Patent Application
20250180544
The present invention relates to in vitro methods for production of Sertoli cells and related organoids.
Infertility is a rapidly rising crisis worldwide. One percent of all reproductive age couples (ages 20-50) globally suffer from infertility, and in roughly 50% of cases the cause is male factor infertility. In the US, 300,000 men have nonobstructive azoospermia (i.e., lacking any germ or mature sperm cells) and most are of unknown genetic origin. Traditional treatment options, such as in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), require sperm, and leave these men with no therapies. In addition to adult infertility, each year in the US ˜10,000 prepubertal boys develop cancers and require gonadotoxic treatments, such as chemotherapy and radiation (1). Furthermore, ˜1,000 pediatric patients with blood and immune deficiencies and autoimmune disorders receive myeloablative conditioning prior to bone marrow transplantation, which is also gonadotoxic (2). These treatments—with alkylating chemotherapeutic agents, total body irradiation (3), and gonadal radiation (4)—put patients at a significant risk of infertility due to damage to both somatic and germ cell populations in the testis. However, with the advancements in medicine 85% of these children will be cured and, upon reaching adulthood they desire to have their own children (5). Since gametes are needed for any reproductive therapy, developing methods to reconstitute germ cell development in vitro or in vivo for these patients remains a clinical challenge, and will continue to be the focus of the reproductive biology community for the coming decades. Successful generation of patient-derived, testis-like somatic cells in vitro will provide a novel cell-based therapy for genetic and iatrogenic (i.e., induced by treatment) forms of male infertility. Such a treatment is also applicable to restore endocrine function in aging men, those with hypogonadism, or patients with gender dysphoria. Further, if in vitro derived cells or cell communities (sometimes called organoids) can recapitulate the natural spermatogenesis processes, they can be used as a new experimental system to screen for new classes of male contraceptives.
The current race for cell-based treatment has centered on the development of germ cell-like precursors known as primordial germ cell-like cells (PGCLCs). While PGCLCs have been derived in vitro from mouse and human embryonic stem cells (ESCs), they cannot be maintained in vitro (6). Only when transplanted into mouse neonatal testis can these mouse PGCLCs develop into sperm, which can then be used to make live born pups. Furthermore, when female and male mouse PGCLCs were reconstituted with fetal gonadal tissue in vitro, successful reconstitution of the oogenesis and spermatogenesis program was possible, but the frequency of live born pups obtained from the in vitro derived germ cells is roughly ˜ 1-3% (6-9). These preliminary findings are exciting and underscore the importance of somatic cells for the execution of the gametogenesis program. In similar experiments using human or rhesus PGCLCs with mouse embryonic gonadal tissue from three research groups it was shown that when the female and male human hPGCLCs or male rhesus PGCLCs were combined with mouse fetal ovarian tissue or testis tissue, respectively, the PGCLC initiated differentiation, but failed to initiate meiosis (9-11). These observations indicate that somatic and germ cell sources must be compatible (i.e., from the same species) to initiate meiosis.
Utilizing somatic cells from human fetal testis to aid the progression of in vitro derived human PGCLCs has ethical and technical limitations related to fetal tissue-based research. Therefore, to generate testis-like somatic cells without relying on fetal tissue is of clinical significance. This has previously been attempted by reprogramming human fibroblasts into Sertoli- or Leydig-like cells using a combination of well-chosen transcription factors. Despite expressing a handful of known markers for Sertoli and Leydig cells, the extent to which the in vitro derived cells resemble in vivo cells or recapitulate endogenous functions remain difficult to assess. Furthermore, given the requirement for transgenesis for efficient induction, and for the continuous expression of transcription factors for maintenance of Sertoli and Leydig cell fates, the clinical utility of such cells is unclear.
The present invention relates to in vitro methods for production of Sertoli cells and related organoids.
In some preferred embodiments, the present invention provides in vitro methods for production of Sertoli cells from vertebrate pluripotent stem cells comprising: deriving genital ridge cells from pluripotent stem cells: treating the genital ridge cells with a base medium comprising fibroblast growth factor 9 (FGF9), insulin and/or IGF1 so that the genital ridge cells differentiate into Sertoli cells.
In some preferred embodiments, the step of deriving genital ridge cells further comprises: providing vertebrate pluripotent stem cells in a maintenance medium comprising a ROCK inhibitor; at day 0, removing the maintenance medium comprising a ROCK inhibitor and culturing the vertebrate pluripotent stem cells with the base medium comprising CHIR99021 so that the vertebrate pluripotent stem cells differentiate into presomitic mesoderm cells: on about day 4, removing the base medium comprising CHIR99021 and culturing the presomitic mesoderm cells in base medium comprising fibroblast growth factor 9 (FGF9) and heparin so that the presomitic mesoderm cells differentiate into intermediate mesoderm cells: on about day 7, removing the medium comprising FGF9 and heparin and culturing the intermediate mesoderm cells in base medium so that the intermediate mesoderm cells differentiate into genital ridge cells.
In some preferred embodiments, the step of treating the genital ridge cells with a culture medium comprising insulin and/or IGF1 so that the genital ridge cells differentiate into Sertoli cells further comprises on about day 10, removing the base medium and culturing the genital ridge cells in base medium comprising insulin-like growth factor 1 (IGF1) and insulin so that the genital ridge cells differentiate into artificial Sertoli cells.
In some preferred embodiments, step of treating the genital ridge with a base medium comprising fibroblast growth factor 9 (FGF9), insulin and/or IGF1 so that the genital ridge cells differentiate into Sertoli cells further comprises treating the genital ridge cells with epidermal growth factor (EGF), bone morphogenetic protein 4 (BMP4). IWR1, or combinations thereof. In some preferred embodiments, the step of treating the genital ridge with a base medium comprising fibroblast growth factor 9 (FGF9), insulin and/or IGF1 so that the genital ridge cells 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.
In some preferred embodiments, the vertebrate pluripotent stem cells are human embryonic stem cells (hESC).
In some preferred embodiments, the present invention provides in vitro methods for production of Sertoli cells from vertebrate pluripotent stem cells comprising: deriving anterior intermediate mesoderm cells from pluripotent stem cells: treating the anterior intermediate mesoderm cells with a base medium comprising insulin and/or IGF1 so that the anterior intermediate mesoderm cells differentiate into Sertoli cells.
In some preferred embodiments, the step of deriving anterior intermediate mesoderm cells further comprises: providing vertebrate pluripotent stem cells in a maintenance medium comprising a ROCK inhibitor: at day 0, removing the maintenance medium comprising a ROCK inhibitor and culturing the vertebrate pluripotent stem cells with the base medium comprising CHIR99021 so that the vertebrate pluripotent stem cells differentiate into presomitic mesoderm cells; and on about day 4, removing the base medium comprising CHIR99021 and culturing the presomitic mesoderm cells in base medium comprising fibroblast growth factor 9 (FGF9) and heparin so that the presomitic mesoderm cells differentiate into intermediate mesoderm cells.
In some preferred embodiments, the step of treating the intermediate mesoderm cells with a culture medium comprising insulin and/or IGF1 so that the intermediate mesoderm cells differentiate into Sertoli cells further comprises on about day 7, removing the medium comprising FGF9 and heparin and culturing the intermediate mesoderm cells in base medium comprising insulin-like growth factor 1 (IGF1) and insulin so that the intermediate mesoderm cells differentiate into artificial Sertoli cells.
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 combinations 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 genital ridge cells with follicle stimulating hormone (FSH) and/or luteinizing hormone (LH) and/or testosterone or a combination thereof. In some preferred embodiments, the one or more hormones are added on about day 9.
In some preferred embodiments, the vertebrate pluripotent stem cells are human embryonic stem cells (hESC).
In some preferred embodiments, the step of deriving anterior intermediate mesoderm cells further comprises: providing vertebrate pluripotent stem cells in a maintenance medium comprising LIF; at day 0, removing the maintenance medium comprising LIF and culturing the vertebrate pluripotent stem cells with the base medium comprising Activin A and bFGF so that the vertebrate pluripotent stem cells differentiate into epiblast cells; and on about day 2, removing the base medium comprising Activin A and bFGF and culturing the epiblast cells in base medium comprising Activin A and RA so that the epiblast cells differentiate into intermediate mesoderm cells.
In some preferred embodiments, the step of treating the intermediate mesoderm cells with a culture medium comprising insulin and/or IGF1 so that the intermediate mesoderm cells differentiate into Sertoli cells further comprises on about day 4, removing the medium comprising Activin A and RA and culturing the intermediate mesoderm cells in base medium comprising insulin-like growth factor 1 (IGF1) and insulin so that the intermediate mesoderm cells differentiate into artificial Sertoli cells. In some preferred embodiments, the vertebrate pluripotent stem cells are mouse embryonic stem cells (mESC).
In some preferred embodiments, the artificial Sertoli cells produced by any of the foregoing methods express at least one of the markers selected from the group consisting of EMX2, WT, SOX9, and LHX9. In some preferred embodiments, the artificial Sertoli cells express at least two of the markers selected from the group consisting of EMX2, WT, SOX9, and LHX9. In some preferred embodiments, the artificial Sertoli cells express at least three of the 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.
In some preferred embodiments, the base medium utilized in the foregoing methods is an APEL2 medium. In some preferred embodiments, the maintenance medium is a mTESR medium. In some preferred embodiments, the base medium is a DMEM medium.
In some preferred embodiments, the base medium comprising insulin-like growth factor 1 (IGF1) and insulin further in any of the foregoing methods comprises one or more of retinoic acid, PDG2, and FGF9, and preferably a combination thereof.
In some preferred embodiments, the vertebrate pluripotent stem cells utilized in any of the foregoing methods comprise an exogenous gene.
In some preferred embodiments, the methods described above further comprise the step of culturing the artificial Sertoli cells under conditions such that the artificial Sertoli cells form an artificial Sertoli cell organoid. In some preferred embodiments, the cells are first dissociated. In some preferred embodiments, the dissociated cells are further cultured in an aggrewell plate.
In some preferred embodiments, the artificial Sertoli cell organoid is characterized by having a tubule structure. In some preferred embodiments, the artificial Sertoli cell organoid is further characterized by comprising smooth muscle actin.
In some preferred embodiments, the methods described above further comprise the step of isolating the artificial Sertoli cells or artificial Sertoli cell organoid.
In some preferred embodiments, the methods described above further comprise transplanting the isolated artificial Sertoli cells or artificial Sertoli cell organoid into a mammal.
In some preferred embodiments, the methods described above further comprise contacting the artificial Sertoli cells or artificial Sertoli cell organoid with a test reagent and assaying the effect of the test reagent on the artificial Sertoli cells or artificial Sertoli cell organoid.
In some preferred embodiments, the patient prior to gonadotoxic treatment (chemotherapy and radiation) preserves testicular tissue or somatic cells so that germline stem cells may be produced or isolated in the future. In some preferred embodiments, the germ line stem cells obtained from the tissue can be expanded using the in vitro derived cells of the present invention. In some preferred embodiments, the methods described above further comprise obtaining fibroblast tissue to reprogram to induced pluripotent stem cells from a patient which a can be used to make autologous artificial Sertoli cells. In some preferred embodiments, these differentiated cells can be combined with germline stem cells: either primordial germ cell like cells (PGCLC), prospermatogonia (proSSC), or neonatal/adult spermatogonial stem/progenitor cells (SSC/SPCs). In some preferred embodiments, the stem cells can either proliferate or differentiate into spermatogonia or later germ cell stages. In some preferred embodiments, the methods further comprise transferring the expanded stem cells or differentiated spermatogonia back to a patient in need thereof.
In some preferred embodiments, the present invention provides a cell culture comprising artificial Sertoli cells produced by any of the methods described above.
In some preferred embodiments, the present invention provides isolated artificial Sertoli cells produced by any of the foregoing methods.
In some preferred embodiments, the present invention provides an artificial Sertoli cell organoid produced by any of the methods described above.
In some preferred embodiments, the present invention provides methods comprising: providing artificial Sertoli cells or organoids as described above; contacting the artificial Sertoli cells or organoids with a test reagent; and assaying the effect of the test reagent on the artificial Sertoli cells or organoids.
In some preferred embodiments, the present invention provides methods comprising: providing artificial Sertoli cells or organoids as described above; and transplanting the artificial Sertoli cells or organoids into a subject.
In some preferred embodiments, the present invention provides methods comprising: providing artificial Sertoli cells or organoids as described above: obtaining stem cells or tissue comprising stem cells from a patient; and culturing the stem cells or tissue comprising stem cells from the patient the artificial Sertoli cells. In some preferred embodiments, the stem cells or tissue comprising stem cells are obtained from the patient prior to a gonadotoxic treatment and stored. In some preferred embodiments, the germ line stem cells obtained from the tissue can be expanded using the in vitro derived cells of the present invention. In some preferred embodiments, the methods described above further comprise obtaining fibroblast tissue to reprogram to induced pluripotent stem cells from a patient which a can be used to make autologous artificial Sertoli cells. In some preferred embodiments, these differentiated cells can be combined with germline stem cells: either primordial germ cell like cells (PGCLC), prospermatogonia (proSSC), or neonatal/adult spermatogonial stem/progenitor cells (SSC/SPCs). In some preferred embodiments, the stem cells can either proliferate or differentiate into spermatogonia or later germ cell stages. In some preferred embodiments, the methods further comprise transferring the expanded stem cells or differentiated spermatogonia back to a patient in need thereof.
In some preferred embodiments, the present invention provides kits comprising multiple vessels, wherein at least one vessel contains base medium comprising fibroblast growth factor 9 (FGF9) and heparin, and wherein at least one vessel contains base medium comprising insulin-like growth factor 1 (IGF1) and insulin. In some preferred embodiments, the kits further comprise at least one vessel comprising an inhibitor of ROCK I and/or ROCK II in maintenance medium. In some preferred embodiments, the kits further comprise at least one vessel comprising CHIR99021. In some preferred embodiments, the kits further comprise at least one vessel comprising BMP4. In some preferred embodiments, the kits further comprise at least one vessel comprising EGF. In some preferred embodiments, the kits further comprise at least one vessel comprising IWR1. In some preferred embodiments, the kits further comprise at least one vessel comprising FSH. In some preferred embodiments, the kits further comprise at least one vessel comprising LH. In some preferred embodiments, the kits further comprise at least one vessel comprising testosterone. In some preferred embodiments, the kits further comprise instructions for performing a method as described above.
As used herein the term “stem cell” (“SC”) refers to cells that can self-renew and differentiate into multiple lineages. A stem cell is a developmentally pluripotent or multipotent cell. A stem cell can divide to produce two daughter stem cells, or one daughter stem cell and one progenitor (“transit”) cell, which then proliferates into the tissue's mature, fully formed cells. Stem cells may be derived, for example, from embryonic sources (“embryonic stem cells”) or derived from adult sources. For example, U.S. Pat. No. 5,843,780 to Thompson describes the production of stem cell lines from human embryos. PCT publications WO 00/52145 and WO 01/00650 describe the use of cells from adult humans in a nuclear transfer procedure to produce stem cell lines.
Examples of adult stem cells include, but are not limited to, hematopoietic stem cells, neural stem cells, mesenchymal stem cells, and bone marrow stromal cells. These stem cells have demonstrated the ability to differentiate into a variety of cell types including adipocytes, chondrocytes, osteocytes, myocytes, bone marrow stromal cells, and thymic stroma (mesenchymal stem cells): hepatocytes, vascular cells, and muscle cells (hematopoietic stem cells): myocytes, hepatocytes, and glial cells (bone marrow stromal cells) and, indeed, cells from all three germ layers (adult neural stem cells).
As used herein, the term “totipotent cell” refers to a cell that is able to form a complete embryo (e.g., a blastocyst).
As used herein, the term “pluripotent cell” or “pluripotent stem cell” refers to a cell that has complete differentiation versatility, e.g., the capacity to grow into any of the mammalian body's approximately 260 cell types. A pluripotent cell can be self-renewing and can remain dormant or quiescent within a tissue. Unlike a totipotent cell (e.g., a fertilized, diploid egg cell), a pluripotent cell, even a pluripotent embryonic stem cell, cannot usually form a new blastocyst.
As used herein, the term “induced pluripotent stem cells” (“iPSCs”) refers to a stem cell induced from a somatic cell, e.g., a differentiated somatic cell, and that has a higher potency than said somatic cell. iPS cells are capable of self-renewal and differentiation into mature cells.
As used herein, the term “multipotent cell” refers to a cell that has the capacity to grow into a subset of the mammalian body's approximately 260 cell types. Unlike a pluripotent cell, a multipotent cell does not have the capacity to form all of the cell types.
As used herein, the term “progenitor cell” refers to a cell that is committed to differentiate into a specific type of cell or to form a specific type of tissue.
As used herein, the term “embryonic stem cell” (“ES cell” or ESC”) refers to a pluripotent cell that is derived from the inner cell mass of a blastocyst (e.g., a 4- to 5-day-old human embryo) and has the ability to yield many or all of the cell types present in a mature animal.
As used herein the term “feeder cells” refers to cells used as a growth support in some tissue culture systems. Feeder cells may be embryonic striatum cells or stromal cells. As used herein, the term “chemically defined media” refers to culture media of known or essentially-known chemical composition, both quantitatively and qualitatively. Chemically defined media is free of all animal products, including serum or serum-derived components (e.g., albumin).
As used herein, the term “serum-free media” refers to culture media that is devoid of serum, but not necessarily of other undefined components.
Methods, kits, compositions, and systems are provided for culturing pluripotent stem cells to produce populations of cells comprising artificial Sertoli cells. In particular, culture conditions are provided that result in the generation of artificial Sertoli cells from a starting culture of human pluripotent stem cells.
These methods overcome the limitations noted in the Background and leverage genetic, evolutionary, and molecular insights gained from scRNAseq data that the inventors collected across developmental stages and multiple species to develop a new, highly efficient, directed somatic cell differentiation protocol. The inventors have tuned many parameters of the protocols on a mouse ESC and two human ESC lines and analysed scRNAseq data to confirm the progression through expected cell states along the developmental trajectories. Benchmarking and classification of the in vitro derived cell states has relied on the wealth of in vivo markers that have been previously described during gonadal differentiation (see
Somatic cells of the testis are central to testis tissue homeostasis and men's reproductive and overall health. The somatic cells provide a series of unknown growth factors and cytokines that are necessary for guiding germ cell development in vivo and required for the complete reconstitution of germline development for females in vitro or promoting the differentiation of male primordial germ cell like cells (PGCLC) to spermatogonia.
When working with mice, co-culturing of in vitro derived PGCLC with fetal somatic cells isolated from littermates or allogenic embryonic gonads is feasible, but this is costly in non-human primates and ethically inconceivable in human. Thus, an improved understanding of the somatic cell specification program and generation of alternative somatic cell sources will prove critical for reconstituting somatic cells in a petri-dish, replacement of damaged cells in vivo in response to inherited/natural occurring genetic mutations, iatrogenic agents because of cancer therapy, or to rejuvenate the aging gonads. To address this gap, the present inventors utilized single cell RNAseq data to provide a method for the generation of human artificial Sertoli cells from ESCs using the differentiation schema described below.
The methods systems, systems and kit of the present invention find use with a variety of pluripotent cells. Suitable pluripotent stem cells include, but are not limited to, embryonic stem cells, adult stem cells, and induced pluripotent stem cells. In some preferred embodiments, the pluripotent stem cells are vertebrate pluripotent stem cells. In some particularly preferred embodiments, the pluripotent stem cells are human embryonic stem cells (hESCs). In other particularly preferred embodiments, the pluripotent stem cells are mouse embryonic stem cells (mESCs).
In some preferred embodiments, the pluripotent stem cells may be genetically modified by methods known in the art so that they comprise and express one or more exogenous genes.
A concern in the culture of human ES cells is to remove, to the extent possible, undefined constituents and constituents of animal origin from ES cell culture conditions. Standardizing culture conditions minimizes the normal variations in biological materials to which the cells are exposed. Further, by avoiding the use of materials, cells, exudates or constituents of animal origin, one can avoid possible cross-species viral transmission through the culture system. Thus, utilization of chemically defined media (CDM) that avoid the use of animal products provides a baseline culture condition upon which differentiation factors may be added with predictable effects.
CDM (e.g., for hESCs) may include maintenance or basal media containing salts, vitamins, glucose and amino acids. For maintenance of human stem cells prior to the differentiation protocol, a mTeSR medium such as mTeSR1™ from StemCell Technologies may be utilized. In some embodiments, the maintenance medium preferably comprises a ROCK inhibitor such as Y27632. Maintenance medium for mouse stem cells may preferably be GMEM from ThermoFisher Scientific, preferably supplemented with LIF (Leukemia Inhibitory Factor) and optionally knockout serum. The basal differentiation medium can be any of a number of commercially available media. In some preferred embodiments, a combination of Dulbecco's Modified Eagle Medium and Hams F12 medium, sold as a combination (DMEM/F12: Invitrogen) may be utilized. In other preferred embodiments, an APEL medium may be utilized, for example, STEMdiff™ APEL™ medium from StemCell Technologies. STEMdiff™ APEL™ Medium is a serum-free and animal component-free medium specifically developed to support hPSC differentiation. It was first described for the induction of hemato-endothelial cells, when supplemented with VEGF, BMP-4, SCF, and Activin A, but it has also been proven to be an effective basal medium for hPSC differentiation to other lineages, including cardiomyocytes. In other preferred embodiments, an mTeSR medium may be utilized for maintenance of stem cells.
Differentiation into Artificial Sertoli Cells
The present invention provides methods and reagents for producing artificial Sertoli cells from pluripotent stems cells. The present invention is not limited to the use of any particular pluripotent stem cells or chemically defined media. The methods described herein for the production of artificial Sertoli cells are described in relation to events occurring at various time points. It will be recognized that the methods may be varied by making alterations to the described time schedules. “Day 0” as used herein refers to the day and time that the pluripotent stem cells are removed from a maintenance medium and exposed to a differentiation medium. The differentiation timeline is then defined from the Day 0 starting point. When the term “about” X days is utilized, it refers to the number of days from the Day 0 starting time point plus or minus 12 hours. For example, “on about Day 4” means 96 hours (i.e., 4 days) from the Day 0 starting point plus or minus 12 hours. If the Day 0 stating time was 9:00 AM, “about Day 4” then refers to 96 hours from that time point plus or minus 12 hours.
The first step in a method for producing human artificial Sertoli cells according to the invention comprises providing pluripotent hESC as described above. In some preferred embodiments, the pluripotent stem cells are provided in a stem cell maintenance medium. In some preferred embodiments, the stem cell maintenance medium is a chemically defined medium such as an mTeSR medium. In some preferred embodiments, the stem cell maintenance medium comprises a ROCK inhibitor. In some preferred embodiments, the ROCK inhibitor is Y27632.
The second step of the method of the present invention comprises removing the pluripotent stem cells from the maintenance medium and culturing the pluripotent stem cells in a basal medium supplemented with agents suitable for directing the pluripotent stem cells to a presomitic mesoderm lineage. In some preferred embodiments, the basal medium is a chemically defined medium. In some particularly preferred embodiments, the basal medium is an APEL medium such as STEMdiff™ APEL™ medium from StemCell Technologies. In some particularly preferred embodiments, the basal medium is supplemented with from between 0.5 to 15 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 therein) CHIR99021. This step defines Day 0 of the process. The base medium with supplements is preferably changed daily.
The third step of the method of the present invention comprises on about or at day 4 culturing the presomitic mesoderm cells produced in step 2 in a basal medium supplemented with agents for directing the presomitic mesoderm cells to form intermediate mesoderm. In some preferred embodiments, the basal medium is a chemically defined medium. In some particularly preferred embodiments, the basal medium is an APEL medium such as STEMdiff™ APEL™ medium from StemCell Technologies. In some particularly preferred embodiments, the basal medium is supplemented with from between 20 and 500 ng/ml (50, 100, 150, 200, 250, 300, 350, 400, 450 ng/ml and values and ranges therein) FGF 9. In some particularly preferred embodiments, the basal medium is further supplemented with from between 0.1 and 10 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 therein) heparin. The base medium with supplements may preferably be changed every two days.
The fourth step of the method of the present invention comprises on about or at day 7 culturing the intermediate mesoderm cells produced in step 3 in a basal medium to direct the intermediate mesoderm cells to form genital ridge cells. In some preferred embodiments, the basal medium is a chemically defined medium. In some particularly preferred embodiments, the basal medium is an APEL medium such as STEMdiff™ APEL™ medium from StemCell Technologies. In some particularly preferred embodiments, the basal medium is not supplemented with additional differentiation agents during this step. The base medium may preferably be changed every two days.
Optionally, the fourth step may be skipped, and the intermediate mesoderm cells may be cultured in the medium described in Step 5 on or about day 7.
The fifth step of the method of the present invention comprises on about or at day 10 (or about day 7 if the fourth step is skipped, see
However, in other additional embodiments, the base medium may be supplemented with one or more other agents. In some embodiments, the base medium may be further supplemented with from between 0.01 and 10 μM (0.05, 0.1, 1.0, 5.0, 8.0 UM and values and ranges therein) retinoic acid (RA). In some embodiments, the base medium may be further supplemented with from between 50 and 1000 ng/ml (100, 200, 300, 400, 500, 600, 700, 800, 900 and values and ranges therein) PGD2. In some embodiments, the base medium may be further supplemented with from between 50 and 500 ng/ml (100, 200, 300, 400 and values and ranges therein) FGF9. In some further preferred embodiments, the base medium used in step 5 may be further be supplemented with from 5 to 100 mg/ml (10, 20, 30, 40, 50, 60, 70, 80, 90 and values and ranges therein) bone morphogenetic protein 4 (BMP4). In some further preferred embodiments, the base medium used in step 5 may be further be supplemented with from 10 to 200 mg/ml (30, 50, 70, 100, 150 and values and ranges therein) epidermal growth factor (EGF). In some still further preferred embodiments, the base medium used in step 5 may be further be supplemented with from 0.5 to 10 μM (1.0, 2.0, 3.0, 4.0 5.0, 6.0, 7.0, 8.0, 9.0 μM and values and ranges therein) IWR1. In some preferred embodiments, a combination of insulin and IGF1 in the above ranges is utilized. In some preferred embodiments, a combination of insulin, IGF1, and FGF9) in the above ranges is utilized. In some preferred embodiments, a combination of insulin. IGF1. FGF9, RA and PGD2 in the above ranges is utilized. In some preferred embodiments, a combination of insulin, IGF1, FGF9, BMP4, EGF and/or IWR1 in the above ranges is utilized. In some preferred embodiments, a combination of insulin, IGF1, FGF9, BMP4, EGF, RA, IWR1, and PGD2 in the above ranges is utilized. The base medium with supplements may preferably be changed every two days.
In some further preferred embodiments, the base 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 at about day 9. In some preferred embodiments, the base medium (which may preferably comprise one or more of insulin, IGF1, FGF9, BMP4, EGF, RA, IWR1 and PGD2 in the above ranges) is further supplemented with from 5 to 100 mg/ml (10, 20, 30, 40, 50, 60, 70, 80, 90 and values and ranges therein) luteinizing hormone (LH). In some preferred embodiments, the base medium (which may preferably comprise one or more of insulin, IGF1, FGF9, BMP4, EGF, RA, IWR1 and PGD2 in the above ranges) is further supplemented with from 20 to 300 mg/ml (50, 100, 150, 200, 250 and values and ranges therein) follicle stimulating hormone (FSH). In some preferred embodiments, the base medium (which may preferably comprise one or more of insulin, IGF1, FGF9, BMP4, EGF, RA. IWR1, and PGD2 in the above ranges) is further supplemented with from between 0.01 and 10 μM (0.05, 0.1, 1.0, 5.0, 8.0 μM and values and ranges therein) testosterone. In some preferred embodiments, the base medium (which may preferably comprise one or more of insulin, IGF1, FGF9, BMP4, EGF, RA, IWR1 and PGD2 in the above ranges) with LH. FSH and testosterone in the ranges described herein.
In some preferred embodiments, the cultures of step 5 for hESC are maintained until artificial Sertoli cells and/or organoids are derived or until about day 13 and most preferably until about days 18 to 22. In some preferred embodiments, a sixth step of the methods of the present invention comprises dissociation of the differentiated cells. In some preferred embodiments, the differentiated cells are harvested for dissociation at from about day 10 to day 20, preferably about from day 15 to day 18, and most preferably on or about day 18. In some preferred embodiments, the cells are plated into an aggrewell (Aggrewell 400, 24 well plate (Stemcell Technologies Cat #34421)) and the organoids allowed to form. In some preferred embodiments, the organoids are collected on or about days 20 to 24, and most preferably on days 20 to 22.
The first step in a method for producing mouse artificial Sertoli cells according to the invention comprises providing pluripotent mESC as described above. In some preferred embodiments, the pluripotent stem cells are provided in a stem cell maintenance medium. In some preferred embodiments, the stem cell maintenance medium is GMEM supplemented with LIF and serum.
The second step of the method of the present invention comprises removing the pluripotent stem cells from the maintenance medium and culturing the pluripotent stem cells in a basal medium supplemented with agents suitable for directing the pluripotent stem cells to epiblast. This is defined as Day 0. In some preferred embodiments, the basal medium is DMEM/F12 and Neurobasal Medium (both from ThermoFisher Scientific) in a ratio of from 2:1 to 1:2 and most preferably at a ratio of about 1:1. In some preferred embodiments, the basal medium is supplemented with N2 supplement, B-27 supplement, and Knockout Serum Replacer (KSR: all from ThermoFisher Scientific), Activin A and bFGF. The supplemented basal medium is referred to as priming medium. In some particularly preferred embodiments, the basal medium is supplemented with from 1 to 10 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 therein) N2 supplement. In some particularly preferred embodiments, the basal medium is supplemented with from 1 to 20 μl/ml (1.0, 5.0, 10.0, 20.0 μl/ml and ranges and values therein) B-27 supplement. In some particularly preferred embodiments, the basal medium is supplemented with from 1 to 20 μl/ml (1.0, 5.0, 10.0, 20.0 μl/ml and ranges and values therein) KSR. In some particularly preferred embodiments, the basal medium is supplemented with from 1 to 20 ng/ml (1.0, 5.0, 10.0, 20.0 ng/ml and ranges and values therein) Activin A. In some particularly preferred embodiments, the basal medium is supplemented with from 1 to 20 ng/ml (1.0, 5.0, 10.0, 20.0 ng/ml and ranges and values therein) bFGF.
The third step of the method of the present invention comprises removing the epiblasts from the priming medium used in step 2 at about day 2 and culturing the epiblasts in a basal medium supplemented with agents suitable for directing the epiblast cells to form anterior intermediate mesoderm. 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 referred to as differentiation medium. In some particularly preferred embodiments, the basal medium is supplemented with from 1 to 20 μl/ml (1.0, 4.0, 5.0, 10.0, 20.0 μl/ml and ranges and values therein) KSR. In some particularly preferred embodiments, the basal medium is supplemented with from 1 to 20 ng/ml (1.0, 5.0, 10.0, 20.0 ng/ml and ranges and values therein) Activin A. In some particularly preferred embodiments, the basal medium is supplemented with from 10 to 200 nM (10.0, 50.0, 100.0, 200.0 nM) and ranges and values therein) RA.
The fourth step of the method of the present invention comprises removing the anterior intermediate mesoderm cells from the differentiation medium used in step 3 on about day 4 and culturing the anterior intermediate mesoderm cells in a basal medium supplemented with agents suitable for directing the anterior intermediate mesoderm cells to form Sertoli cells and/or testis organoids comprising 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 aggregation medium. In some particularly preferred embodiments, the basal medium is supplemented with from 1 to 20 μl/ml (1.0, 4.0, 5.0, 10.0, 20.0 μl/ml and ranges and values therein) KSR. In some particularly preferred embodiments, the basal medium is supplemented with from between 5 and 100 nM (10, 17, 20, 30, 40, 50, 60, 70, 80, 90 nM and values and ranges therein) IGF1. In some particularly preferred embodiments, the basal medium is further supplemented with from between 10 and 500 nM (20, 50, 100, 200, 300, 400 nm and values and ranges therein) insulin. It is contemplated that the inclusion of IGF1 and insulin in the base medium is sufficient to drive differentiation of the anterior intermediate mesoderm cells to form artificial Sertoli cells and organoids comprising the cells. However, in other additional embodiments, the base medium may be supplemented with one or more other agents. In some embodiments, the base medium may be further supplemented with from between 10.0 and 200 nM (10.0, 20.0, 50.0, 100.0 and 200.0 nM and values and ranges therein) RA. In some embodiments, the base medium may be further supplemented with from between 50 and 1000 ng/ml (100, 200, 300, 400, 500, 600, 700, 800, 900 ng/ml and values and ranges therein) PGD2. In some embodiments, the base medium may be further supplemented with from between 50 and 500 ng/ml (100, 200, 300, 400 and values and ranges therein) FGF9. In some preferred embodiments, the base medium may be further supplemented with from between 1 and 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 therein) Y-27632. In some further preferred embodiments, the base medium used in step 5 may be further be supplemented with from 5 to 100 mg/ml (10, 20, 30, 40, 50, 60, 70, 80, 90 and values and ranges therein) bone morphogenetic protein 4 (BMP4). In some further preferred embodiments, the base medium used in step 5 may be further be supplemented with from 10 to 200 mg/ml (30, 50, 70, 100, 150 and values and ranges therein) epidermal growth factor (EGF). In some further preferred embodiments, the base medium used in step 5 may be further be supplemented with from 0.5 to 10 μM (0.5, 1, 2, 4, 5, 7, 10 and values and ranges therein) of IWR1. The base medium with supplements may preferably be changed every two days.
In some preferred embodiments, the cultures of step 4 for mESC are maintained until artificial Sertoli cells and/or organoids are derived or until about day 8. In some preferred embodiments, the methods of the present invention comprise dissociation of the differentiated mESC cells after step 4. In some preferred embodiments, the cells are plated into an aggrewell (Aggrewell 400, 24 well plate (Stemcell Technologies Cat #34421)) and the organoids allowed to form. In some preferred embodiments, the organoids are collected on or about days 6 to 10, and most preferably on about day 8.
In some preferred embodiments, the artificial Sertoli cells produced by the methods described above express at least one of the markers selected from the group consisting of EMX2, WT, SOX9, and LHX9. In some preferred embodiments, the artificial Sertoli cells express at least two of the markers selected from the group consisting of EMX2, WT, SOX9, and LHX9. In some preferred embodiments, the artificial Sertoli cells express at least three of the 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.
In some preferred embodiments, the cultured are maintained until artificial Sertoli cells form organoids. The artificial Sertoli organoids preferably are three dimensional organoids having a roughly spherical shape. In some preferred embodiments, the organoids are characterized by comprising tubule structures. In some preferred embodiments, the organoids are characterized by comprising smooth muscle actin.
In some preferred embodiments, the artificial Sertoli cells or organoids may be harvested or isolated from the cultures for further use.
In some preferred embodiments, methods, reagents, and kits described herein, as well as the artificial Sertoli cells and organoids generated therewith, find use in various research, diagnostic, clinical, and therapeutic applications. In some embodiments, artificial Sertoli cells or organoids are used for direct transplantation into a subject (e.g., for the treatment of infertility, etc.). In some embodiments, artificial Sertoli cells generated by methods herein are useful for diagnostic, prognostic, and/or therapeutic uses.
In some embodiments, the isolated artificial Sertoli cells or organoids may be directly transplanted in a subject. If appropriate, cells are co-administered with one or more pharmaceutical agents or bioactives that facilitate the survival and function of the transplanted cells.
In some embodiments, human organoids are transplanted into mice for additional differentiation and/or maturation of the cells in the organoids. In other embodiments, the organoids are preferably combined with in vivo or in vitro derived germ cells to achieve human germline stem cell expansion and further promote differentiation. It is contemplated that these methods will result in production of haploid round or elongating spermatids which find use in assisted reproductive technologies such as IVF/ICSI.
In some embodiments, the Sertoli cells or organoids of the invention may be used to co-culture gamete stem cells such as primordial germ cell like cells, prospermatogonia or spermatogonial stem/progenitor cells (SSC/SPCs) from a patient. In some preferred embodiments, the cells are cultured so that the stem cells from the patient differentiate into spermatogonia. In some preferred embodiments, the gamete stem cells or cells derived from the gamete stem cells such as spermatogonia are transplanted back into the patient or a patient in need thereof. In some preferred embodiments, the patient has previously undergone a gonadotoxic treatment, including but not limited to chemotherapy and/or radiation. In some preferred embodiments, the stem cells or tissue comprising stem cells are obtained from the patient prior to a gonadotoxic treatment. In some preferred embodiments, prior to gonadotoxic treatment (chemotherapy and radiation), the testicular tissue or somatic cells of the subject are preserved so that germline stem cells may be produced or isolated in the future. In some preferred embodiments, the germ line stem cells obtained from the tissue can be expanded using the in vitro derived cells of the present invention. In some preferred embodiments, the methods described above further comprise obtaining fibroblast tissue to reprogram to induced pluripotent stem cells from a patient which a can be used to make autologous artificial Sertoli cells. In some preferred embodiments, these differentiated cells can be combined with germline stem cells: either primordial germ cell like cells (PGCLC), prospermatogonia (proSSC), or neonatal/adult spermatogonial stem/progenitor cells (SSC/SPCs). In some preferred embodiments, the stem cells can either proliferate or differentiate into spermatogonia or later germ cell stages. In some preferred embodiments, the methods further comprise transferring the expanded stem cells or differentiated spermatogonia back to a patient in need thereof.
In some embodiments, the artificial Sertoli cells or organoids may be provided on a support material. Support materials suitable for use for purposes of the present invention include tissue templates, conduits, barriers, and reservoirs useful for tissue repair. In particular, synthetic and natural materials in the form of foams, sponges, gels, hydrogels, textiles, and nonwoven structures, which have been used in vitro and in vivo to reconstruct or regenerate biological tissue, as well as to deliver chemotactic agents for inducing tissue growth, are suitable for use in practicing the methods of the present invention. See, for example, the materials disclosed in U.S. Pat. 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. Published Application 2004/0062753 A1, U.S. Pat. Nos. 4,557,264 and 6,333,029.
Cells generated with methods and reagents herein may be implanted as dispersed cells or formed into implantable clusters. In some embodiments, cells are provided in biocompatible degradable polymeric supports; porous, permeable, or semi-permeable non-degradable devices: or encapsulated (e.g., to protect implanted cells from host immune response, etc.). Cells may be implanted into an appropriate site in a recipient. Suitable implantation sites may include, for example, the testes or subcutaneously.
In some embodiments, cells or cell clusters are encapsulated for transplantation into a subject. Encapsulation techniques are generally classified as microencapsulation, involving small spherical vehicles, and macroencapsulation, involving larger flat-sheet and hollow-fiber membranes (Uludag, H. et al. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64, herein incorporated by reference in its entirety). Methods of preparing microcapsules include those disclosed by Lu M Z, et al. Biotechnol Bioeng. 2000, 70: 479-83; Chang T M and Prakash S, Mol Biotechnol. 2001, 17: 249-60; and Lu M Z. et al, J. Microencapsul. 2000, 17: 245-51.; herein incorporated by reference in their entireties. For example, microcapsules may be prepared by complexing modified collagen with a ter-polymer shell of 2-hydroxy ethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 μm. Such microcapsules can be further encapsulated with additional 2-5μιη ter-polymer shells in order to impart a negatively charged smooth surface and to minimize plasma protein absorption (Chia, S. M. et al. Multi-layered microcapsules for cell encapsulation Biomaterials. 2002 23: 849-56; herein incorporated by reference in its entirety). In some embodiments, microcapsules are based on alginate, a marine polysaccharide (Sambanis, Diabetes Technol. Ther. 2003, 5: 665-8; herein incorporated by reference in its entirety) or its derivatives. For example, microcapsules can be prepared by the polyelectrolyte complexation between the polyanions sodium alginate and sodium cellulose sulphate with the polycation poly(methylene-co-guanidine) hydrochloride in the presence of calcium chloride.
In some embodiments, cells generated using methods and reagents described herein are microencapsulated for transplantation into a subject (e.g., to prevent immune destruction of the cells). Microencapsulation of cells (e.g., pancreatic lineage cells, beta-like cells, etc.) provides local protection of implanted/transplanted cells from immune attack (e.g., along with or without the use of systemic immune suppressive drugs). In some embodiments, cells and/or cell clusters are microencapsulated in a polymeric, hydrogel, or other suitable material, including but not limited to: poly(orthoesters), poly(anhydrides), poly(phosphoesters), poly(phosphazenes), polysaccharides, polyesters, poly(lactic acid), poly(L-lysine), poly(glycolic acid), poly(lactic-co-glycolic acid), poly(lactic acid-co-lysine), poly(lactic acid-graft-lysine), polyanhydrides, poly(fatty acid dimer), poly(fumaric acid), poly(sebacic acid), poly(carboxyphenoxy propane), poly(carboxyphenoxy hexane), poly(anhydride-co-imides), poly(amides), poly(ortho esters), poly(iminocarbonates), poly(urethanes), poly(organophasphazenes), poly(phosphates), poly(ethylene vinyl acetate), poly(caprolactone), poly(carbonates), poly(amino acids), poly(acrylates), polyacetals, poly(cyanoacrylates), poly(styrenes), poly(vinyl chloride), poly(vinyl fluoride), poly(vinyl imidazole), chlorosulfonated polyolefins, polyethylene oxide, polystyrene, polysaccharides, alginate, hydroxypropyl cellulose (HPC), N-isopropylacrylamide (NIP A), polyethylene glycol, polyvinyl alcohol (PVA), polyethylenimine, chitosan (CS), chitin, dextran sulfate, heparin, chondroitin sulfate, gelatin, etc., and their derivatives, co-polymers, and mixtures thereof. In some embodiments, cell are microencapsulated in an encapsulant comprising or consisting of alginate. Cells may be embedded in a material or within a particle (e.g., nanoparticle, microparticle, etc.) or other structure (e.g., matrix, nanotube, vesicle, globule, etc.). In some embodiments, microencapsulating structures are modified with immune-modulating or immunosuppressive compounds to reduce or prevent immune response to encapsulated cells. For example, pancreatic lineage cells are encapsulated within an encapsulant material (e.g., alginate hydrogel) that has been modified by attachment of an immune-modulating agent (e.g., the immune modulating chemokine, CXCL12 (also known as SDF-1). In some embodiments, such an immune modulating agent is a T-cell chemorepellent and/or a pro-survival factor.
In some embodiments, cells generated using methods and reagents described herein are macroencapsulated for transplantation into a subject. Macroencapsulation of cells, for example, within a permeable or semipermeable chamber, provides local protection of implanted/transplanted cells from immune attack (e.g., along with or without the use of systemic immune suppressive drugs), prevents spread of cells to other tissues or areas of the body, and/or allows for efficient removal of cells. Suitable devices for macroencapsulation include those described in, for example, U.S. Pat. No. 5,914,262: Uludag, et al, Advanced Drug Delivery Reviews, 2000, pp. 29-64, vol. 42, herein incorporated by reference in their entireties.
Other encapsulation (micro or macro) devices and methods may find use in embodiments described herein. For example, methods and devices described in U.S. Pub No. 20130209421, U.S. Pat. No. 8,785,185, each of which are herein incorporated by reference in their entireties, are within the scope of embodiments described herein.
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.
In some embodiments, the Sertoli cells or organoids of the present invention may be used for fertility restoration. In some preferred embodiments, endogenous defective somatic cells in the testis are combined or replaced by the Sertoli cells or organoids of the present invention. In other preferred embodiments, the organoids are transplanted into a subject to allow spermatogenesis to occur at an ectopic location (e.g., a subcutaneous location) other than the testis.
In further embodiments, populations of artificial Sertoli cells and organoids may be used to prepare antibodies and cDNA libraries that are specific for the differentiated phenotype. General techniques used in raising, purifying and modifying antibodies, and their use in immunoassays and immunoisolation methods are described in Handbook of Experimental Immunology (Weir & Blackwell, eds.): Current Protocols in Immunology (Coligan et al, eds.); and Methods of Immunological Analysis (Masseyeff et al, eds., Weinheim: VCH Verlags GmbH). General techniques involved in preparation of mR A and cDNA libraries are described in R A Methodologies: A Laboratory Guide for Isolation and Characterization (R. E. Farrell, Academic Press, 1998): cDNA Library Protocols (Cowell & Austin, eds., Humana Press); and Functional Genomics (Hunt & Livesey, eds., 2000). Relatively homogeneous cell populations are particularly suited for use in drug screening and therapeutic applications.
In some embodiments, the artificial Sertoli cells and organoids generated by methods provided herein are used to screen for agents (e.g., small molecule drugs, peptides, polynucleotides, and the like) or environmental conditions (such as culture conditions or manipulation) that affect the cells. Particular screening applications relate to the testing of pharmaceutical compounds in drug research and to agents for use in cryopreservation of gametes including sperm. Assessment of the activity of candidate pharmaceutical compounds generally involves combining the cells with the candidate compound, determining any change in the morphology, marker phenotype, or metabolic activity of the cells that is attributable to the compound (compared with untreated cells or cells treated with an inert compound), and then correlating the effect of the compound with the observed change. Any suitable assays for detecting changes associated with test agents may find use in such embodiments. The screening may be done, for example, either because the compound is designed to have a pharmacological effect on Sertoli cell types, because a compound designed to have effects elsewhere may have unintended side effects, or because the compound is part of a library screen for a desired effect. Two or more drugs can be tested in combination (by combining with the cells either simultaneously or sequentially), to detect possible drug-drug interaction effects. In some applications, compounds are screened for cytotoxicity.
In some embodiments, methods and systems are provided for assessing the safety and efficacy of drugs that act upon Sertoli cells, or drugs that might be used for another purpose but may have unintended effects upon Sertoli cells. In some embodiments, cells described herein find use in high throughput screening (HTS) applications. In some embodiments, a HTS screening platform is provided (e.g., cells and plates) that allows for the rapid testing of large number (e.g., 1×103, 1×104, 1×105, 1×106 or more) of agents (e.g., small molecule compounds, peptides, etc.). In some embodiments, artificial Sertoli cells or organoids generated using methods and reagents described herein are utilized for therapeutic delivery to a subject. Cells may be placed directly in contact with subject tissue or may be otherwise sealed or encapsulated (e.g., to avoid direct contact). In embodiments in which cells are encapsulated, exchange of nutrients, gases, etc. between the encapsulated cells and the subject tissue is allowed. In some embodiments, cells are implanted/transplanted on a matrix or other delivery platform.
In some embodiments, the methods and kits described herein are useful for identifying additional factors, reagents, and methods for the generation of artificial Sertoli cells or other cell types. The methods used herein may be used to screen factors, reagents and/or conditions for their effect of differentiation. In some embodiments, any screening performed in this or other embodiments discussed herein may be high-throughput screening.
The following example provides a description of the reagents and protocols for producing artificial Sertoli cells according to the present invention from mouse Embryonic Stem Cells (mESC).
Growing mESCs
Growth medium: can be prepared and stored in 4° C. Don't keep longer than a month (date bottle).
Growth medium composition for 100 ml-Glasgow's MEM (GMEM) 92 ml. 10 ml fetal bovine serum ES cell qualified, 1 ml of Nonessential amino acid, 1 ml of sodium pyruvate, 1 ml of Penicillin-Streptomycin, 100 μl of 2-mercaptoethanol, 100 μl LIF (final is 1000 unit/ml).
20% Dimethyl Sulfoxide, 80% fetal bovine serum ES cell qualified was prepared and stored in −20° C. freezer.
Priming medium: can be prepared and stored in 4° C. Don't keep longer than a month (date bottle).
Priming medium composition for 100 ml medium: add 46.9 ml of Neurobasal Medium, 46.9 ml of DMEM-F12 Medium, 500 μl of N2 supplement. 1 ml B27 supplement, 500 μl of Glutamax, 100 μl 2-Mercaptoethanol, 1 ml Sodium pyruvate, 1 ml knockout serum replacer, 1 ml of non-essential amino acids, 1 ml of Penicillin-Streptomycin. The additional growth factors, 10 ng/ml Activin A and 10 ng/ml bFGF are mixed in medium just before the addition of medium into the cells.
A small amount of basal AIM differentiation media can be prepared and stored in 4° C. Don't keep longer than a month (date bottle).
AIM differentiation medium composition for 100 ml of medium: add 92.9 ml of DMEM-F12 medium, 1 ml of Sodium pyruvate, 4 ml of knockout serum replacer, 1 ml of non-essential amino acids, 100 μl of 2-Mercaptoethanol, 1 ml of Penicillin-Streptomycin. Addition of growth factors 10 ng/ml Activin A and 100 nM RA are mixed in medium right before the addition of medium into the cells.
The starting cells need to be in undifferentiated and in the growth phase. To make starting cells are in same growth phase consistently between experiments, Harvest cells from 80% confluent plate (at least one passage after thawing).
Note: cells were collected every day after second day of differentiation for RNA isolation. qPCR was done for multiple AIM markers, gonadal markers, pluripotency marker Oct4, posterior intermediate mesoderm marker HoxD11. The data was confirmed by immunostaining of AIM as well as gonadal markers Wt1, gonadal marker Sox9.
Organoid differentiation medium: A small amount of basal organoid differentiation media can be prepared and stored in 4° C. Don't keep longer than a month (date bottle).
Combination 1: Organoid differentiation medium composition for 100 ml of medium: add 92.9 ml of DMEM-F12 medium, 1 ml of Sodium pyruvate, 4 ml of knockout serum replacer, 1 ml of non-essential amino acids, 100 μl of 2-Mercaptoethanol, 1 ml of 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 was mixed right before adding the media in the cells.
Combination 2: Organoid differentiation medium composition for 100 ml of medium: add 92.9 ml of DMEM-F12 medium, 1 ml of Sodium pyruvate, 4 ml of knockout serum replacer, 1 ml of non-essential amino acids, 100 μl of 2-Mercaptoethanol, 1 ml of Penicillin-Streptomycin. Additional factors, 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, 5 PM Y-27632 was mixed right before adding the media in the cells.
Combination 3: Organoid differentiation medium composition for 100 ml of medium: add 92.9 ml of DMEM-F12 medium, 1 ml of Sodium pyruvate, 4 ml of knockout serum replacer, 1 ml of non-essential amino acids, 100 μl of 2-Mercaptoethanol, 1 ml of Penicillin-Streptomycin. Additional factors, 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, 5 M Y-27632 was mixed right before adding the media in the cells.
Before starting the organoid differentiation, the aggrewell 400-24 well plate was prepared as follow.
1. Generation and validation of mouse testis-like organoids from mESC. We have leveraged genetic and scRNAseq data from mice to develop and optimize a mouse ESC differentiation protocol to produce cells resembling many of the testicular cell types. Our approach entails iteratively titrating cells' seeding density and testing many combinations of growth factors, cytokines, and small molecules at multiple concentrations and durations, until we produced cells that recapitulate those along the differentiation trajectory observed in vivo (
Briefly, treating mouse ESCs with Activin A and bFGF for two days leads to a suppression of many pluripotency marker (Nanog, Sox2, Rex1, Klf1,
To confirm that the changes in RNA are evident on protein levels, we performed immunofluorescence imaging using a subset of markers analyzed by qPCR. As expected, OCT4, a marker for stemness, is expressed in day 0 but repressed in day 4 (
2. The mouse in vitro derived testis somatic-like cells resemble gonadal progenitors identified in E10.5-11.5 gonads. For mouse ESC experiments, cells from days 0), 2, 4, and 8 of differentiation were molecularly barcoded, captured, and sequenced in the same run to avoid batch effect. Unsupervised clustering of ˜5.000 cells revealed 8 clusters: C1-C8 (
3. The mouse testis somatic like cells can support the maintenance of pro-spermatogonia in vitro and possibly their differentiation as well. To determine whether the in vitro derived somatic like cells can support germ cell maintenance and differentiation, we performed a similar differentiation protocol described above except we collected and dissociated organoids at day 6 and re-aggregated the in vitro derived somatic cells with roughly 4% pro-spermatogonia (Oct4-egfp+ cells) from the postnatal days (PND) 1-2 testis (
Based on our current data, we have very strong molecular and histological evidence suggesting that our 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.
4. Efforts to continue to refine the robustness of our somatic cell differentiation protocol. Although our original cocktail in
The following example provides a description of the reagents and protocols for producing artificial Sertoli cells according to the present invention from human Embryonic Stem Cells (hESC). The protocol can be preferably be continued to at least 13 days and up to 22 days.
We have begun optimizing the differentiation protocol using two male human ESC lines (U6 and H1). The differentiation schema corresponding to the protocol above is described in
To assess the developmental stage of cultured cells we performed scRNAseq analysis across the days of differentiation. We identified 13 clusters (
Despite this exciting progress, the human differentiation protocol generated mainly progenitor-like cells, few of which advance further to become terminally differentiated gonadal cells. Indeed, cells in our late-stage culture resembled in vivo somatic cells seen at 6-7 weeks of gestation. To drive differentiation further in human cell culture we opted to supplement the ALL-GF differentiation cocktail with hormones Testosterone. LH and FSH (
Next, we tested the ability of these in vitro derived cells to home germ cells and allow their survival. We generated PGCLC (SOX17+; TFAP2C+) and combined with our in vitro derived somatic cells at day 8, and like mouse we find that the germ cells get incorporated into the organoids and they can continue to proliferate for at least 5 days in culture (
Taken together, our data demonstrate the successful generation of human somatic cells using two ESC lines. Furthermore, our in vitro derived somatic cells can home germ cells and support their proliferation in the absence of a PGC culture cocktail.
To have more clinical relevance, we next tested whether our ESC differentiation schema can be directly applied to differentiate induced pluripotent stem cells into testis somatic cells. We applied the exact regimen used for ESC, but unfortunately the efficiency was significantly lower. Unexpectedly, we realized that our initial differentiation step for IPSC required a higher concentration of Chiron (8 μM) than what is used for ESC (3 μM), and the ideal stopping point for the differentiation is 10 days. Beyond the 10 day gonadic differentiation we are decreasing the number of gonadal cells (
All publications, patents, patent applications and accession numbers mentioned in the above specification are herein incorporated by reference in their entirety. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications and variations of the described compositions and methods of the invention will be apparent to those of ordinary skill in the art and are intended to be within the scope of the following claims.
The present application claims priority to U.S. Provisional Patent Application Ser. No. 63/312,528, filed Feb. 22, 2022 the contents of which are hereby incorporated by reference in its entirety.
This invention was made with government support under HD091949 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2023/013608 | 2/22/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63312528 | Feb 2022 | US |
