entirety). In some embodiments, the microcapsules are based on the marine polysaccharide alginate (Sambanis, Diabetes Technol. Ther. 2003, 5: 665-8; incorporated herein by reference in its entirety) or derivatives thereof. For example, microcapsules can be prepared by polyelectrolyte complexation of the polyanions sodium alginate and sodium cellulose sulfate with the polycation poly(methylene-co-guanidine) hydrochloride in the presence of calcium chloride.

[0096] In some embodiments, cells produced using the 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., cells of pancreatic lineage, beta-like cells, etc.) provides local protection of the transplanted / implanted cells from immune attack (e.g., with or without the use of systemic immunosuppressants). In some embodiments, the cells and / or cell clusters are microencapsulated in a polymer, hydrogel, or other suitable material, including, but not limited to: poly(orthoester), poly(anhydride), poly(phosphoester), poly(phosphazene), polysaccharide, polyester, poly(lactic acid), poly(L-lysine), poly(glycolic acid), poly(lactic-co-glycolic acid), poly(lactic-co-lysine), poly(lactic-graft-lysine), polyanhydride, poly(fatty acid dimer), poly(fumaric acid), poly(sebacic acid), poly(carboxyphenoxypropane), poly(carboxyphenoxyhexane), poly(anhydride-co-imide). , poly(amides), poly(orthoesters), poly(iminocarbonates), poly(urethanes), poly(organophasphazenes), poly(phosphates), poly(ethylene vinyl acetate), poly(caprolactone), poly(carbonates), poly(amino acids), poly(acrylates), polyacetals, poly(cyanoacrylates), poly(styrene), poly(vinyl chloride), poly(vinyl fluoride), poly(vinylimidazole), chlorosulfonated polyolefins, polyethylene oxide, polystyrene, polysaccharides, alginates, hydroxypropyl cellulose (HPC), N-isopropylacrylamide (NIPA), polyethylene glycol, polyvinyl alcohol (PVA), polyethyleneimine, chitosan (CS), chitin, dextran sulfate, heparin, chondroitin sulfate, gelatin, and the like, and their derivatives, copolymers, and mixtures thereof. In some embodiments, the cells are microencapsulated in an encapsulating material that comprises or consists of alginate.The cells may be embedded in a material or within particles (e.g., nanoparticles, microparticles, etc.) or other structures (e.g., matrices, nanotubes, vesicles, globules, etc.). In some embodiments, the microencapsulation structures are modified with immunomodulatory or immunosuppressive compounds to reduce or prevent immune responses against the encapsulated cells. For example, cells of the pancreatic lineage are encapsulated within an encapsulation material (e.g., an alginate hydrogel) that has been modified by attaching an immunomodulatory agent (e.g., CXCL12 (also known as SDF-1), an immunomodulatory chemokine). In some embodiments, such an immunomodulatory agent is a T cell chemokine and / or a survival promoting factor.

[0097] In some embodiments, the cells produced using the methods and reagents described herein are macroencapsulated for transplantation into a subject.For example, macroencapsulation of cells in a permeable or semi-permeable chamber provides local protection of transplanted / implanted cells from immune attack (e.g., with or without the use of systemic immunosuppressants), prevents the spread of cells to other tissues or regions of the body, and / or allows efficient removal of cells.Devices suitable for macroencapsulation include, for example, those described in U.S. Patent No. 5,914,262; Uludag et al., Advanced Drug Delivery Reviews, 2000, pp.29-64, vol.42 (incorporated herein in their entirety by reference).

[0098] Other encapsulation (micro or macro) devices and methods may find use with the embodiments described herein. For example, the methods and devices described in U.S. Patent Application Publication No. 20130209421, U.S. Patent No. 8,785,185 (incorporated herein by reference in their entirety) are within the scope of the embodiments described herein.

[0099] In some embodiments, the Sertoli cells or organoids of the present invention may be used for hormone therapy. In some preferred embodiments, the organoids are encapsulated and subcutaneously implanted in the subject.

[0100] In some embodiments, the Sertoli cell or organoid of the present invention can be used for fertility restoration.In some preferred embodiments, the endogenous defective somatic cells in testis are combined or replaced with the Sertoli cell or organoid of the present invention.In other preferred embodiments, the organoid is transplanted into subject so that spermatogenesis occurs in the ectopic place other than the testis (for example, subcutaneously).

[0101] In further embodiments, artificial Sertoli cell and organoid populations can be used to prepare antibodies and cDNA libraries that are specific for differentiated phenotypes.The 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).The general techniques involved in preparing mRNA and cDNA libraries are described in RA Methodologies: A Laboratory Guide for Isolation and Characterization (RE Farrell, Academic Press, 1998); cDNA Library Protocols (Cowell & Austin, eds., Humana Press); and Functional Genomics (Hunt & Livesey, eds., 2000). Relatively homogenous cell populations are particularly suitable for use in drug screening and therapeutic applications.

[0102] In some embodiments, the artificial Sertoli cells and organoids produced by the methods provided herein are used to screen for agents (e.g., small molecule drugs, peptides, polynucleotides, etc.) or environmental conditions (e.g., culture conditions or manipulations) that affect cells. In particular, screening applications relate to the testing of pharmaceutical compounds in drug research, and agents used in cryopreservation of gametes, including sperm. Evaluating the activity of a candidate pharmaceutical compound generally involves combining cells with the candidate compound, determining changes in cell morphology, marker phenotype, or metabolic activity caused by the compound (compared to untreated cells or cells treated with an inactive compound), and then correlating the effects of the compound with the observed changes. Any suitable assay for detecting changes associated with a test drug may be used in such embodiments. Screening may be performed, for example, either because the compound is designed to have a pharmacological effect on Sertoli cell types, because a compound designed to have an effect elsewhere may have unintended side effects, or because the compound is part of a library screen for a desired effect. Two or more drugs may be tested in combination (simultaneously or sequentially, combined with cells) to detect possible drug-drug interaction effects. In some applications, compounds are screened for cytotoxicity.

[0103] In some embodiments, methods and systems are provided for evaluating the safety and efficacy of drugs that act on Sertoli cells, or drugs that may be used for other purposes but that may have unintended effects on Sertoli cells. In some embodiments, the cells described herein find use in high throughput screening (HTS) applications. In some embodiments, cells that are large numbers (e.g., 1×10 3 , l x 10 4 , l x 10 5 , l x 10 6HTS screening platforms (e.g., cells and plates) are provided that allow for rapid testing of drugs (e.g., small molecule compounds, peptides, etc.) of any of a variety of therapeutic agents (e.g., avian, cerebrovascular, vascular endothelial, and endothelial cells) in a variety of therapeutic applications. In some embodiments, artificial Sertoli cells or organoids produced using the methods and reagents described herein are utilized for therapeutic delivery to a subject. The cells may be in direct contact with the target tissue or may otherwise be sealed or encapsulated (e.g., to avoid direct contact). In embodiments in which the cells are encapsulated, exchange of nutrients, gases, etc. between the encapsulated cells and the target tissue is permitted. In some embodiments, the cells are implanted / implanted onto a matrix or other delivery platform.

[0104] In some embodiments, the methods and kits described herein are useful for identifying additional factors, reagents, and methods for generating artificial Sertoli cells or other cell types. The methods used herein can be used to screen factors, reagents, and / or conditions for differentiation effects. In some embodiments, the screening performed in this or other embodiments discussed herein can be high-throughput screening.

[0105] [Example] [Example 1] The following examples describe reagents and protocols for generating artificial Sertoli cells derived from mouse embryonic stem cells (mESCs) according to the present invention.

[0106] [Proliferation of mESCs] (Reagents / Materials) 1. Mouse embryonic stem cells (TG2A) 2.GMEM(ThermoFisher scientific Cat # 1171035) 3. Type B Gelatin Solution (Sigma Aldrich Cat # G1393-100ML) 4.MEM Non-Essential Amino Acids (ThermoFisher Scientific Cat # 11140050) 5. 2-Mercaptoethanol (ThermoFisher scientific Cat # 21985023) 6. Sodium Pyruvate Solution (Sigma Aldrich Cat # S8636-100ML) 7. Embryonic Stem Cell Fetal Bovine Serum, Qualified (ThermoFisher Scientific Cat #16141079) 8.TrypLE Express Enzyme(ThermoFisher scientific Cat #12604013) 9. Leukemia inhibitory factor (Sigma Aldrich Cat #L5283-10μG) 10. Penicillin-streptomycin (5,000 U / mL) (ThermoFisher scientific Cat #15070063) 11.PBS- / -(ThermoFisher scientific Cat #10010023) 12. Dimethyl sulfoxide (Sigma-Aldrich Cat #D8418-100ML) 13. 6-well cell culture plates (ThermoFisher Scientific Cat #140675)

[0107] Growth medium: may be prepared and stored at 4°C. Do not store for more than one month (dated bottles). Composition of 100 ml of growth medium: Glasgow MEM (GMEM) 92 ml, qualified bovine fetal serum ES cells 10 ml, non-essential amino acids 1 ml, sodium pyruvate 1 ml, penicillin-streptomycin 1 ml, 2-mercaptoethanol 100 μl, LIF 100 μl (final 1000 units / ml).

[0108] 2X Freezing Buffer ES cells were prepared in 20% dimethyl sulfoxide and 80% qualified fetal bovine serum and stored in a -20°C freezer.

[0109] Maintaining ESC (1) Before seeding the cells, add 2 ml / well of 0.1% gelatin solution and incubate at room temperature overnight. (2) Thawing of TG2A cells Remove a 1 ml vial of frozen cells (frozen in 1:1 freezing buffer and growth medium) from liquid nitrogen and immediately thaw in a 37°C water bath. (3) Wash the cells by adding 2 ml of growth medium to the thawed vial and centrifuge at 1000 RPM for 5 minutes. (4) Aspirate the medium and dilute the pelleted cells with 2 ml of growth medium. (5) Aspirate the gelatin from the plates and seed approximately 500,000 cells in 2 ml of growth medium per well of a 6-well plate. (6) Complete medium change every 24 hours. (7) When the cells reach approximately 80% confluency, passage the cells onto new plates. (a) To passage the cells, aspirate the medium and rinse the cells with 1 ml of PBS per well. (b) Aspirate the PBS. (c) Add 300 μl of TrypLE expression enzyme per well and incubate at 37° C. in a CO 2 incubator for 4 minutes. (d) Remove the cells from the incubator and add 2 mL of growth medium to quench the TrypLE expression enzyme. (e) Centrifuge the cells at 1000 RPM for 5 minutes. Aspirate off the fluid and dilute the cells with 2 ml of growth medium. (f) Gelatin-coated 6-well cell culture plates are seeded with approximately 500,000 cells per well in 2 ml of growth medium. (g) Complete medium change every 24 hours. (h) After reaching approximately 80% confluence, repeat the same process as in point 7 once more.

[0110] [Differentiation of TG2A cells into anterior intermediate mesoderm-like cells] (Reagents / Materials) (1) Neurobasal medium (ThermoFisher scientific Cat # 21103049) (2) DMEM-F12 medium (ThermoFisher Scientific Cat #) (3) 2-Mercaptoethanol (ThermoFisher scientific Cat # 21985023) (4) Sodium pyruvate solution (Sigma Aldrich Cat # S8636-100ML) (5) MEM non-essential amino acids (ThermoFisher scientific Cat # 11140050) (6)N-2 supplement (ThermoFisher scientific Cat # 17502048) (7)B-27 Refill (ThermoFisher scientific Cat # 17504044) (8)Glutamax(100X)(ThermoFisher scientific Cat # 35050061) (9) Knockout serum replacement solution (ThermoFisher Scientific Cat # 10828010) (10) Penicillin-streptomycin (5,000 U / mL) (ThermoFisher scientific Cat # 15070063) (11)TrypLE Express Enzyme(ThermoFisher scientific Cat # 12604013) (12) Activin A (R&D systems Cat # 338-AC-010) (13)bFGF(Proteintech Cat #HZ-1285) (14) Retinoic Acid (Sigma-Aldrich Cat # R2625) (15) 6-well cell culture plate (ThermoFisher Scientific Cat # 140675) (16) ROCK inhibitor Y-27632 (Enzo Life Sciences Cat # ALX-270-333) (17)PBS- / -(ThermoFisher scientific Cat # 10010023)

[0111] Priming medium: can be prepared and stored at 4°C. Do not store for more than one month (dated bottles). Priming medium composition for 100 ml medium: 46.9 ml Neurobasal medium, 46.9 ml DMEM-F12 medium, 500 μl N2 supplement, 1 ml B27 supplement, 500 μl Glutamax, 100 μl 2-mercaptoethanol, 1 ml sodium pyruvate, 1 ml knockout serum supplement, 1 ml non-essential amino acids, 1 ml penicillin-streptomycin.Additional growth factors, 10 ng / ml activin A and 10 ng / ml bFGF, are mixed in the medium just prior to adding the medium to the cells.

[0112] AIM differentiation medium A small amount of basal AIM differentiation medium can be prepared and stored at 4°C. Do not store for more than one month (dated bottles).

[0113] AIM Differentiation Medium Composition for 100 ml medium: Add 92.9 ml DMEM-F12 medium, 1 ml sodium pyruvate, 4 ml knockout serum supplement, 1 ml non-essential amino acids, 100 μl 2-mercaptoethanol, 1 ml penicillin-streptomycin.Additional growth factors, 10 ng / ml activin A and 100 nM RA, are mixed into the medium just before adding the medium to the cells.

[0114] (1) Proliferation of cells for differentiation Starting cells should be undifferentiated and in proliferative phase. To ensure starting cells are consistently in the same proliferative phase between experiments, harvest cells from an 80% confluent plate (at least one passage after thawing). (a) Aspirate the medium and wash the cells with 1 ml of PBS- / - in a 6-well plate. (b) PBS- / - is aspirated and cells are incubated with 300 μl of TrypLE expression solution per well of a 6-well plate for 5 minutes in a CO2 incubator. (c) Remove the cells from the incubator and add 2 mL of growth medium to quench the TrypLE expression enzyme. (d) The cells are centrifuged at 1000 RPM for 5 minutes, the fluid aspirated, and the pelleted cells diluted with growth medium. (e) Approximately 300,000 cells are seeded per well of a gelatin-coated 6-well plate in 2 ml of growth medium. (f) Every 24 hours, the complete medium is replaced with fresh growth medium. (g) 48 hours after plating, the cells are ready to begin differentiation.

[0115] (2) Initiation of differentiation (a) Media was aspirated from cells grown for 48 hours and washed with 1 ml of PBS- / - in a 6-well plate. (b) PBS- / - is aspirated and cells are incubated with 300 μl of TrypLE expression solution per well of a 6-well plate for 5 minutes in a CO2 incubator. (c) Remove the cells from the incubator and add 2 mL of growth medium to quench the TrypLE expression enzyme. (d) The cells are centrifuged at 1000 RPM for 5 minutes, the fluid aspirated, and the palletized cells are diluted with priming medium. (e) Approximately 30,000 cells per well of a gelatin-coated 6-well plate are seeded in 2 ml of priming medium and cultured in a CO2 incubator. (f) After 24 hours of incubation, the priming complete medium is replaced. Since the cells are loosely attached to the surface, the medium is replaced very carefully without disturbing the plate too much. (g) After 48 h total incubation in priming medium, replace the complete medium with 2 ml of AIM differentiation medium per well of a 6-well plate. Since the cells are loosely attached to the surface, replace the medium very carefully without disturbing the plate too much. (h) At 72 h total incubation, replace medium with 3 ml of AIM differentiation medium and 5 μM Y-27632 per well of a 6-well plate. (i) After 96 h total incubation, the cells are ready for dissociation and organoid generation.

[0116] NOTE: Cells were harvested daily after day 2 of differentiation for RNA isolation. qPCR was performed for multiple AIM markers, gonadal marker, pluripotency marker Oct4, and posterior intermediate mesoderm marker HoxD11. Data were confirmed by immunostaining for AIM as well as gonadal marker Wt1, and gonad marker Sox9.

[0117] [Creation of testicular organoids using AIM cells differentiated for 4 days] (material) (1) DMEM-F12 medium (ThermoFisher Scientific Cat #) (2) 2-Mercaptoethanol (ThermoFisher scientific Cat # 21985023) (3) Sodium pyruvate solution (Sigma Aldrich Cat # S8636-100ML) (4) MEM non-essential amino acids (ThermoFisher scientific Cat # 11140050) (5)Glutamax(ThermoFisher scientific Cat # 35050061) (6) Knockout serum replacement solution (ThermoFisher Scientific Cat # 10828010) (7) Penicillin-streptomycin (5,000 U / mL) (ThermoFisher scientific Cat # 15070063) (8)FGF9(R&D systems Cat # 273-F9-025) (9) Retinoic Acid (Sigma-Aldrich Cat # R2625) (10)IGF1(Sigma-Aldrich cat # I3769) (11) Insulin (Sigma-Aldrich cat #I9278) (12) Prostaglandin D2 (Cayman Chemical Cat # 12010) (13) ROCK inhibitor Y-27632 (Enzo Life Sciences Cat # ALX-270-333) (14) Matrigel Basement Membrane Matrix (Corning 354234) (15)TrypLE Express Enzyme(ThermoFisher scientific Cat # 12604013) (16) Aggrewell 400, 24-well plate (Stemcell Technologies Cat # 34421) (17) Anti-adherence Cleaning Solution (Stemcell Technologies Cat # 07010) (18) BMP4 (R&D 314-BP) (19)IWR1(Sigma-Aldrich Cat # 681669) (20)EGF(Sigma-Aldrich Cat # E4127)

[0118] Organoid Differentiation Medium: Basal Organoid Differentiation Medium can be prepared in small quantities and stored at 4°C. Do not store for more than one month (dated bottles).

[0119] Combination 1: Organoid Differentiation Medium composition for 100 ml of medium: 92.9 ml DMEM-F12 medium, 1 ml sodium pyruvate, 4 ml knockout serum supplement, 1 ml non-essential amino acids, 100 μl 2-mercaptoethanol, 1 ml penicillin-streptomycin.Additional factors, 1% Matrigel, 100 nM retinoic acid, 17 nM IGF1, 100 nM insulin, 200 ng / ml FGF9, 500 ng / ml PGD2, 5 μM Y-27632, mixed immediately before adding medium to the cells.

[0120] Combination 2: Organoid differentiation medium for 100 ml of medium: 92.9 ml DMEM-F12 medium, 1 ml sodium pyruvate, 4 ml knockout serum supplement, 1 ml non-essential amino acids, 100 μl 2-mercaptoethanol, 1 ml penicillin-streptomycin. Additional factors include 1% Matrigel, 100 nM retinoic acid, 17 nM IGF1, 100 nM insulin, 1 ng / ml FGF9, 500 ng / ml PGD2, 20 ng / ml BMP4, 50 ng / ml EGF, and 5 μM Y-27632, mixed immediately before adding medium to the cells.

[0121] Combination 3: Organoid differentiation medium for 100 ml of medium: 92.9 ml DMEM-F12 medium, 1 ml sodium pyruvate, 4 ml knockout serum supplement, 1 ml non-essential amino acids, 100 μl 2-mercaptoethanol, 1 ml penicillin-streptomycin. Additional factors include 1% Matrigel, 100 nM retinoic acid, 17 nM IGF1, 100 nM insulin, 1 ng / ml FGF9, 500 ng / ml PGD2, 20 ng / ml BMP4, 50 ng / ml EGF, 2 μM IWR1, and 5 μM Y-27632, mixed immediately before adding medium to the cells.

[0122] [Preparing plates for organoid differentiation] Prior to initiating organoid differentiation, Aggrewell 400-24 well plates were prepared as follows. (a) Add 500 μl of anti-adherent wash solution to each well of an Aggrewell 400-24 well plate and incubate for 30 minutes at room temperature. (b) Aspirate the anti-adherent wash solution and wash thoroughly with 1 ml PBS- / - per well. (c) Aspirate PBS- / - and add 500 μl of organoid differentiation medium per well of an Aggrewell 400-24 well plate. (d) Centrifuge the plate at 500 RCF for 5 minutes to remove any air in the wells. The plate is now ready for cell seeding.

[0123] (1) Organoid differentiation (a) Aspirate the AIM differentiation medium and wash the cells three times with 2 ml of PBS- / - per well of a 6-well plate from AIM cells differentiated on day 4. (b) Add 500 μl of TrypLE Express Enzyme to each well of a 6-well plate and incubate in a CO2 incubator for 5 minutes. (c) Remove the cells from the incubator and add 2 mL of growth medium per well of a 6-well plate to quench the TrypLE expression enzyme. (d) The cells are centrifuged at 1000 RPM for 5 minutes, the above fluid is aspirated and the palletized cells are diluted in organoid differentiation medium. (e) Approximately 300,000 cells were mixed in 500 μl of organoid differentiation medium and added to one well of a pre-prepared Aggrewell plate. (f) Incubate the plate in a CO2 incubator for 10 minutes to allow the cells to settle into the wells. (g) Centrifuge the Aggrewell plate again at 500 RCF for 5 minutes. (h) Culture the cells in a CO2 incubator. (i) The forming organoids were left for 2 days, after which half of the medium was replaced with fresh organoid differentiation medium every day. (j) Organoids were harvested from day 0 to a total of days 6 and 8 for RNA isolation and qPCR analysis of different terminal differentiation markers. (k) Data were confirmed by whole-mount immunostaining for progenitor cell marker CoupTf2, Sertoli cell markers Gata4, Sox9, and Wt1, and Leydig cell marker Cyp17a1.

[0124] [result] 1. Generation and validation of mouse testis-like organoids from mESCs Leveraging mouse genetic and scRNAseq data, we developed and optimized a mouse ESC differentiation protocol that produces cells resembling many types of testicular cells. Our approach involves iteratively titrating cell seeding densities and testing numerous combinations of growth factors, cytokines, and small molecules at multiple concentrations and durations, allowing cells to recapitulate the differentiation trajectories observed in vivo (Figure 1). During this protocol optimization effort, we harvested cells at various time points and monitored the expression of multiple markers shown in Figure 1 to determine whether the in vitro derived cells were undergoing the expected differentiation stages: epidermal state (OCT4, FGF5, FGF8, and DNMT3b), premesoderm (T and TBX6), premesoderm (LHX1, GATA3), gonad primordium (WT1, GATA4, EMX2, LHX9), and Sertoli cells (Sox9). + , Gata4 + , SF1 + , WT1 + , AMHR2 + We applied a similar strategy for mouse and human ESCs, establishing two subtly different cocktails: terminally differentiated testicular somatic cells such as ES cells (SF1, 3BHSD, STAR), Leydig cells (SF1, 3BHSD, STAR), and myoid cells (SMA, TAGLN).

[0125] Briefly, treatment of mouse ESCs with Activin A and bFGF for 2 days suppresses many pluripotency markers (Nanog, Sox2, Rex1, Klf1; Figure 2B, left) while maintaining Oct4 expression and increasing Fgf5, Fgf8, and Bracury(T) (Figure 2B, right). These transcriptional changes are consistent with ESCs transitioning towards epiblast cells (Figure 1). Upon reaching the epiblast state, cells were treated with Activin A and retinoic acid for 3–5 days (Figure 2A). During the 3–5 day differentiation window, early mesoderm markers such as OSR1 were decreased, whereas anterior intermediate mesoderm and gonadal primordium markers, such as Gata4, Pax2, WT1, Sox9, and Lhx1, were enriched (Figure 2C). Importantly, our differentiation protocol limited the generation of posterior intermediate mesodermal progenitors (HOXD11+ cells) that give rise to the kidney. Although these preliminary findings at day 5 were exciting, monolayer cells rarely formed organoids spontaneously. Therefore, to promote organoid formation and further progress beyond the gonadal primordium to advanced gonadal cell fates, we dissociated cells on day 4 or 5 and replicated ~300K cells in 800-microwell Aggrewells (Figure 2A). Plated cells were supplemented with Fgf9, Igf1, insulin, Pgd2, and RA (Figure 2A). Within 24 h after plating, single cell suspensions formed spheroids / organoids and were maintained in culture for an additional 48–96 h. RT-qPCR analysis of organoids at days 6–8 revealed an increase in stromal progenitor cell markers (Tcf21, Nr2f2, Wt1) and Sertoli cell markers (Wt1, Sox9, Fshr), as well as identification of Leydig cell markers (Cyp17a1, StAr, and LHR) (Figure 2D).

[0126] To confirm that the RNA changes were also evident at the protein level, we performed immunofluorescence imaging with a subset of the markers analyzed by qPCR. As expected, the stem cell marker OCT4 was expressed at day 0 but was suppressed at day 4 (Figure 3A). Meanwhile, day 4 cells began to express gonadal primordium markers such as Wt1, CoupTF2 (Nr2F2), and Sox9, and maintained expression of Gata4 (Gata4 is expressed in mouse ESCs but not in humans) (Figure 3B). After dissociation and aggregation, retinoic acid was required for efficient differentiation and complete suppression of the pluripotency program (data not shown), as well as for the generation of ordered tubular structures within the organoids (Figure 3C). This tubular organization was observed in approximately 70% of the generated organoids in more than 10 repeated experiments. In the center of the tubular tissue, several Sertoli cell-like markers, such as Gata4, Sox9, AMH, and SF1, were detected, and these cells secreted extracellular matrix (lamin) and formed tight function (Zo1; Figure 3C), similar to in vivo cells.

[0127] 2. Testicular somatic-like cells induced in vitro in mice are similar to gonadal progenitor cells identified in the E10.5-11.5 gonads For mouse ESC experiments, cells at days 0, 2, 4, and 8 of differentiation were molecular barcoded, captured, and sequenced in the same run to avoid batch effects. Unsupervised clustering of approximately 5,000 cells resulted in eight clusters: C1-C8 (Figure 4A). Genes highly expressed in C1 cells include pluripotency factors such as Oct4, Sox2, and Nanog (Figure 4C), consistent with this cluster being ESC. C2 maintains high Oct4 levels and expresses DNMT3b, Fgf5 / 8, but is depleted in Nanog, consistent with epiblast cells. C3-8 cells are molecularly heterogeneous and enriched in anterior intermediate mesoderm and gonad primordium cell populations (Figure 4C). Importantly, C1 cells are primarily induced on day 0, while clusters 2, 3, and 4-8 are induced on days 2, 4, and 8, respectively (Figure 4B, 4F). Comparing our in vitro derived somatic cells with those reported for in vivo cells (12,13), our day 0-2-4-8 cells and E10.5-E13.5 embryonic gonad cells progress in parallel (Figure 4E), and our eight cell clusters progressing from day 0-8 are consistent with the gonadal progenitors, stromal progenitors, and pre-Sertoli cells of the in vivo data (Figure 4F). Importantly, our differentiation protocol is gonadal cell specific, and we did not detect transcription factors for alternative urogenital cell fates, such as kidney (Hoxd11, Six2, nphs2), adrenal (Sult2a1; Arhgap), ovarian (Foxf1), or granulosa cells (Foxl2) (Figure 4D).

[0128] 3. Mouse testicular somatic-like cells can maintain and possibly support the differentiation of prespermatogonia in vitro To investigate whether the in vitro induced somatic-like cells could support germ cell maintenance and differentiation, organoids were harvested and dissociated on day 6 and cultured to identify in vitro induced somatic cells and approximately 4% of prespermatogonia (Oct4-EGFP) cells harvested from postnatal day (PND) 1–2 testes. +A similar differentiation protocol as described above was performed, except that the somatic and germ cell organoids were reaggregated from day 0 ESCs (Figure 5). The combined somatic and germ cell organoids were maintained in culture for an additional 2, 4, and 6 days (10–16 days total from day 0 ESCs). Importantly, these in vitro assembled organoids are not only able to incorporate prospermatogonia within these structures and maintain prospermatogonia germ cells (Oct4-EGFP+ / Plzf) (Figure 6), but also potentially capable of prespermatogonia differentiation, as evidenced by the appearance of Oct4-Egfp-dim cells as well as Stra8 and Sycp3 cells after a few days in culture. Building on this initial success, ongoing experiments are fine-tuning meiotic progression and completion.

[0129] Our data provide very strong molecular and histological evidence suggesting that testis-like organoids resemble fetal-like gonadal tissue. Importantly, these cells support spermatogonia- and primordial germ cell-like cell homing, proliferation, and differentiation, and we are currently optimizing meiotic progression.

[0130] 4. Continuing efforts to refine the robustness of somatic cell differentiation protocols Although our original cocktail shown in Figure 2A worked well and consistently, off-target differentiated cells (such as kidney cells) can be detected at low frequencies in organoids. To increase purity and suppress off-target differentiation, we screened 20 more drugs in the baseline medium of ALL-GF. When we added BMP4+EGF1 to the original cocktail of ALL-GF with decreasing FGF9 concentration (referred to as cocktail 3), we were able to significantly improve testis-like differentiation and at the same time completely block off-target changes (Figure 7A). Specifically, all gonadal markers (Lhx9, Pdgra, Sma, Nr5a1, Tcf21, Gata3, Nr2f2, Wt1, Gata4, Fshr, Amhr, Inhbb, Inha, Dhh and Sox9) are elevated in cocktail 3 compared to ALL GF. Importantly, this cocktail suppresses gene expression in kidney and granulosa cells (female lineage). Consistent with the improved RNA expression, many protein markers were also more robustly expressed by immunofluorescence (Figure 8). Furthermore, these cells are superior for expanding germline stem cell progenitors in vitro (Figure 9).

[0131] [Example 2] The following example describes reagents and protocols for producing artificial Sertoli cells according to the invention from human embryonic stem cells (hESCs), which can preferably last for at least 13 days and up to 22 days.

[0132] (reagent) STEMdiff APEL medium (Stem Cell Technologies, Cat. No. 05270 or No. 05275) ·DMEM / F-12 (ThermoFisher scientific, Cat.No.11320-082) ·DMSO (Sigma Aldrich, Cat.No:D5879) Dulbecco's Phosphate Buffered Saline (DPBS) (ThermoFisher Scientific, Cat. No. 14190-144) Accutase (Cat No: 07920) CHIR99021 (R&D, Cat No: 9902): Briefly centrifuge the stock solution (10 mM) tube before opening. Reconstitute 10 mg of CHIR99021 in 2.149 ml of DMSO to make a 10 mM stock. Aliquot this solution in 20 μL portions, label as "Ch", and store at -20°C. Do not reuse after 24 hours after thawing. Y27632 ROCK inhibitor (Enzo, Cat No.: ALX-270-333): Dissolve 1 mg of stock solution (5 mM) in 625 uL of sterile TC water. Aliquot into 20 μL. Label as "Y". Store at -20°C at Stem Cell Core. Dilute in 10 mL of medium to a final concentration of 10 μM. Use immediately after thawing. Do not reuse after 24 hours after thawing. Heparin (Sigma Aldrich, Cat. No.: H4784-250Mg): Stock solution (1mg / ml) Adjust to 1mg / ml with ultrapure water and filter sterilize through a 0.22μm syringe-driven filter unit of polyethersulfone (PES). Heparin solution can be stored at 4℃ for more than 12 months. Aliquot into 40μl. Store at 4℃. · FGF9 (R&D, Cat No.: 273-F9-025): Stock FGF9 (100 μg / ml). Briefly centrifuge before opening the tube. Reconstitute to 100 μg / ml in filtered DPBS containing 0.1% (wt / vol) human serum albumin. Aliquot in 20 μl and store at -80°C for up to 6 months. Store stocks at -80°C. Once thawed, FGF9 can be stored at 4°C for up to 2 weeks. · RA (Sigma, Cat No: R2625-5mg): Stock solution (0.5mM) Weigh out 1mg of RA and dissolve in 3ml of DMSO to give a stock solution of 1.11mM concentration. Serially dilute this solution to 0.5mM stock solution and store at -20°C in 50μl or 100μl aliquots. Use the 0.5mM stock solution in cell culture media based on the appropriate concentration desired for the experiment. Do not reuse after 24 hours after thawing. · Insulin (Sigma, Cat No: I9278): Obtained in liquid form from the stock (1.7 mM) vendor. · IGF1-50μg (Sigma, Cat No: I3769): Stock solution (10mM) Dissolve 50μg of IGF1 in 658μl of 0.2% acetic acid to give a stock concentration of 10mM. Aliquot into 10μl and store at -80℃. Take 10μl of 10mM and dissolve in 1ml of 0.2% acetic acid to give a concentration of 100μM in cell culture media based on the appropriate concentration desired for the experiment. Once thawed, can be stored at 4℃ for 1 month. PGD2 (Cayma, Cat No: 12010): Stock solution (2 mg / ml) Dissolve PGD2 in 200 ul of PBS 7.4 to prepare a 2 mg / ml stock solution, dispense into 5 ul portions, and store at -80°C. Do not reuse within 24 hours after thawing. · Matrigel (Corning, Cat No: 354234, Lot No: 9133008): Stock solution (2 mg / ml or 1 mg / ml) Dilute Matrigel with DMEM / F12 medium to a concentration of 2 mg / ml or 1 mg / ml, aliquot it, and store it at -20℃ until use. Epidermal growth factor, EGF (Sigma, Cat No. E4127-.1mg) derived from mouse submandibular gland: Stock solution (100μg / ml) 0.1mg of EGF was dissolved in 10ml of 0.1%BSA / PBS, then dispensed into 1ml portions and stored at -20℃ until use. IWR1 (Sigma: cat no. 681669-10mg) stock solution (10mg / ml) Dissolve 10mg / ml in DMSO and store at -20℃ until use. · BMP4 (R&D: Cat No. 314-bp-500): Stock solution (200μg / ml) Dissolve 200μg / mL in 4mM sterile HCl containing 0.1% BSA / H2O, prepare aliquots, and store at -20℃ until use.

[0133] (material) Nunc™ 4-well dish for IVF (Fisher, Cat No. 144444) ·Nunc Multidish Nunclon Delta SI-24 well (Fisher, Cat No. 142475) Coverslips 12mm (Hampton Research, Cat No. HR3-277) ·Benchtop centrifuge (Eppendrof, 5417c) Biological safety cabinet CO2 incubator (Heraeus) Conical tube 15ml (Falcon, Cat No. 352096) Conical tube 50ml (Falcon, Cat No. 352070) Freezer container (Nalgene, Mr.Frosty) Inverted confocal tissue culture microscope (KL1500CD, Lieca) Laser confocal microscope (Nikon Eclipse Ti2) Pipette (Gilson) ·Automatic cell counter (Biorad, TC20) Serological pipettes (Fisher, Cat No. 13-678-11D, 13-676-10J, 13-676-10R) Stericup 0.22μm filter unit (Millipore, 03290) Sterifilter pipette tips (Genesie Scientific, 24-815, 24-804, 24-830, 26-401) Sterile microcentrifuge tubes (Fisher, Cat No. 05-408-120)

[0134] (Cover slip washing method) Incubate the coverslips in xylene in a 50 mL conical tube for 2-3 hours. Discard the xylene and rinse with acetone. Discard the acetone and add fresh acetone to a 50 mL conical tube and shake for 2-3 hours. Discard the acetone. If desired, autoclave on liquid cycle in water. Rinse the coverslips twice with 100% ethanol. Store the coverslips in fresh 100% ethanol at 4°C.

[0135] Day 1: Plating ESCs in single cell suspension for differentiation (Coating plates for cell pellets (RNAseq analysis) or staining coverslips) After washing, transfer the coverslips with forceps into a 24- or 4-well plate and dry in a sterile biosafety cabinet hood until the alcohol has completely evaporated. Prior to plating the cells, coat the coverslips with Matrigel in the plate for 1 hour. Coat 24-well or 4-well plates with Matrigel for 1 hour before plating the cells.

[0136] (Monolayer cell suspension) Wash the 60mm Petri dish to remove any differentiating colonies. Wash with 2 ml of DPBS buffer. Add 0.5ml of Accutase and place in incubator for 5-10 minutes. Add 2 to 5 ml of DMEM / F12 medium to stop dissociation. Centrifuge at 1000 rpm for 3 minutes to pellet the cells. · Resuspend in 5 ml mTESR medium containing 10 μM Rock inhibitor and count the cells. Plate 10,000 cells per well in a 24-well Matrigel-coated plate.

[0137] (ESC cell harvest on day 0 as control): · hESC cells are harvested on day 0. Wash the cells with 0.5 ml of DPBS buffer per well of the 24-well plate. Add 0.5ml of Accutase and place the plate in an incubator for 3-5 minutes. Add 1 ml of DMEM / F12 medium to stop the digestion reaction. Centrifuge at 10,000g for 2 minutes. Aspirate the medium. -Snap freeze in liquid N2 and store at -80°C until further use. · Cells were fixed with 4% PFA for 20 min, washed three times with DPBS, and stored in DPBS at 4°C until use.

[0138] (cells used to initiate differentiation) Remove the mTESR medium containing the ROCK inhibitor and add 0.5 ml of APEL2 medium + CHIR 3 μM. Change the medium every 2 days.

[0139] (Day 2) The medium is replaced with APEL2 medium + CHIR 3 μM.

[0140] (Day 4: premesoderm) · Replace the medium with 200ng / ml FGF9 + 1μg / ml heparin. Change the medium every 2 days.

[0141] (Day 6) · Replace the medium with 200ng / ml FGF9 + 1μg / ml heparin.

[0142] (Day 7 (intermediate mesoderm)) Some wells are harvested on day 7 for analysis. Wash the cells with 0.5 ml of DPBS buffer per well of the 24-well plate. Add 0.5ml of Accutase and place the plate in an incubator for 3-5 minutes. · Stop the reaction by adding 1 ml of DMEM / F12 medium. Centrifuge at 10,000g for 2 minutes. Aspirate the above medium. · Snap freeze in liquid N2 and store at -80°C until further use (intermediate mesoderm). · Cells were fixed with 4% PFA for 20 min, washed three times with DPBS, and stored in DPBS at 4°C until use. · Continue the differentiation protocol in the remaining wells: in these wells, remove growth factors and replace medium with APEL only.

[0143] (Day 9) · Replace only the APEL medium mentioned above.

[0144] (10th day (genital ridge)) Some wells are harvested on day 10 for analysis. Wash the cells with 0.5 ml of DPBS buffer per well of the 24-well plate. Add 0.5ml of Accutase and place the plate in an incubator for 3-5 minutes. · Stop the reaction by adding 1 ml of DMEM / F12 medium. Centrifuge at 10,000g for 2 minutes. Aspirate the medium. -Snap freeze in liquid N2 and store at -80°C until further use. · Cells were fixed with 4% PFA for 20 min, washed three times with DPBS, and stored in DPBS at 4°C until use. The remaining wells receive different combinations of growth factors (medium is changed every 2 days): Combo: APEL+17nM IGF1+100nM insulin Combo: APEL+17nM IGF1+100nM insulin+200ng / ml FGF9. Combo:APEL+0.1μM RA+100nM insulin+17nM IGF1+500ng / ml PGD2+200ng / ml FGF9+100ng / ml FSH+20ng / ml LH+1uM testosterone. Combo:APEL+0.1μM RA+100nM Insulin+17nM IGF1+500ng / ml PGD2+1ng / ml FGF9+10ng / ml BMP4+50ng / ml EGF+100ng / ml FSH+20ng / ml LH+1μM Testosterone. Combo:APEL+0.1μM RA+100nM Insulin+17nM IGF1+500ng / ml PGD2+1ng / ml FGF9+10ng / ml BMP4+50ng / ml EGF+2μM IWR1+100ng / ml FSH+20ng / ml LH+1μM Testosterone.

[0145] (Day 12) Change medium every 2 days and add growth factors: Combo: APEL+17nM IGF1+100nM insulin Combo: APEL+17nM IGF1+100nM insulin+200ng / ml FGF9. Combo:APEL+0.1μM RA+100nM insulin+17nM IGF1+500ng / ml PGD2+200ng / ml FGF9+100ng / ml FSH+20ng / ml LH+1μM testosterone. Combo:APEL+0.1μM RA+100nM Insulin+17nM+IGF1+500ng / ml PGD2+1ng / ml FGF9+10ng / ml BMP4+50ng / ml EGF+100ng / ml FSH+20ng / ml LH+1uM Testosterone. Combo:APEL+0.1μM RA+100nM Insulin+17nM+IGF1+500ng / ml PGD2+1ng / ml FGF9+10ng / ml BMP4+50ng / ml EGF+2μM IWR1+100ng / ml FSH+20ng / ml LH+1μM Testosterone.

[0146] (Day 14) Some wells are harvested on day 14 for analysis. Wash the cells with 0.5 ml of DPBS buffer per well of the 24-well plate. Add 0.5ml of Accutase and place the plate in an incubator for 3-5 minutes. · Stop the reaction by adding 1 ml of DMEM / F12 medium. Centrifuge at 10,000g for 2 minutes. Aspirate the medium. -Snap freeze in liquid N2 and store at -80°C until further use. · Cells were fixed with 4% PFA for 20 min, washed three times with DPBS, and stored in DPBS at 4°C until use.

[0147] (Differentiation state). The remaining wells will have the medium changed and growth factors added every 2 days. Combo: APEL+17nM IGF1+100nM insulin Combo: APEL+17nM IGF1+100nM insulin+200ng / ml FGF9. Combo:APEL+0.1μM RA+100nM insulin+17nM IGF1+500ng / ml PGD2+200ng / ml FGF9+100ng / ml FSH+20ng / ml LH+1μM testosterone. Combo:APEL+0.1μM RA+100nM Insulin+17nM IGF1+500ng / ml PGD2+1ng / ml FGF9+10ng / ml BMP4+50ng / ml EGF+100ng / ml FSH+20ng / ml LH+1μM Testosterone. Combo:APEL+0.1μM RA+100nM Insulin+17nM IGF1+500ng / ml PGD2+1ng / ml FGF9+10ng / ml BMP4+50ng / ml EGF+2μM IWR1+100ng / ml FSH+20ng / ml LH+1μM Testosterone.

[0148] (Day 16) Change medium every 2 days and add growth factors: Combo: APEL+17nM IGF1+100nM insulin Combo: APEL+17nM IGF1+100nM insulin+200ng / ml FGF9. Combo:APEL+0.1μM RA+100nM insulin+17nM IGF1+500ng / ml PGD2+200ng / ml FGF9+100ng / ml FSH+20ng / ml LH+1μM testosterone. Combo:APEL+0.1μM RA+100nM Insulin+17nM IGF1+500ng / ml PGD2+1ng / ml FGF9+10ng / ml BMP4+50ng / ml EGF+100ng / ml FSH+20ng / ml LH+1uM Testosterone. Combo:APEL+0.1μM RA+100nM Insulin+17nM IGF1+500ng / ml PGD2+1ng / ml FGF9+10ng / ml BMP4+50ng / ml EGF+2μM IWR1+100ng / ml FSH+20ng / ml LH+1μM Testosterone.

[0149] (Day 18 (differentiated cells)) · Cells are harvested on day 18. · Wash the cells with 0.5 ml of DPBS buffer per well of the 24-well plate. Add 0.5ml of Accutase and place the plate in an incubator for 3-5 minutes. · Stop the reaction by adding 1 ml of DMEM / F12 medium. Centrifuge at 10,000g for 2 minutes. Aspirate the medium. -Snap freeze in liquid N2 and store at -80°C until further use (differentiated state). · Fix cells with 4% PFA for 20 min and wash three times with DPBS.

[0150] (Cover Slip Staining Protocol) Fix cells with 4% PFA for 20 minutes. Wash three times for 5 min with PBS- / - and store at 4°C in PBS- / - until staining. · Wash quickly with PBS- / - before staining three times. Add 200 μl to a 24-well plate and incubate at room temperature in a lidded humidity chamber for 1 hour. Blocking buffer: PBS+ / +, 0.5% Triton-X 100, 5% donkey or goat serum, and 10% Na-Azide (8.85ml PBS+ / +, 50μl Triton-x 100+1ml donkey serum or goat serum+100μl Na-Azide). Add 200ul of primary Ab and leave overnight in a 4℃ humidor with a lid. · Dilute the above blocking buffer to 50% for primary antibody buffer (PBS+ / +, 0.25% Triton-x, 2.5% donkey or goat serum + 50ul NA-Azide). The next day, wash the slides with PBS- / - and 0.5% Triton-X for 10 min each. Add 200μl of secondary Ab with DAPI and leave in a covered humidity chamber at room temperature for 1.5 hours. (PBS+ / +, 1ml donkey serum + 100ul Na-Azide) Wash with PBS- / - for 10 min each. Mount on coverslips with Vecta Shield.

[0151] [result] We began optimizing the differentiation protocol with two male human ESC lines (U6 and H1). The differentiation schema corresponding to the above protocol is shown in Figure 10A. Briefly, the protocol describes the differentiation of hESCs into presomal mesoderm (BRA / T, TBX6, GATA3), anterior mesoderm (LHX1, GATA3, PAX2), and gonadal primordium cells (GAT4, WT1, EMX2), as well as into Sertoli cells (SOX9, WT1, SRY, Inhibin B, FSHR) and interstitial cells (WT1, SF1, COUPTFII) with a differentiation efficiency of 40-50%. Unlike mouse, differentiation of human ESCs takes time (~18 days), and only a few organoids spontaneously self-organized by day 18 in 2D culture. We dissociated the cells on day 18, re-plated them in Aggrawel, and were able to generate hundreds of organoids by day 22. These organoids express multiple gonadal and Sertoli markers, but unlike mouse ESC-derived organoids, they formed few organized tubules (Figure 11). Encouragingly, the human differentiation scheme shows little off-target expression. To assess the developmental stage of cultured cells, we performed scRNAseq analysis across days of differentiation. We identified 13 clusters (Fig. S12A), with “early” clusters at d4 or d7 and others appearing “late” at d10, d18, or d22 (Fig. S12B; left panel). We compared cells per day or cluster with in vivo scRNAseq data of embryonic gonadal cells recently reported by two other groups (14, 15). First, early and late enriched cell clusters in our hESC cultures correlated increasingly with several differentiated cell types identified by the Clark group (14), such as Sertoli interstitial precursors, interstitial Leydig cell precursors, and smooth muscle subtypes (Fig. S12B; right panel). Second, when we compared our daily cell centroids to the cell types identified in vivo by the Roser group (15), we observed that cells emerging at later stages of culture (e.g., days 18 and 22) showed increasing similarity to bone marrow epithelial-like, mesenchymal stromal, and supporting stromal cells (Figure S12C). Although many of the annotated cell names differ in the Clark and Roser dataset, the comparative analysis presented above strongly suggests that our in vitro differentiation protocol advanced ESCs and generated multiple spermioid cell progenitors.

[0152] Despite this exciting progress, human differentiation protocols have mainly produced progenitor-like cells, some of which have progressed further to become terminally differentiated gonadal cells. Indeed, the cells in our late-stage cultures resembled in vivo somatic cells found at 6-7 weeks of gestation. To drive further differentiation in human cell cultures, we decided to supplement the ALL-GF differentiation cocktail with the hormones testosterone, LH, and FSH (Figure 13A). Surprisingly, while the addition of hormones to mouse cells did not affect differentiation at day 8 (not shown), it significantly improved human differentiation efficiency, and accelerated differentiation rates allowed the detection of many terminal differentiation markers by day 13 (Figure 13A). RT-qPCR analysis of gonadal, Sertoli, and Leydig cell markers showed that they were increased in the presence of hormones at days 10 and 13 in both U6 and H1 embryonic stem cells (Figure 13B). Furthermore, immunofluorescence staining of both U6 and H1 confirmed that testicular somatic cell-like markers were more strongly induced in the organoids (Figure 14C). Based on these findings, we decided to add hormones to human organoid differentiation. Immunofluorescence analysis of organoids on day 22 confirmed the presence of many testicular markers.

[0153] We next tested the ability of these in vitro-derived cells to host and allow the survival of germ cells. We generated PGCLCs (SOX17+; TFAP2C+) and combined them with in vitro-derived somatic cells on day 8. We found that germ cells were incorporated into the organoids, as in mice, and continued to proliferate for at least 5 days in culture (Figure 15).

[0154] Taken together, our data demonstrate that we have successfully generated human somatic cells using two ESC lines, and furthermore, our in vitro derived somatic cells can host and support the proliferation of germ cells in the absence of the PGC culture cocktail.

[0155] To be more clinically relevant, we next tested whether the ESC differentiation schema could be directly applied to the differentiation of induced pluripotent stem cells into testicular somatic cells. We applied exactly the same regimen used for ESCs, but unfortunately the efficiency was significantly lower. Unexpectedly, the initial differentiation step of IPSCs required a higher concentration of Chiron (8 μM) than that used for ESCs (3 μM), and we found that the ideal stopping point of differentiation was 10 days. Beyond 10 days of gonadal differentiation, the number of gonad cells decreased (Figure 16).

[0156] [References] 1. Jemal, A. et al. Cancer statistics, 2004. CA Cancer J Clin 54, 8-29, doi:10.3322 / canjclin.54.1.8 (2004). 2.Gratwohl, A. et al. Hematopoietic stem cell transplantation: a global perspective. JAMA 303, 1617-1624, doi:10.1001 / jama.2010.491 (2010). 3.Wallace, WH Oncofertility and preservation of reproductive capacity in children and young adults. Cancer 117, 2301-2310, doi:10.1002 / cncr.26045 (2011). 4.Lambertini, M. et al. Cancer and fertility preservation: international recommendations from an expert meeting. BMC Med 14, 1, doi:10.1186 / s12916-015-0545-7 (2016). 5.Nieman, C. L. et al. Fertility preservation and adolescent cancer patients: lessons from adult survivors of childhood cancer and their parents. Cancer Treat Res 138, 201-217, doi:10.1007 / 978-0-387-72293-1_15 (2007). 6.Hayashi, K., Ohta, H., Kurimoto, K., Aramaki, S. & Saitou, M. Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 146, 519-532, doi:10.1016 / j.cell.2011.06.052 (2011). 7.Hayashi, K. et al. Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice. Science 338, 971-975, doi:10.1126 / science.1226889 (2012). 8.Ishikura, Y. et al. in vitro reconstitution of the whole male germ-cell development from mouse pluripotent stem cells. Cell Stem Cell, doi:10.1016 / j.stem.2021.08.005 (2021). 9.Yamashiro, C. et al. Generation of human oogonia from induced pluripotent stem cells in vitro. Science 362, 356-360, doi:10.1126 / science.aat1674 (2018). 10.Sosa, E. et al. Differentiation of primate primordial germ cell-like cells following transplantation into the adult gonadal niche. Nat Commun 9, 5339, doi:10.1038 / s41467-018-07740-7 (2018). 11.Hwang, Y. S. et al. Reconstitution of prospermatogonial specification in vitro from human induced pluripotent stem cells. Nat Commun 11, 5656, doi:10.1038 / s41467-020-19350-3 (2020). 12.Stevant, I. et al. Dissecting Cell Lineage Specification and Sex Fate Determination in Gonadal Somatic Cells Using Single-Cell Transcriptomics. Cell reports 26, 3272-3283 e3273, doi:10.1016 / j.celrep.2019.02.069 (2019). 13.Stevant, I. et al. Deciphering Cell Lineage Specification during Male Sex Determination with Single-Cell RNA Sequencing. Cell reports 22, 1589-1599, doi:10.1016 / j.celrep.2018.01.043 (2018). 14.Guo, J. et al. Single-cell analysis of the developing human testis reveals somatic niche cell specification and fetal germline stem cell establishment. Cell Stem Cell 28, 764-778 e764, doi:10.1016 / j.stem.2020.12.004 (2021). 15.Luz Garcia-Alonso et al. Single-cell roadmap of human gonadal development. Nature, doi:doi: 10.1038 / s41586-022-04918-4.(2022). 16.Shen, Y.-c. et al. TCF21+ mesenchymal cells contribute to testis somatic cell development, homeostasis, and regeneration in mice. Nature Communications 12, 3876, doi:10.1038 / s41467-021-24130-8 (2021). 17.Yokonishi, T., McKey, J., Ide, S. & Capel, B. Sertoli cell ablation and replacement of the spermatogonial niche in mouse. Nat Commun 11, 40, doi:10.1038 / s41467-019-13879-8 (2020).

[0157] All publications, patents, patent applications and accession numbers mentioned in the above specification are incorporated herein by reference in their entirety.Although the present invention has been described in connection with specific embodiments, it should be understood that the invention claimed should not be unduly limited to such specific embodiments.Indeed, various modifications and variations of the described compositions and methods of the present invention are obvious to those skilled in the art and are intended to be within the scope of the following claims. [Brief description of the drawings]

[0158] [Figure 1] Schematic diagram showing the developmental process of testicular somatic cells and key markers that identify key stages. [Figure 2A-C] In vitro generation of testicular cells and the resulting organoids. (A) Schematic of the experimental method used to generate the organoids. (B-D) Data from RT-qPCR analysis of different markers along the differentiation time course. [Figure 2D] As mentioned above. [Figure 3A-B] Micrographs showing the expression dynamics of pluripotency and gonadal markers during our mouse ESC differentiation trajectory. [Figure 3C] As mentioned above. [Figure 4A-F]In vitro derived somatic cells resemble embryonic gonad cells in vivo. A) Single cell RNA-seq time course analysis of in vitro differentiation (days 0, 2, 4, 8) identified 8 clusters visualized in UMAP space. Note: Shown is one experiment, but 9 successful in vitro differentiations. B) Cells harvested on days 0, 2, 4, and 8 contribute to 8 clusters, showing emergence of new cell types at later days. (C). Expression profile of selected specific and non-specific (D) gonadal differentiation markers. (E) Correlation of our scRNAseq daily centroids with published in vivo daily cell centroids. (F) Daily cell counts per cluster in our data (left) and correlation of our 8 cluster centroids with 4 in vivo cell types (right). [Diagram 5] Schematic diagram showing the strategy of combining in vitro derived somatic cells with in vivo derived Oct4-egfp precursor spermatogonia. [Figure 6] Micrographs showing that our in vitro induced somatic cells entrap progenitor spermatogonia and promote their differentiation in vitro. In vitro induced somatic-like cells are mixed with purified Oct4+ prospermatogonia harvested from the testes of PND1-2 Oct4-egfptg / + mice. During the experiment, a subset of Oct4-egfp+ prospermatogonia maintains expression of a known undifferentiated spermatogonia marker (known as PLZF), but after 4 days of co-culture, a subset of cells transitions to an OCT4-dim or OCT4-low state and acquires strong expression of Stra8. [Figure 7] Improved somatic cell differentiation protocol. A) Schematic of somatic cell generation including the most promising four drug combination. B) Expression of testicular somatic cell markers in all three cocktails described in 7A. C) Renal differentiation markers. D) Granulosa cell markers. [Figure 8] Photomicrographs showing representative staining patterns for gonadal markers obtained from ALL-GF organoids compared with other culture conditions. [Figure 9]Graph of data showing that testicular cells derived from cocktail #3 improve stem cell expansion on days 3 and 5 of in vitro co-culture. Germ cell numbers are determined by quantifying the number of EGFP+ germline stem cells. The starting numbers of somatic and germ cells are the same for all combinations. [Figure 10] Generation of human testicular somatic-like cells. A) Optimized differentiation scheme used for H1 and U6 hESC lines. B) Graph showing tracking of relative RNA expression of several cell type specific markers over the differentiation trajectory. [Figure 11] Micrographs showing that our differentiation protocol induces expression of key protein markers across the differentiation trajectory, with results similar for H1 and U6 lines. [Figure 12] Data show that scRNAseq analysis identified 13 clusters, a subset of which emerged at later stages of culture and resembled known differentiated cells observed in vivo in human gonadal tissue. [Figure 13] Improved differentiation efficiency of human somatic-like cells in the presence of hormones. A) Modified differentiation scheme. B) qPCR expression of gonadal, Sertoli, Leydig, and off-target markers. [Figure 14] Representative images of gonadal markers at day 22 from both H1 ESC lines. [Figure 15] Photomicrograph showing in vitro derived organoids consisting of germ and somatic cells after 5 days of mixed culture. [Figure 16A] Graphs showing data related to validation of iPSC differentiation schema and gonadal cell generation. [Figure 16B] As mentioned above.

Claims

Claim 1: An in vitro method for producing Sertoli cells from vertebrate pluripotent stem cells, preferably wherein said vertebrate pluripotent stem cells are human embryonic stem cells (hESCs), said method comprising the steps of: Inducing gonadal ridge cells from pluripotent stem cells; treating the genital ridge cells with a basal medium containing fibroblast growth factor 9 (FGF9), insulin, and / or IGF1, thereby causing the genital ridge cells to differentiate into Sertoli cells; Preferably, the step of inducing the genital ridge cells further comprises the steps of: providing vertebrate pluripotent stem cells in a maintenance medium containing a ROCK inhibitor; on day 0, removing the maintenance medium containing a ROCK inhibitor and culturing the vertebrate pluripotent stem cells in the basal medium containing CHIR99021, thereby allowing the vertebrate pluripotent stem cells to differentiate into premesodermal cells; On about day 4, removing the basal medium containing CHIR99021 and culturing the promesodermal cells in a basal medium containing fibroblast growth factor 9 (FGF9) and heparin, thereby allowing the promesodermal cells to differentiate into intermediate mesodermal cells; and / or on about day 7, removing the medium containing FGF9 and heparin and culturing the cells; and / or Preferably, the step of treating the genital ridge cells with a culture medium containing fibroblast growth factor 9 (FGF9), insulin and / or IGF1 to differentiate the genital ridge cells into Sertoli cells further comprises, on about day 7 to 10, removing the basal medium and culturing the genital ridge cells in a basal medium containing insulin-like growth factor 1 (IGF1), insulin and FGF9 to differentiate the genital ridge cells into artificial Sertoli cells; and / or Preferably, the step of treating the genital ridge cells with a basal medium containing fibroblast growth factor 9 (FGF9), insulin and / or IGF1 to cause the genital ridge cells to differentiate into Sertoli cells further comprises treating the genital ridge cells with epidermal growth factor (EGF), bone morphogenetic protein 4 (BMP4), IWR1, or a combination thereof; and / or Preferably, the step of treating the genital ridge cells with a basal medium containing fibroblast growth factor 9 (FGF9), insulin and / or IGF1 to cause the genital ridge cells to differentiate into Sertoli cells further comprises treating the genital ridge cells with follicle-stimulating hormone (FSH) and / or luteinizing hormone and / or testosterone or a combination thereof. Claim 2: An in vitro method for producing Sertoli cells from vertebrate pluripotent stem cells, preferably wherein said vertebrate pluripotent stem cells are human embryonic stem cells (hESCs), said method comprising the steps of: Inducing pre-intermediate mesoderm cells from pluripotent stem cells; treating the pre-intermediate mesoderm cells with a basal medium containing insulin and / or IGF1, thereby causing the pre-intermediate mesoderm cells to differentiate into Sertoli cells; The step of inducing anterior intermediate mesoderm cells further comprises the steps of: providing vertebrate pluripotent stem cells in a maintenance medium containing a ROCK inhibitor; On day 0, removing the maintenance medium containing a ROCK inhibitor and culturing the vertebrate pluripotent stem cells in the basal medium containing CHIR99021, thereby allowing the vertebrate pluripotent stem cells to differentiate into premesodermal cells; and and / or, on about day 4, removing the basal medium containing CHIR99021 and culturing the premesodermal cells in a basal medium containing fibroblast growth factor 9 (FGF9) and heparin, thereby allowing the premesodermal cells to differentiate into intermediate mesodermal cells. and / or the step of treating the intermediate mesodermal cells with a culture medium containing insulin and / or IGF1 to cause the intermediate mesodermal cells to differentiate into Sertoli cells further comprises, on about day 7, removing the medium containing FGF9 and heparin and culturing the intermediate mesodermal cells in a basal medium containing insulin-like growth factor 1 (IGF1) and insulin to cause the intermediate mesodermal cells to differentiate into artificial Sertoli cells; the step of treating the pre-intermediate mesoderm cells with a basal medium containing insulin and / or IGF1 further comprises treating the genital ridge cells with FGF9, epidermal growth factor (EGF), bone morphogenetic protein 4 (BMP4), IWR1, or a combination thereof; and / or The method, wherein the step of treating the pre-intermediate mesoderm cells with a basal medium containing insulin and / or IGF1 further comprises treating the gonadal ridge cells with follicle-stimulating hormone (FSH) and / or luteinizing hormone and / or testosterone or a combination thereof.

3. The step of inducing anterior intermediate mesoderm cells further comprises the steps of: providing vertebrate pluripotent stem cells in a maintenance medium comprising LIF; On day 0, removing the maintenance medium containing LIF and culturing the vertebrate pluripotent stem cells in the basal medium containing activin A and bFGF, thereby allowing the vertebrate pluripotent stem cells to differentiate into epidermal cells; and and / or on about day 2, removing the basal medium comprising activin A and bFGF and culturing the epidermal cells in a basal medium comprising activin A and RA, thereby allowing the epidermal cells to differentiate into intermediate mesodermal cells; The method of claim 2, wherein the step of treating the intermediate mesoderm cells with a culture medium containing insulin and / or IGF1 to cause the intermediate mesoderm cells to differentiate into Sertoli cells further comprises removing the medium containing activin A and RA on about day 4 and culturing the intermediate mesoderm cells in a basal medium containing insulin-like growth factor 1 (IGF1) and insulin so that the intermediate mesoderm cells differentiate into artificial Sertoli cells.

4. The method described in claim 2 or 3, wherein the vertebrate pluripotent stem cells are mouse embryonic stem cells (mESCs).

5. the artificial Sertoli cells express at least one marker selected from the group consisting of EMX2, WT, SOX9, and LHX9; or the artificial Sertoli cells express at least two markers selected from the group consisting of EMX2, WT, SOX9, and LHX9; or the artificial Sertoli cells express at least three markers selected from the group consisting of EMX2, WT, SOX9, and LHX9; or The method of claim 1, wherein the artificial Sertoli cells express the markers EMX2, WT, SOX9 and LHX9.

6. The basal medium is APEL2 medium; or The method according to any one of claims 1 to 2, wherein the maintenance medium is mTESR medium.

7. The method according to any one of claims 1 to 2, wherein the basal medium is a DMEM medium.

8. The basal medium containing insulin-like growth factor 1 (IGF1) and insulin is ２ The method of any one of claims 1 to 2, further comprising one or more of, preferably a combination of, FGF1 and FGF2.

9. further comprising culturing the artificial Sertoli cells under conditions such that the artificial Sertoli cells form artificial Sertoli cell organoids, preferably characterized by having tubule structures, and / or preferably the artificial Sertoli cell organoids are further characterized by comprising smooth muscle actin; and optionally The method according to any one of claims 1 to 2, further comprising the step of isolating the artificial Sertoli cells or artificial Sertoli cell organoids.

10. A cell culture comprising artificial Sertoli cells produced by the method of any one of claims 1 to 2.

11. 10. An isolated artificial Sertoli cell produced by the method of claim 9.

12. 10. An artificial Sertoli cell organoid produced by the method of claim 9.

13. A method comprising the steps of: Providing artificial Sertoli cells or artificial Sertoli cell organoids isolated by the step of isolating the artificial Sertoli cells or artificial Sertoli cell organoids according to the method of claim 9; contacting the artificial Sertoli cells or organoids with a testing reagent; and Evaluating the effect of the test reagent on the artificial Sertoli cells or organoids.

14. An artificial Sertoli cell as described in claim 11 for use in transplantation into a subject.

15. An artificial Sertoli cell organoid as described in claim 12 for use in transplantation into a subject.

16. The following steps: Providing artificial Sertoli cells or artificial Sertoli cell organoids isolated by the step of isolating the artificial Sertoli cells or artificial Sertoli cell organoids according to the method of claim 9; and Culturing said artificial Sertoli cells with stem cells or stem cell-containing tissue from a patient, preferably wherein said stem cells are prespermatogonia or spermatogonial stem / progenitor cells (SSC / SPC), and preferably The method wherein said prespermatogonial stem cells differentiate into spermatogonia.

17. Stem cells or differentiated spermatogonia as defined in claim 16 for use in retransplantation into a patient in need thereof, preferably comprising: Stem cells or differentiated spermatogonia, wherein said patient has previously undergone gonadotoxic treatment, preferably said gonadotoxic treatment is selected from the group consisting of chemotherapy and radiation therapy.

18. 17. The method of claim 16, wherein the stem cells or tissue containing stem cells are from a patient prior to gonadotoxic treatment.

19. A kit comprising a plurality of containers, at least one container contains a basal medium comprising fibroblast growth factor 9 (FGF9) and heparin; at least one container contains a basal medium comprising insulin-like growth factor 1 (IGF1) and insulin; the kit preferably further comprises at least one container containing an inhibitor of ROCK I and / or ROCK II in a maintenance medium; and / or the kit preferably further comprises at least one container having CHIR99021; and / or The kit preferably further comprises at least one container having BMP4; and / or The kit preferably further comprises at least one container with EGF; and / or the kit preferably further comprises at least one container with FSH; and / or The kit preferably further comprises at least one container with LH; and / or the kit preferably further comprises at least one container with testosterone; and / or The kit preferably further comprises at least one container having IWR1; and / or The kit preferably further comprises instructions for carrying out the method according to any one of claims 1-2.

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