Method for producing leydig-like cells derived from human pluripotent stem cells

Abstract

Provided is a simpler, more efficient method for producing Leydig-like cells derived from human pluripotent stem cells, said method capable of reducing the risk of biological contamination and the like and involving a cell culturing method with which mass culturing is possible. Provided is a method in which a process for producing Leydig-like cells derived from human pluripotent stem cells includes a culturing process in which adherent culturing is performed after suspension culturing inside a closed-system cell culture vessel having, on the interior thereof, a culture surface on which suspension cells can be subjected to suspension culturing, and a culture surface to which adherent cells can be adhered.

Description

Method for producing Leydig-like cells derived from human pluripotent stem cells

[0001] The present invention relates to a method for producing Leydig-like cells derived from human pluripotent stem cells, more specifically to a cell culture method in the production process, and further to human pluripotent stem cell-derived Leydig-like cells and cell populations thereof produced by the method.

[0002] This application claims priority from Japanese Patent Application No. 2024-153591, which is incorporated herein by reference.

[0003] In recent years, attention has been focused on age-related male hypogonadism (LOH), a condition characterized by a variety of symptoms resulting from an age-related decline in male hormones. Hormone replacement therapy using testosterone has been used to address this condition. However, hormone replacement therapy produces hormone levels that differ from physiological secretion patterns and has a short duration of action, necessitating repeated administration, which places a significant burden on patients. There is a need for new treatment methods that do not require repeated administration and that reduce the time and effort required for clinic visits and the pain associated with injections, thereby improving quality of life (QOL).

[0004] The hypothalamus produces gonadotropin-releasing hormone (GnRH). The anterior pituitary gland produces luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in response to GnRH production. Leydig cells in the testes are said to produce 5-10 mg / day of testosterone in response to LH. Testosterone is synthesized from cholesterol via several intermediate compounds, including dehydroepiandrosterone (DHEA) and androstenedione.

[0005] Efforts are being made to develop methods for inducing differentiation of pluripotent stem cells (PSCs), which possess both self-renewal and pluripotency, into various cell types. Among these pluripotent stem cells, methods using embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) have attracted considerable attention. Various attempts have been made to induce differentiation into steroid hormone-producing cells of the testes and adrenal glands by stably transfecting various mesenchymal stem cells with the transcription factor SF-1 (steroidogenic factor-1) and adding cyclic adenosine monophosphate (cAMP) to the culture medium. SF-1 is a member of the nuclear receptor family of intracellular transcription factors and is encoded by the NR5A1 (nuclear receptor subfamily 5, group A, member 1) gene.

[0006] A method for inducing steroid hormone-producing cells by introducing SF-1 into two types of human ES cells (H9 and KhES1) and one type of human iPS cell line (201B7) has been reported. In this study, human ES cells and human iPS cells were induced to differentiate into mesodermal cells using BIO (6-bromoindirubin-3'-oxime) or a GSK-3 (glycogen synthase kinase 3) inhibitor. The mesodermal cells were sorted using flow cytometry. SF-1 was then introduced into the mesodermal cells using the SF-1 expression plasmid pCMFlag-hsNR5A1, allowing them to express SF-1. The cells were then cultured in a medium containing 8-Br-cAMP (8-Bromo-cAMP) to differentiate into steroid hormone-producing cells (Non-Patent Document 1). However, although the production of progesterone and cortisol is mentioned, there is no report on the production of testosterone, and the expression of markers that selectively indicate Leydig cells (INSL3, 17βHSD3, LHCGR, etc.) has not been demonstrated.

[0007] A method has been disclosed in which mouse-derived pluripotent stem cells (ES cells) are induced to differentiate into mesenchymal stem cells, and then conditionally express SF-1 to differentiate into steroid hormone-producing cells (Patent Document 1). However, there is no report on the production of hormones such as testosterone and cortisol, and no indication of the expression of markers that indicate Leydig cells.

[0008] A study has shown that mouse SF-1 was introduced into mouse ES cells (OriCellStrain C57BL / 6 mESCs) using a lentiviral vector to generate an SF-1+ ESC line (ESC-SF-1). The line was then cultured in a medium containing 8-Br-cAMP and forskolin, which induced differentiation into progenitor Leydig cells (PLCs). This study demonstrated that treatment of ESC-SF-1 with 8-Br-cAMP and forskolin induced differentiation into PLCs more effectively than treatment with 8-Br-cAMP alone. The resulting PLCs were then transplanted into rats as Leydig-like cells, and markers for Leydig cells were measured in serum samples (Non-Patent Document 2).

[0009] A method for producing human pluripotent stem cell-derived Leydig-like cells has been disclosed, in which human pluripotent stem cells are expressing NR5A1, INSL3, and at least one marker selected from 17βHSD3 and LHCGR (Patent Document 2). Another method for producing human pluripotent stem cell-derived Leydig-like cells expressing HSD17B3, INSL3, LHCGR, etc. has been disclosed (Non-Patent Document 3). A method for producing human pluripotent stem cell-derived Leydig-like cells has been disclosed, which includes culturing human pluripotent stem cells under culture conditions containing a cytokine cocktail to induce forced expression of NR5A1, and adjusting the timing of cAMP addition and removal, suspension culture, adherent culture, etc. (Patent Document 3). However, improvements are needed to address the complexity of cell transfer and medium change during differentiation induction during the culture process in the production of human pluripotent stem cell-derived Leydig-like cells. Furthermore, mass culture is also desired for practical application.

[0010] Endocrinology, 153(9): 4336-4345 (2012)STEM CELLS AND DEVELOPMENT, 24(4): 459-470 (2015)Endocrinology, 2021, Vol.162, No.12, 1-11,https: / / doi.org / 10.1210 / endocr / bqab202

[0011] Japanese Patent Publication No. 2011-15630 International Publication No. WO2018 / 088240 (Patent No. 6979702) International Publication No. WO2023 / 157727

[0012] The objective of the present invention is to introduce a cell culture process that is simpler and more efficient in the differentiation induction process of human pluripotent stem cells in the process of producing Leydig-like cells derived from human pluripotent stem cells, reduces the risk of biological contamination, etc., and allows for mass culture.

[0013] As a result of extensive research to solve the above problems, the present inventors discovered that the above problems can be solved by introducing a culture step in which suspension culture is performed in a closed culture vessel and then adhesion culture is performed in the process of producing Leydig-like cells derived from human pluripotent stem cells, and thus completed the present invention. More specifically, this is achieved by introducing a culture step using a closed culture vessel that includes a culture surface inside the vessel on which suspension cells can be cultured in suspension and a culture surface on which adherent cells can adhere.

[0014] That is, the present invention comprises the following: 1. A method for producing Leydig-like cells derived from human pluripotent stem cells, the method comprising a culture step of using a closed culture vessel to culture Leydig-like cells in suspension and then performing adherent culture in the culture vessel during the production of Leydig-like cells from human pluripotent stem cells. 2. The method for producing Leydig-like cells derived from human pluripotent stem cells according to the preceding paragraph 1, wherein the closed culture vessel contains a culture surface within the vessel that allows suspension cells to be cultured in suspension and a culture surface within the vessel that allows adherent cells to be adherent. 3. The method for producing Leydig-like cells derived from human pluripotent stem cells according to the preceding paragraph 2, characterized in that a closed culture vessel is used in which the culture surface within the vessel that allows suspension culture and the culture surface within the vessel that allows adherent cells to be cultured on either culture surface while the vessel is stationary. 4. The method for producing Leydig-like cells derived from human pluripotent stem cells according to the preceding paragraph 2, characterized in that a closed culture vessel is used in which the culture surface within the vessel that allows suspension culture of suspension cells has a well-like shape that allows cell clumps to be cultured in a suspended state. 5. A method for producing human pluripotent stem cell-derived Leydig-like cells according to the preceding paragraph 1, wherein the closed culture vessel has a medium exchange port. 6. A method for producing human pluripotent stem cell-derived Leydig-like cells according to the preceding paragraph 1, wherein the closed culture vessel is a bag-shaped culture vessel made of a soft packaging material. 7. A method for producing human pluripotent stem cell-derived Leydig-like cells according to the preceding paragraph 1, wherein the culture step of performing suspension culture within the closed culture vessel and then adhesion culture comprises a step of culturing cells on a culture surface inside the vessel where suspension cells can be cultured in suspension, and then inverting the cell suspension onto a culture surface where adherent cells can be cultured in adhesion. 8. A method for producing human pluripotent stem cell-derived Leydig-like cells according to the preceding paragraph 1, comprising a step of performing suspension culture from human pluripotent stem cells until they start to form embryoid bodies, and then performing adhesion culture. 9. 9. A method for producing human pluripotent stem cell-derived Leydig-like cells according to the preceding item 8, comprising the step of forcibly expressing NR5A1 in human pluripotent stem cells during the process leading up to the initiation of embryoid body formation from the human pluripotent stem cells. 10. A method for producing human pluripotent stem cell-derived Leydig-like cells according to the preceding item 9, comprising the steps of forcibly expressing NR5A1 in human pluripotent stem cells in the presence of one or more members selected from the group consisting of a WNT canonical pathway activator, BMP4, and VEGF, and then performing suspension culture for 2 to 10 days, followed by adhesion culture.11. A method for producing human pluripotent stem cell-derived Leydig-like cells according to the preceding item 10, comprising the step of removing any WNT canonical pathway activator, BMP4, and VEGF present in the adhesion culture step, and then adding cAMP. 12. A method for producing human pluripotent stem cell-derived Leydig-like cells according to the preceding item 11, comprising the step of removing cAMP from the culture system between days 2 and 40 after the start of the cAMP treatment. 13. A method for producing human pluripotent stem cell-derived Leydig-like cells according to the preceding item 10, comprising the step of removing any WNT canonical pathway activator, BMP4, and VEGF present in the adhesion culture step, and then treating the cells with forskolin. 14. Human pluripotent stem cell-derived Leydig-like cells produced by the method according to the preceding item 1. 15. 15. The human pluripotent stem cell-derived Leydig-like cells according to item 14 above, which are Leydig-like cells that continuously produce testosterone for at least 60 days or more. 16. The human pluripotent stem cell-derived Leydig-like cells according to item 14 above, which have been passaged at least once. 17. A human pluripotent stem cell-derived Leydig-like cell population comprising the human pluripotent stem cell-derived Leydig-like cells according to item 14 above.

[0015] According to the method for producing human pluripotent stem cell-derived Leydig-like cells of the present invention, by introducing a culture process in which suspension culture is performed in a closed culture vessel and then adhesion culture is performed, medium exchange is facilitated compared to conventional production methods in which culture is performed in an open culture vessel and the culture vessel is replaced when transitioning from suspension culture to adhesion culture. Furthermore, there is no need to transfer cells from suspension culture dishes to adhesion culture dishes during differentiation induction, thereby reducing the risk of biological contamination. The produced Leydig-like cells can stably and continuously secrete testosterone comparable to conventional methods.

[0016] 1 is a diagram showing one embodiment of a closed culture vessel used in the present invention.

[0023] FIG. 1 is a diagram illustrating a method for producing human pluripotent stem cell-derived Leydig-like cells. (Example 1) This diagram shows the production process up to day 6, from embryoid body (EB) formation from human pluripotent stem cells (iPS cells) in the method for producing human pluripotent stem cell-derived Leydig-like cells. (Example 1) This diagram is a photograph showing the shape of EBs when cultured using the culture plate used in Patent Document 3 as a comparative example and the closed culture vessel used in this example. (Example 1) This diagram shows the production process up to day 17 of culture of human pluripotent stem cells (iPS cells) in the method for producing human pluripotent stem cell-derived Leydig-like cells. (Example 1) This diagram is a photograph showing the shape of EBs on day 6 of culture before and after inversion of the closed culture vessel. (Example 1) This photograph shows the shape of cells cultured using the closed culture vessel of Example 1 and cells cultured by the method of Example 1 of Patent Document 3 as a comparative example, and also shows the testosterone concentration in the culture supernatant. (Experimental Example 1-1) This is a photograph showing the results of confirming the expression of HSD17B3, a Leydig cell marker, by cell immunostaining for the cultured cells on the 18th day of culture prepared in Example 1. (Experimental Example 1-2) This is a diagram showing the results of measuring the testosterone concentration in the culture supernatant when the cultured cells using the closed culture vessel of Example 1 and the cells cultured by the method of Example 1 of Patent Document 3 as a comparative example were cultured for a long period of time. (Experimental Example 1-3)

[0017] The present invention relates to a method for producing human pluripotent stem cell-derived Leydig-like cells, more specifically to a cell culture method in the step of producing the human pluripotent stem cell-derived Leydig-like cells, and further to human pluripotent stem cell-derived Leydig-like cells and cell populations thereof produced by the method.

[0018] As used herein, the term "Leydig-like cells" refers to cells obtained by inducing differentiation of pluripotent stem cells, and is used to distinguish them from naturally occurring "Leydig cells." The "Leydig-like cells derived from human pluripotent stem cells" of the present invention refer to cells obtained by inducing differentiation of human pluripotent stem cells, which have the ability to produce testosterone and / or express Leydig cell markers. Examples of Leydig cell markers include at least one marker selected from HSD17B3, StAR, CYP17A1, and CYP11A1, with HSD17B3 being the most preferred marker. Cells that are particularly recognized to have the ability to produce testosterone are also referred to herein as "testosterone-producing cells."

[0019] As used herein, "pluripotent stem cells" refer to undifferentiated cells that possess both the ability to self-renew, allowing them to proliferate while maintaining an undifferentiated state, and the ability to differentiate into all three germ layer lineages, allowing them to differentiate into all three germ layer lineages. Examples of such cells include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). The method of the present invention uses human pluripotent stem cells. While human iPS cells share similar properties to human ES cells, differences have been reported between them and mouse ES cells, including transcription factor networks, epigenetics, and responsiveness to extracellular factors (STEM CELLS 2010; 28: pp. 419-430; Jikken Igaku 2012; 30, No. 10: pp. 1544-1548; Igaku no Ayumi 2011; 239, No. 14: pp. 1247-1251). Therefore, in this specification, human pluripotent stem cells and non-human animal pluripotent stem cells are considered to be distinguished from each other.

[0020] As used herein, "ES cells" refer to pluripotent stem cells isolated as an undifferentiated stem cell population by transferring a cell cluster called the inner cell mass inside a blastocyst-stage embryo to in vitro culture. Many human ES cell lines have already been established and are available from ES Cell International, Inc., Wisconsin Alumni Research Foundation, the National Stem Cell Bank (NSCB), and other sources.

[0021] As used herein, "iPS cells" refers to induced pluripotent stem cells that can be reprogrammed without the use of oocytes, embryos, or ES cells by introducing several genes into somatic cells, and that have pluripotency and proliferation capabilities similar to ES cells (K. Takahashi and S. Yamanaka (2006) Cell, 126:663-676; K. Takahashi et al. (2007), Cell, 131:861-872; J. Yu et al. (2007), Science, 318:1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26:101-106 (2008); International Publication WO 2007 / 069666). Applicable iPS cells may be human iPS cells produced by publicly known or future methods. Furthermore, there are no particular limitations on the cells from which iPS cells are derived.

[0022] The culture method and medium used for culturing pluripotent stem cells such as ES cells or iPS cells are not particularly limited, and may be any known or future developed medium or technology. Examples of media that can maintain the undifferentiated state and pluripotency of ES cells or iPS cells, or media suitable for inducing differentiation, include commercially available basal media for mammalian cells, such as DMEM and / or DMEM / F12, supplemented with serum or serum replacement solution (KnockOut). TMExamples of media that can be used include those containing ES cell suspensions containing ES cells such as ES cells containing ...

[0023] As used herein, the term "closed culture vessel" refers to a sealed culture vessel that allows cells to be cultured without opening or closing the vessel and has a structure that allows the culture medium to be exchanged via a tube or the like that is connected to the vessel through an opening (see Figure 1). The use of a closed culture vessel is particularly advantageous from a hygienic standpoint, for example, it can reduce the risk of biological contamination during culture or when exchanging the culture medium, and is advantageous in that it can reduce the burden on the culture environment.

[0024] The "closed culture vessel" used in the present invention is advantageous in that it allows suspension culture in the closed culture vessel followed by adhesion culture without replacing the culture vessel. Such a closed culture vessel has both a suspension culture surface and an adhesion culture surface, and is equipped with an internal culture surface on which suspension cells can be cultured in suspension (hereinafter also referred to as a "non-adhesive culture surface") and a culture surface on which adherent cells can adhere (hereinafter also referred to as an "adhesive culture surface"). The non-adhesive culture surface and the adhesion culture surface inside the vessel are arranged so that cells can be cultured on either culture surface when the vessel is stationary. For example, as shown in FIG. 1, a closed culture vessel may have a bag-like shape, with the non-adhesive culture surface located on the bottom when stationary, and the adhesion culture surface located on the ceiling. When a cell suspension is introduced and cultured using a vessel of this shape, cells can be cultured in suspension with the non-adhesive culture surface located on the bottom. Then, by inverting the ceiling and bottom, the cell suspension of the suspension-cultured cells comes into contact with the adhesion culture surface, allowing adhesion culture. It is clear that the closed culture vessel shown in FIG. 1 is only one embodiment and should not be limited to the form shown in FIG.

[0025] In the method for producing Leydig-like cells of the present invention, human pluripotent stem cells can be cultured in suspension until they begin to form embryoid bodies (EBs), and then cultured in adherence. In the step of transferring from suspension culture to adherent culture, human pluripotent stem cells can be cultured in suspension in the closed culture vessel until they begin to form EBs, and then the closed culture vessel can be inverted to perform adherent culture without transferring the cell suspension to another culture vessel. Human pluripotent stem cells can be cultured in suspension for 10 days, 8 days, or more preferably 6 days from the start of culture. EBs are formed during the suspension culture process for 2 to 10 days, preferably 6 to 10 days, more preferably 6 to 8 days, and most preferably 6 days from the start of culture.

[0026] Suspension culture of human pluripotent stem cells until the initiation of EB formation is preferably carried out on a culture surface having a well-like depression that allows the formation of small spherical cell clusters (small spheres) or large spherical cell clusters (large spheres). Therefore, the non-adhesive culture surface placed in the closed culture vessel used in the present invention is preferably a well-like surface that allows cell clusters to be cultured in a suspended state. For example, in a suspension culture system, cells in a single cell suspension can be seeded to form uniformly sized EBs. A well-like surface that allows the formation of approximately 50 to 20,000 cells per EB, preferably approximately 50 to 5,000 cells, more preferably approximately 100 to 200 cells, is preferred.

[0027] The closed culture vessel used in the present invention allows the supply and exchange of culture medium necessary for culture from a tube or the like connected to the vessel via an opening (medium exchange port). Furthermore, in order to maintain a gaseous state at a required concentration during culture, gases containing, for example, carbon dioxide may be supplied through the opening. The location of the opening is not particularly limited, as long as it is located in a position that allows suspension culture and / or adherent culture.

[0028] The material of the closed culture vessel is not particularly limited as long as it allows for suspension culture and adherent culture. Materials that allow for suspension culture or adherent culture in a closed system are preferably materials that allow gas exchange or materials that have been designed to maintain appropriate concentrations of gases such as oxygen and carbon dioxide during culture. For example, soft or hard packaging materials are acceptable. Packaging materials are preferred, and olefin resins such as polyethylene and polypropylene are suitable. Other usable materials include polystyrene, polymethylpentene, cyclic olefin polymers, cyclic olefin copolymers, polyvinyl chloride, polyurethane, polymethyl methacrylate, polyester, polyamide, ionomer, ethylene-α-olefin copolymer, ethylene-vinyl acetate copolymer, ethylene-vinyl alcohol copolymer, ethylene-acrylic acid copolymer, ethylene-methacrylic acid copolymer, ethylene-methyl methacrylate copolymer, polyacrylic acid, polymethacrylic acid, polymethyl acrylate, polymethyl methacrylate, polydimethylsiloxane, fluororesin, silicone resin, polybutadiene resin, and chlorinated polyethylene. Also usable are thermoplastic elastomers such as olefin-based thermoplastic elastomers, vinyl chloride-based thermoplastic elastomers, styrene-based thermoplastic elastomers, urethane-based thermoplastic elastomers, ester-based thermoplastic elastomers, nylon-based thermoplastic elastomers, etc. Also usable are thermosetting elastomers such as urethane rubber, silicone rubber, and fluororubber, and thermosetting resins such as phenol resins, epoxy resins, melamine resins, urea resins, unsaturated polyester resins, alkyd resins, urethane resins, and thermosetting polyimides.

[0029] For suspension culture and / or adherent culture, the culture surface of the culture vessel may be coated and / or surface-treated with a known or future-developed coating agent. The coating agent is not particularly limited. For example, a non-adhesive surface can be formed by using a polyethylene film without surface treatment (untreated state), maintaining a high static water contact angle of 80° to 100° on the surface, thereby creating a non-adhesive surface to which adherent cells do not adhere. Another method involves, for example, surface-treating a polyethylene film to achieve a static water contact angle of approximately 60° to 80°, and then coating this surface with a cell adhesion inhibitor to create a non-adhesive surface to which adherent cells do not adhere. Examples of cell adhesion inhibitors include phospholipid polymers, polyvinyl alcohol derivatives, phospholipid-polymer complexes, polyhydroxyethyl methacrylate, polyvinyl alcohol, agarose, chitosan, polyethylene glycol, albumin, and the like, and these may also be used in combination. The coating agent for the adherent culture surface is also not particularly limited as long as it allows cells to adhere and be cultured. Examples include Matrigel (Matrigel TM BD), collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, or a combination thereof can also be used. The surface of the substrate that serves as the adhesive surface of the culture vessel can be treated with, for example, plasma treatment, excimer treatment, corona treatment, or the like.

[0030] The method of the present invention for producing Leydig-like cells derived from human pluripotent stem cells can be the method described in Patent Document 3. Specifically, the process leading up to the initiation of EB formation from human pluripotent stem cells can include a step of forcibly expressing NR5A1 in human pluripotent stem cells. More specifically, the method can include a step of forcibly expressing NR5A1 in human pluripotent stem cells in the presence of one or more species selected from the group consisting of a WNT canonical pathway activator, BMP4, and VEGF, followed by suspension culture for 2 to 10 days using a closed culture vessel as used in the present invention, followed by adhesion culture. The adhesion culture step can include a step of removing the WNT canonical pathway activator, BMP4, and VEGF present, and adding cAMP.

[0031] In the process of preparing human pluripotent stem cell-derived Leydig-like cells of the present invention, the NR5A1 gene is introduced into human pluripotent stem cells, and embryoid bodies (EBs) are formed. For EB formation, a cytokine cocktail, specifically one or more cytokines selected from the group consisting of a WNT canonical pathway activator, BMP4 (Bone Morphogenetic Protein 4), and VEGF (Vascular Endothelial Growth Factor), can be added to the human pluripotent stem cell culture system. WNT canonical pathway activation refers to activation of the classical Wnt pathway, also referred to as activation of the β-catenin pathway, in which β-catenin controls gene expression. Examples of WNT canonical pathway activators include glycogen synthase kinase 3 (GSK-3) inhibitors, more specifically CHIR99021. The concentration of the WNT canonical pathway activator added is not particularly limited, but can be appropriately selected from a range of up to 300 mM, for example, 1 to 300 mM, 1 to 100 mM, preferably 10 to 50 mM, and more preferably 20 mM. The concentration of BMP4 added is not particularly limited, but can be appropriately selected from a range of up to 2000 μg / mL, for example, 10 to 2000 μg / mL, 10 to 1000 μg / mL, preferably 30 to 500 μg / mL, and more preferably 100 μg / mL. The concentration of VEGF added is not particularly limited, but can be appropriately selected from a range of up to 2000 μg / mL, for example, 10 to 2000 μg / mL, 10 to 1000 μg / mL, preferably 30 to 500 μg / mL, and more preferably 100 μg / mL.

[0032] It is preferable to carry out suspension culture of human pluripotent stem cells for 10 days, 8 days, and more preferably 6 days from the start of culture. EBs are formed during the suspension culture process for 2 to 10 days, preferably 6 to 10 days, more preferably 6 to 8 days, and most preferably 6 days from the start of culture of human pluripotent stem cells.

[0033] As used herein, the "NR5A1 gene" can be identified, for example, by GenBank Accession No. NM_004959 (SEQ ID NO: 1). The NR5A1 gene can be introduced into human pluripotent stem cells by any method known per se or any method to be developed in the future. NR5A1 cDNA can be inserted into an appropriate expression vector containing a promoter that can function in host cells by a method known per se. Specifically, the method described in Patent Document 2 can be applied.

[0034] The present invention includes a step of forcibly expressing the introduced gene in human pluripotent stem cells into which the NR5A1 gene has been introduced. The gene can be expressed using any gene expression system known per se or that will be developed in the future. The timing of gene expression can be controlled using any method known per se or that will be developed in the future. As an experimental system capable of reversibly regulating the expression of a target gene in cells, etc., Tet-On, which expresses genes in a tetracycline-dependent manner, is used. TM / Off TM This expression system allows the expression of the target gene to be regulated by adding or not adding the antibiotic tetracycline or its derivative doxycycline (Dox) to the culture medium. TM According to this system, for example, the Tet repressor (TetR) and Tet operator sequence (tetO sequence) that work in the E. coli tetracycline resistance operon are used, and TetR binds to the tetO sequence in the absence of tetracycline, allowing for forced expression of NR5A1.

[0035] In the method of the present invention for producing Leydig-like cells derived from human pluripotent stem cells, TM System and Tet-On TM Although any of these systems can be used to control the expression of the NR5A1 gene, the Tet-Off system, which forcibly expresses NR5A1 in the absence of tetracycline or doxycycline, is preferred. TM It is preferable to use the Tet-Off system. TMUsing this system, human pluripotent stem cells previously transfected with the NR5A1 gene can be cultured in the presence of tetracycline or doxycycline to inhibit differentiation into human pluripotent stem cell-derived Leydig-like cells. Then, by removing tetracycline or doxycycline at the time when differentiation into Leydig-like cells is required, NR5A1 can be continuously and forcibly expressed. Furthermore, by removing tetracycline or doxycycline during the continuous forced expression of NR5A1, the undesirable effects of these drugs can be avoided, which is advantageous. This allows for more effective control and progression of differentiation into human pluripotent stem cell-derived Leydig-like cells.

[0036] In addition, the examples of Patent Document 2 (International Publication No. WO2018 / 088240) also describe Tet-on / off, but Patent Document 2 describes a Tet-On / Off system that forcibly expresses NR5A1 in the presence of tetracycline or doxycycline. TM The Tet-off system in the examples of Patent Document 2 describes a case in which NR5A1 is not forcibly expressed in the absence of tetracycline or doxycycline, and therefore the Tet-Off system of the present invention is not suitable for this purpose. TM It is different from the system.

[0037] The process for producing human pluripotent stem cell-derived Leydig-like cells of the present invention includes a step of performing suspension culture in the closed culture vessel, followed by adhesion culture of the cells without changing the culture vessel. The adhesion culture refers to culturing the cells in a monolayer state. The adhesion culture step can induce differentiation of human mesodermal cells into Leydig-like cells. In the adhesion culture step, it is preferable to remove the cytokine cocktail (cytokines present in the culture system from one or more cytokines selected from the group consisting of a WNT canonical pathway activator, BMP4, and VEGF) added to the human pluripotent stem cell culture system used in the suspension culture step.

[0038] In the above-mentioned adhesion culture step, it is preferable to further treat the cells with cAMP. When 8-Br-cAMP is used as cAMP, it can be added to the medium at 0.01 to 4 mM, preferably 0.1 to 1 mM. In addition to the cAMP treatment, it is preferable to further stimulate the cells with forskolin. Forskolin can be added to the medium at 0.1 to 100 μM, preferably 2 to 100 μM, and more preferably 5 to 100 μM. cAMP is preferably removed between 8 and 40 days after the start of treatment, preferably by 30 days after the start of culture, more preferably by 20 days after the start of culture, and most preferably on the 17th day after the start of culture. Meanwhile, it is preferable to continue adding forskolin to the medium even after cAMP removal.

[0039] The period of adherent culture is not particularly limited, and can be continued as long as culturing is possible. Furthermore, cells can be subcultured during the adherent culture process. Cell subculture is preferably carried out using a cell separation enzyme such as TrypLE Select, TrypLE Express, or Accutase. Furthermore, treatment with a ROCK inhibitor such as Y27632 is also possible. The seeding density of cells during subculture is not particularly limited as long as the cells are viable, but may be, for example, 1 to 1.0 × 10 6 cells / well, preferably 1000 to 1.0 × 10 5 cells / well, and more preferably about 1.0 × 10 4 cells / well (9.6cm 2 )

[0040] Furthermore, cells cultured in adhesion can be cryopreserved. The method and conditions for cryopreserving cells are not particularly limited as long as they are methods used by those skilled in the art, and any known method or any method to be developed in the future can be applied. For example, it is necessary to use a medium containing a cryoprotectant such as DMSO (dimethyl sulfoxide) or glycerol as a freezing medium, and commercially available medium can also be used. Examples of commercially available medium include STEM-CELLBANKER TM (Zenogen Pharma), RecoveryTM Cell Culture Freezing Medium (ThermoFisher, SCIENTIFIC) or Synth-a-Freeze TM Cryopreservation Medium (ThermoFisher, SCIENTIFIC), etc., can be used. The frozen cells can be maintained and stored at, for example, -80°C, or can be stored at an even lower temperature in liquid nitrogen. The method for thawing frozen cells is not particularly limited as long as it is a method used by those skilled in the art, and any method known per se or any method developed in the future can be applied.

[0041] According to the method of the present invention for producing human pluripotent stem cell-derived Leydig-like cells, differentiation of human pluripotent stem cells into human pluripotent stem cell-derived Leydig-like cells can be induced with a high efficiency of 80% or more, preferably 90% or more, and more preferably 95% or more. The present invention also extends to human pluripotent stem cell-derived Leydig-like cells and human pluripotent stem cell-derived Leydig-like cell populations produced by the methods of the present invention. The human pluripotent stem cell-derived Leydig-like cells of the present invention are capable of continuously producing testosterone for at least 14 days, preferably 22 days or more, and more preferably 32 days or more. It is even believed that testosterone production can be continued for 60 days or more, even 120 days or more. Furthermore, the Leydig-like cell population of the present invention is a human pluripotent stem cell-derived Leydig-like cell population containing testosterone-producing Leydig-like cells at a high ratio of 80% or more, preferably 90% or more, and more preferably 95% or more.

[0042] The human pluripotent stem cell-derived Leydig-like cells prepared by adherent culture in the above step can be further cultured in suspension after recovery. The culture vessel used for suspension culture is not particularly limited as long as it is a culture vessel capable of three-dimensional culture, but the same type of culture vessel used in the step of forming EBs from human pluripotent stem cells or the step of forcibly expressing NR5A1 in human pluripotent stem cells can be used.

[0043] The present invention also encompasses pharmaceutical compositions comprising, as an active ingredient, human pluripotent stem cell-derived Leydig-like cells or cell populations produced by the methods of the present invention. Examples of targets of the pharmaceutical compositions of the present invention include diseases associated with decreased testosterone levels and conditions requiring testosterone supplementation. The present invention also encompasses therapeutic agents for diseases associated with decreased testosterone levels, comprising, as an active ingredient, human pluripotent stem cell-derived Leydig-like cells or cell populations produced by the methods of the present invention. Specific examples include LOH syndrome, Klinefelter's syndrome, testicular trauma, secondary hypogonadism, and cases in which biologically female individuals, such as transgender individuals, desire male hormone supplementation.

[0044] Pharmaceutical compositions or therapeutic agents for diseases associated with decreased testosterone levels containing human pluripotent stem cell-derived Leydig-like cells or cell populations as active ingredients may contain, in addition to the prepared Leydig-like cells, a pharmaceutically acceptable carrier. Examples of carriers include the medium used to culture the cells for the preparation of the Leydig-like cells. Furthermore, the human pluripotent stem cell-derived Leydig-like cells may have been subjected to immunoisolation.

[0045] The present invention also extends to a device for transplanting human pluripotent stem cell-derived endocrine cells. That is, the present invention also extends to a device before the human pluripotent stem cell-derived Leydig-like cell population of the present invention is adherently cultured thereon. As used herein, a "pluripotent stem cell-derived endocrine cell transplantation device" refers to a device into which pluripotent stem cell-derived endocrine cells can be transplanted, and which, when the pluripotent stem cell-derived endocrine cells are adherently cultured on the device, can effectively secrete hormones and other substances, specifically testosterone, produced by the pluripotent stem cell-derived endocrine cells. Materials usable as pluripotent stem cell-derived endocrine cell transplantation devices must be biocompatible, meaning they do not adversely affect the living body. Biocompatible materials are preferably biocompatible, and may be bioabsorbable after the transplanted human pluripotent stem cell-derived Leydig-like cell population has engrafted into the living body. Examples of such materials include synthetic polymers such as polyethylene terephthalate (PET), poly-L-lactic acid (PLLA), polyglycolic acid (PGA), copolymers of lactic acid and glycolic acid (PLGA), and polycaprolactone (PCL). Natural polymers such as collagen, gelatin, glycosaminoglycans, chitin, chitosan, hyaluronic acid, and polypeptides can also be used. The shape of the device for transplanting pluripotent stem cell-derived endocrine cells is not particularly limited, and sheet-like structures such as biocompatible membranes can be used.

[0046] The present invention also encompasses a cell transplantation composition comprising a pluripotent stem cell-derived endocrine cell transplantation device to which a population of human pluripotent stem cell-derived Leydig-like cells of the present invention is attached. The human pluripotent stem cell-derived Leydig-like cells attached to the cell transplantation device may be further subjected to immunoisolation treatment. The cell transplantation composition can be prepared by adherently culturing the population of human pluripotent stem cell-derived Leydig-like cells of the present invention on the cell transplantation device. The shape of the cell transplantation composition is not particularly limited as long as it is a shape that allows cells to be transplanted, and may be, for example, a sheet or capsule shape.

[0047] As used herein, "immunoisolation" refers to a process that allows water, nutrients, and hormones to pass through without interfering with angiogenesis, but blocks immune cells and transplanted cells, preventing the transplanted cells from being rejected in the local environment. Immunoisolation can be achieved by any known method or any method that will be developed in the future. For example, immunoisolation can be achieved by embedding cells in beads or capsules made of alginate gel, agarose, anisotropic materials, polysulfone (PSF), nanofiber mats, polyimide, tetrafluoroethylene / polytetrafluoroethylene (PTFE), ePTFE, polyacrylonitrile, polyethersulfone, acrylic resin, cellulose acetate, cellulose nitrate, polyamide, or hydroxypropylmethylcellulose (HPMC) membranes. Such immunoisolation can avoid problems associated with cell transplantation, such as immune rejection, cell proliferation, and infiltration. The human pluripotent stem cell-derived Leydig-like cells produced by immunoisolation treatment can be used to treat conditions requiring continuous supply of substances produced by human pluripotent stem cell-derived Leydig-like cells, specifically testosterone, to an organism, even if they are not derived from the individual's own cells (autologous cells).

[0048] The present invention also encompasses a method for transplanting a human pluripotent stem cell-derived Leydig-like cell population prepared by the method of the present invention. The transplantation method can be achieved by transplanting the cell population, together with a pluripotent stem cell-derived endocrine cell transplantation device to a desired site in a living body, preferably subcutaneously. Similar to the target of the pharmaceutical composition described above, the human pluripotent stem cell-derived Leydig-like cell population of the present invention can be transplanted to diseases associated with decreased testosterone levels or conditions requiring testosterone supplementation. Specific examples include LOH syndrome, Klinefelter's syndrome, testicular trauma, secondary hypogonadism, and cases in which biologically female individuals, such as transgender individuals, desire male hormone supplementation.

[0049] The present invention also encompasses a method for treating diseases associated with decreased testosterone levels by administering a pharmaceutical composition comprising a population of Leydig-like cells derived from human pluripotent stem cells as an active ingredient, a method for treating diseases associated with decreased testosterone levels by transplanting a cell transplantation composition comprising a device for transplanting pluripotent stem cell-derived endocrine cells to which a population of Leydig-like cells derived from human pluripotent stem cells has attached, or a method for treating symptoms requiring testosterone supplementation.

[0050] EXAMPLES The present invention will be described in detail below with reference to examples to deepen understanding of the present invention, but it goes without saying that the present invention is not limited to these examples.

[0051] Example 1: Method for producing Leydig-like cells In this example, a method for producing Leydig-like cells will be described (see FIG. 2).

[0052] 1. Cells Used In this example, differentiation induction treatment was carried out using iPS cells (FFPB3AB4).

[0053] 2. NR5A1 Gene According to the method described in Example 1 of Patent Document 3, the full-length NR5A1 gene (cDNA) identified by GenBank Accession NM_004959 (SEQ ID NO: 1) was cloned, an NR5A1 gene expression plasmid was prepared, and the gene was introduced into iPS cells (FFPB3AB4). TM NR5A1 was forced to be expressed using the Advanced expression induction system (Clontech).

[0054] 3. Induction of Leydig-like cell differentiation Tet-Off TM Human iPS cells (KW107_121-3_NR5A1_Leydig_P12+2(EX564)) that were forced to express the NR5A1 gene using the system were cultured in a closed culture vessel bag at a density of 3.6 × 10 cells per bag on the culture surface (non-adhesive surface) that allows suspension culture. 6Cells were seeded onto the cells. Culture was performed for 6 days using differentiation-inducing medium (DMEM medium (Invitrogen) containing 15% KSR (ThermoFisher, Scientific)) containing 20 mM CHIR99021, 100 μg / mL BMP4, and 100 μg / mL VEGF as a cytokine cocktail. Approximately 18,000 well-shaped depressions were contained per bag on the non-adhesive surface of the closed culture vessel. After 6 days of culture, one EB was formed from approximately 200 cells per depression (Figure 3). Figure 4 shows the morphology of EBs cultured using the culture plate used in Patent Document 3 and the closed culture vessel used in this example. The EBs produced in this example had a more homogeneous shape than EBs produced by conventional methods.

[0055] The closed culture vessel was then inverted with a medium for adherent culture (1 mM 8-Br-cAMP, 10 μM Y27632, and 100 μM forskolin, but without KSR, containing 10% FBS; DMEM medium (Invitrogen)). The vessel was then inverted so that the culture surface suitable for adherent culture (the adhesive surface) faced the bottom. A cell suspension containing KW107_121-3_NR5A1_Leydig_P12+2 cells, which had formed EBs in the closed culture vessel, was placed on the adhesive surface (Figure 5). Figure 6 shows the morphology of EBs before and after inversion on day 6 of culture. The cells were cultured for an additional 11 days with the cells in contact with the adhesive surface. On day 17, 8-Br-cAMP was removed. The cells were then maintained in adherent culture in DMEM medium (Invitrogen) containing 10% FBS and 100 μM forskolin.

[0056] In the following experimental examples, the properties of the Leydig-like cells prepared by the method of the present invention were confirmed.

[0057] (Experimental Example 1-1) Cell Morphology and Testosterone Concentration in Culture Supernatants In this experimental example, cells prepared by culture in a closed culture vessel according to the method of Example 1 and cells prepared by a conventional method (Example 1 of Patent Document 3) using open culture vessels for suspension culture and adherent culture were observed for cell morphology on day 18 of culture, and testosterone levels in the culture supernatants were measured. Testosterone concentrations were measured by ECLIA (electrochemiluminescence immunoassay). The adherent cells were spindle-shaped, with no morphological differences, and the testosterone concentrations in the culture supernatants were comparable to those obtained by the conventional method (Figure 7).

[0058] (Experimental Example 1-2) Leydig cell marker expression In this experimental example, the expression of HSD17B3, a Leydig cell marker, was confirmed by cell immunostaining in the cultured cells on day 18 of culture prepared in Example 1. As a result, the expression of HSD17B3 was confirmed ( FIG. 8 ).

[0059] (Experimental Example 1-3) Transition of Testosterone Secretion Amounts in Cultured Cells In this Experimental Example, testosterone concentrations in the culture supernatants of cells prepared by the method of Example 1 using closed culture vessels and cells prepared by a conventional method (Example 1 of Patent Document 3) using open culture vessels for suspension culture and adherent culture, respectively, were measured over time. Both cells were KW111_3AB4_2S6_NR5A1_hiPSC-derived Leydig-like cells. Testosterone concentrations were measured using the same method as in Experimental Example 1-1. In the first experiment, measurements were made from days 8 to 22 of culture, and in the second experiment, measurements were made from days 8 to 32 of culture. The results showed that the testosterone concentrations of Leydig-like cells prepared by the method of the present invention were comparable to those of the conventional method (Figure 9).

[0060] As described above in detail, in the process of producing human pluripotent stem cell-derived Leydig-like cells, by using a closed culture vessel and then performing suspension culture in the culture vessel followed by adhesion culture, the medium change process is easier than in conventional production methods in which culture is performed using an open culture vessel and the culture vessel is replaced when switching from suspension culture to adhesion culture, and the risk of biological contamination can be reduced. Furthermore, the produced Leydig-like cells can stably and continuously secrete testosterone comparable to that produced by conventional methods. Furthermore, the method of the present invention allows for easy scale-up of large-scale culture, enabling the mass production of Leydig-like cells for practical use.

Claims

1. A method for producing Leydig-like cells derived from human pluripotent stem cells, comprising a culture step of using a closed culture vessel to culture the cells in suspension and then culturing them in adhesion culture.

2. A method for producing Leydig-like cells derived from human pluripotent stem cells as described in claim 1, wherein the closed culture vessel is a closed culture vessel that includes a culture surface inside the vessel on which floating cells can be cultured in suspension and a culture surface on which adhesive cells can adhere.

3. A method for producing Leydig-like cells derived from human pluripotent stem cells as described in claim 2, characterized in that a closed culture vessel is used in which a culture surface inside the vessel capable of suspension culture and a culture surface capable of adhesive culture are arranged so that cells can be cultured on either culture surface while the vessel is left stationary.

4. A method for producing Leydig-like cells derived from human pluripotent stem cells as described in claim 2, characterized in that a closed culture vessel is used in which the culture surface inside the vessel is in the form of a well, allowing cell aggregates to be cultured in a floating state, allowing floating cells to be cultured.

5. A method for producing Leydig-like cells derived from human pluripotent stem cells according to claim 1, wherein the closed culture vessel has a medium exchange port.

6. A method for producing Leydig-like cells derived from human pluripotent stem cells according to claim 1, wherein the closed culture vessel is a bag-shaped culture vessel made of a soft packaging material.

7. A method for producing Leydig-like cells derived from human pluripotent stem cells according to claim 1, wherein the culture process using a closed culture vessel to perform suspension culture within the culture vessel followed by adhesion culture includes a step of culturing cells on a culture surface inside the vessel where suspension cells can be cultured in suspension, and then inverting the cell suspension onto a culture surface where adherent cells can be cultured in adhesion.

8. A method for producing Leydig-like cells derived from human pluripotent stem cells according to claim 1, comprising the steps of culturing human pluripotent stem cells in suspension until they begin to form embryoid bodies, and then culturing them in adherence.

9. A method for producing Leydig-like cells derived from human pluripotent stem cells as described in claim 8, which includes a step of forcibly expressing NR5A1 in human pluripotent stem cells during the process of initiating embryoid body formation from human pluripotent stem cells.

10. A method for producing Leydig-like cells derived from human pluripotent stem cells according to claim 9, comprising the steps of forcibly expressing NR5A1 in human pluripotent stem cells in the presence of one or more species selected from the group consisting of a WNT canonical pathway activator, BMP4, and VEGF, followed by suspension culture for 2 to 10 days, and then adhesion culture.

11. The method for producing Leydig-like cells derived from human pluripotent stem cells described in claim 10, comprising the step of removing the WNT canonical pathway activator, BMP4, and VEGF present in the adherent culture step, and then adding cAMP.

12. The method for producing Leydig-like cells derived from human pluripotent stem cells according to claim 11, which comprises a step of removing cAMP from the culture system between days 2 and 40 after the start of the treatment with cAMP.

13. The method for producing Leydig-like cells derived from human pluripotent stem cells described in claim 10, comprising the step of removing the WNT canonical pathway activator, BMP4, and VEGF present in the adherent culture step, followed by treating the cells with forskolin.

14. Leydig-like cells derived from human pluripotent stem cells produced by the method of claim 1.

15. The human pluripotent stem cell-derived Leydig-like cells described in claim 14, which are Leydig-like cells that produce testosterone continuously for at least 60 days or more.

16. Human pluripotent stem cell-derived Leydig-like cells according to claim 14, which have been passaged at least once.

17. A human pluripotent stem cell-derived Leydig-like cell population comprising the human pluripotent stem cell-derived Leydig-like cells according to claim 14.

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