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

Abstract

This invention provides a method for producing human pluripotent stem cell-derived Leydig-like cells that is simpler, more efficient, reduces the risk of biological contamination, and allows for mass culture of cells. [Solution] The method for producing human pluripotent stem cell-derived Leydig-like cells includes a closed culture vessel containing a culture surface on which suspension cells can be cultured and a culture surface on which adherent cells can adhere, and the culture step involves performing suspension culture in the culture vessel followed by adherent culture.

Description

[Technical Field]

[0001] This invention relates to a method for producing human pluripotent stem cell-derived Leydig-like cells, and more specifically, to a cell culture method in the production step. Furthermore, it relates to human pluripotent stem cell-derived Leydig-like cells produced by the method described above, and to a cell population thereof. [Background technology]

[0002] In recent years, late-onset hypogonadism (LOH syndrome), a condition characterized by various symptoms resulting from a decline in male hormones with age, has attracted attention. Testosterone replacement therapy (HRT) is currently used to treat this condition. However, HRT results in hormone levels that differ from the physiological secretion pattern and has a short duration of action, requiring repeated administration and placing a significant burden on patients. There is a strong desire for the development of new treatment methods that eliminate the need for such repeated administration, reduce the time and effort spent on hospital visits, and alleviate pain associated with injections, thereby improving the quality of life (QOL).

[0003] The hypothalamus produces gonadotropin-releasing hormone (GnRH). The anterior pituitary gland responds to GnRH production by producing luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Leydig cells in the testes are said to respond to LH by producing 5-10 mg / day of testosterone. Testosterone is synthesized from cholesterol via several intermediate compounds, including dehydroepiandrosterone (DHEA) and androstenedione.

[0004] Attempts are being made to develop methods for inducing differentiation into various cell types using pluripotent stem cells, which possess self-renewal and multipotency. Among pluripotent stem cells, methods using embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) are attracting particular attention. Various attempts have been made to differentiate various mesenchymal stem cells into steroid hormone-producing cells of the testes and adrenal glands by stably introducing the transcription factor SF-1 (Steroidogenic factor-1) and adding cAMP (cyclic adenosine monophosphate) 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.

[0005] There are reports on a method for introducing SF-1 into two types of human ES cells (H9 strain and KhES1 strain) and one type of human iPS cell line (201B7 strain) to induce differentiation into steroid hormone-producing cells. In this method, each type of human ES cell and human iPS cell is differentiated into mesodermal cells using BIO (6-bromoindirubin-3'-oxime) or GSK-3 (Glycogen synthase kinase 3) inhibitors. The mesodermal cells are then sorted using flow cytometry, and SF-1 is introduced into these mesodermal cells using a plasmid containing SF-1 expression pCMFlag-hsNR5A1, and expression is achieved. Furthermore, the cells are cultured in a medium containing 8-Br-cAMP (8-Bromo-cAMP) to differentiate them into steroid hormone-producing cells (Non-Patent Literature 1). However, while the production of progesterone and cortisol is mentioned, there is no report on testosterone production, and the expression of markers that selectively indicate Leydig cells (such as INSL3, 17βHSD3, and LHCGR) is not shown.

[0006] A method has been disclosed for differentiating mouse-derived pluripotent stem cells (ES cells) into mesenchymal stem cells, and then conditionally expressing SF-1 to differentiate them into steroid hormone-producing cells (Patent Document 1). However, there are no reports on the production of hormones such as testosterone or cortisol, nor is the expression of markers that serve as indicators for Leydig cells shown.

[0007] There is a report demonstrating that mouse ES cells (OriCellStrain C57BL / 6mESCs) were introduced with a lentiviral vector to create SF-1+ESC cells (ESC-SF-1), which were then cultured in a medium containing 8-Br-cAMP and forskolin to induce differentiation into progenitor Leydig cells (PLCs). This report shows that treating ESC-SF-1 with 8-Br-cAMP and Forskolin was more effective in inducing differentiation into PLCs than treating with 8-Br-cAMP alone. Furthermore, it has been reported that the obtained PLCs were transplanted into rats as Leydig-like cells, and markers indicating Leydig cells were measured from serum samples, etc. (Non-Patent Literature 2).

[0008] A method for producing human pluripotent stem cell-derived Leydig-like cells expressing NR5A1, INSL3, and at least one marker selected from 17βHSD3 and LHCGR has been disclosed (Patent Document 2). A method for producing human pluripotent stem cell-derived Leydig-like cells expressing HSD17B3, INSL3, and LHCGR has also been disclosed (Non-Patent Document 3). A method for producing human pluripotent stem cell-derived Leydig-like cells has been disclosed, which includes a step of culturing human pluripotent stem cells under culture conditions with a cytokine cocktail added and forcing NR5A1 expression, and adjusting the timing of cAMP addition and removal, suspension culture, adherent culture, etc. (Patent Document 3). However, improvements are desired in the culture step of the human pluripotent stem cell-derived Leydig-like cell production process, as it involves the complexity of transferring cells and changing the culture medium during differentiation induction. Furthermore, large-scale culture is desired for practical application. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Endocrinology, 153(9): 4336-4345 (2012) [Non-Patent Document 2] STEM CELLS AND DEVELOPMENT, 24(4): 459-470(2015) [Non-Patent Document 3] Endocrinology, 2021, Vol.162, No.12,1-11, https: / / doi.org / 10.1210 / endocr / bqab202 [Patent Documents]

[0010] [Patent Document 1] Japanese Patent Publication No. 2011-15630 [Patent Document 2] International Publication WO2018 / 088240 (Patent No. 6979702) [Patent Document 3] International Publication WO2023 / 157727

Summary of the Invention

Problems to be Solved by the Invention

[0011] In the process of producing Riedel-like cells derived from human pluripotent stem cells, in the differentiation induction step of human pluripotent stem cells, it is an object of the present invention to introduce a cell culture process that is simpler, more efficient, can suppress risks such as biological contamination, and enables mass culture.

Means for Solving the Problems

[0012] As a result of intensive research to solve the above problems, the present inventors have found that in the process of producing Riedel-like cells derived from human pluripotent stem cells, by introducing a culture process in which suspension culture is performed in a closed culture vessel and then adherent culture is performed, the above problems can be solved, and the present invention has been completed. More specifically, it is by introducing a culture process using a closed culture vessel that includes a culture surface on which floating cells can be suspended and a culture surface on which adherent cells can adhere inside the vessel.

[0013] That is, the present invention comprises the following. 1. A method for producing Riedel-like cells derived from human pluripotent stem cells, comprising a culture process in which suspension culture is performed in a closed culture vessel and then adherent culture is performed in the closed culture vessel in the process of producing Riedel-like cells from human pluripotent stem cells. 2. The method for producing Riedel-like cells derived from human pluripotent stem cells according to item 1 above, wherein the closed culture vessel is a closed culture vessel that includes a culture surface on which floating cells can be suspended and a culture surface on which adherent cells can adhere inside the vessel. 3. The method for producing Riedel-like cells derived from human pluripotent stem cells according to item 2 above, characterized in that a closed culture vessel is used in which the culture surface on which floating culture can be performed and the culture surface on which adhesion can occur inside the vessel are arranged so that cells can be cultured on either culture surface in a state where the vessel is stationary. 4. A method for producing human pluripotent stem cell-derived Lydig-like cells according to item 2 above, characterized by using a closed culture container in which the culture surface on which floating cells inside the container can be cultured in suspension culture has a well-like form in which cell aggregates can be cultured in a floating state. 5. A method for producing human pluripotent stem cell-derived Lydig-like cells according to item 1 above, wherein the closed culture container has a medium exchange port. 6. A method for producing human pluripotent stem cell-derived Lydig-like cells according to item 1 above, wherein the closed culture container is a bag-shaped culture container made of a soft packaging material. 7. A culture process that includes performing adherent culture after suspension culture in the culture container using a closed culture container, which includes a step of culturing cells on a culture surface on which floating cells inside the container can be cultured in suspension culture, and then inverting the cell suspension onto a culture surface on which adherent cells can be cultured for culture. A method for producing human pluripotent stem cell-derived Lydig-like cells according to item 1 above. 8. A method for producing human pluripotent stem cell-derived Lydig-like cells according to item 1 above, including a step of performing suspension culture until the formation of embryoid bodies is initiated from human pluripotent stem cells, and then performing adherent culture. 9. A method for producing human pluripotent stem cell-derived Lydig-like cells according to item 8 above, including a step of forcibly expressing NR5A1 in human pluripotent stem cells in the process until the formation of embryoid bodies is initiated from human pluripotent stem cells. 10. A method for producing human pluripotent stem cell-derived Lydig-like cells according to item 9 above, including a step of forcibly expressing NR5A1 in human pluripotent stem cells in the presence of one or more selected from the group consisting of a WNT canonical pathway activator, BMP4, and VEGF, performing suspension culture for 2 to 10 days, and then performing adherent culture. 11. A method for producing human pluripotent stem cell-derived Lydig-like cells according to item 10 above, including a step of removing the existing WNT canonical pathway activator, BMP4, and VEGF and then adding cAMP in the step of the adherent culture. 12. A method for producing human pluripotent stem cell-derived Lydig-like cells according to item 11 above, including a step of removing cAMP from the culture system between the 2nd and 40th days after the start of treatment with the cAMP. 13. A method for producing human pluripotent stem cell-derived Leydig-like cells as described in paragraph 10, further comprising the step of removing the WNT canonical pathway activator, BMP4, and VEGF present in the adhesion culture step, followed by treatment with forskolin. 14. Leydig-like cells derived from human pluripotent stem cells, prepared by the method described in item 1 above. 15. Human pluripotent stem cell-derived Leydig-like cells as described in item 14 above, wherein the human pluripotent stem cell-derived Leydig-like cells continuously produce testosterone for at least 60 days. 16. Human pluripotent stem cell-derived Leydig-like cells described in item 14 above, after being passaged at least once. 17. A population of human pluripotent stem cell-derived Leydig-like cells, including the human pluripotent stem cell-derived Leydig-like cells described in item 14 above. [Effects of the Invention]

[0014] The present invention's method for producing human pluripotent stem cell-derived Leydig-like cells introduces a culture step in which cells are cultured in a closed culture vessel in suspension mode before being moved to adherent culture mode. This makes it easier to change the culture medium compared to conventional methods that use open culture vessels and require changing the culture vessel when transitioning from suspension to adherent culture mode. Furthermore, there is no need to transfer cells from suspension to adherent culture dishes during differentiation induction, thus reducing the risk of biological contamination. The produced Leydig-like cells can stably and continuously secrete testosterone, comparable to that produced by conventional methods. [Brief explanation of the drawing]

[0015] [Figure 1] This figure shows one embodiment of a closed-system culture vessel used in the present invention. [Figure 2] This diagram illustrates a method for producing Leydig-like cells derived from human pluripotent stem cells. (Example 1) [Figure 3]This figure shows the steps involved in the production of human pluripotent stem cell-derived Leydig-like cells, from human pluripotent stem cells (iPS cells) to embryoid body (EB) formation up to day 6. (Example 1) [Figure 4] This is a photographic diagram showing the shape of EB when cultured using the culture plate used in Patent Document 3 as a comparative example and the closed-system culture vessel used in this example. (Example 1) [Figure 5] This figure shows the steps involved in the preparation of human pluripotent stem cells (iPS cells) up to 17 days of culture in a method for producing Leydig-like cells derived from human pluripotent stem cells. (Example 1) [Figure 6] This is a photographic diagram showing the shape of the closed culture vessel before and after inversion of EB on day 6 of culture. (Example 1) [Figure 7] This is a photographic diagram showing the shape of cells cultured using a closed-system culture vessel in Example 1 and cells cultured using the method described in Example 1 of Patent Document 3 as a comparative example, and the testosterone concentration in the culture supernatant is shown. (Experimental Example 1-1) [Figure 8] This photograph shows the results of confirming the expression of the Leydig cell marker HSD17B3 in cultured cells prepared in Example 1 after 18 days of culture, using cell immunostaining. (Experimental Example 1-2) [Figure 9] This figure shows the results of measuring the testosterone concentration in the culture supernatant after long-term culture of cells cultured using the closed-system culture vessel of Example 1 and cells cultured using the method of Example 1 in Patent Document 3 as a comparative example. (Experimental Examples 1-3) [Modes for carrying out the invention]

[0016] The present invention relates to a method for producing human pluripotent stem cell-derived Leydig-like cells, and more specifically, to a cell culture method in the process of producing the human pluripotent stem cell-derived Leydig-like cells. Furthermore, it relates to human pluripotent stem cell-derived Leydig-like cells produced by the above method and the cell population thereof.

[0017] In this specification, "Leydig-like cells" refers to cells obtained by differentiation induction treatment of pluripotent stem cells, and is used to distinguish them from naturally occurring "Leydig cells." The "human pluripotent stem cell-derived Leydig-like cells" of the present invention refer to cells obtained by differentiation induction treatment of human pluripotent stem cells that 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. Furthermore, cells that exhibit testosterone production ability are also referred to as "testosterone-producing cells" in this specification.

[0018] In this specification, "pluripotent stem cells" refer to undifferentiated cells that possess both "self-regeneration ability," which allows them to proliferate while maintaining an undifferentiated state, and "differentiation pluripotency," which allows them to differentiate into all three germ layer lineages. Examples include ES cells (Embryonic Stem Cells) and induced pluripotent stem cells (iPS cells). The method of the present invention uses human pluripotent stem cells. Human iPS cells are very similar in properties to human ES cells, but differences have been reported between them and mouse ES cells, for example, in transcription factor networks, epigenetics, and responsiveness to extracellular factors (STEM CELLS 2010; 28: p.419-430, Experimental Medicine 2012; 30, No.10: p.1544-1548, Progress in Medicine 2011; 239, No.14: p.1247-1251). Accordingly, in this specification, human pluripotent stem cells and pluripotent stem cells from non-human animals are considered separately.

[0019] In this specification, "ES cells" generally refer to pluripotent stem cells isolated as an undifferentiated stem cell population from a cell mass called the inner cell mass located inside a blastocyst-stage embryo, which is then cultured in vitro. Many human ES cell lines have already been established and can be obtained from ES Cell International, the Wisconsin Alumni Research Foundation, the National Stem Cell Bank (NSCB), and other sources.

[0020] In this specification, "iPS cells" refers to induced pluripotent stem cells that possess pluripotency and proliferative capacity similar to ES cells, achieved by introducing several genes into somatic cells to induce the reprogramming of differentiated cells without using oocytes, embryos, or 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 known production methods or methods to be developed in the future. Furthermore, there are no particular restrictions on the cells used to produce the iPS cells.

[0021] The culture method for pluripotent stem cells such as ES cells or iPS cells, and the culture medium used for culture, are not particularly limited and can be any known or future culture mediums or technologies. As a culture medium capable of maintaining the undifferentiated and pluripotent state of ES cells or iPS cells, and suitable for differentiation induction, for example, a commercially available mammalian cell basal medium such as DMEM and / or DMEM / F12 can be used with serum or serum replacement (KnockOut TMSerum Replacement (KSR, ThermoFisher, SCIENTIFIC), and a mixture containing bFGF, etc., commercially available primate ES cell culture media, primate ES cell proliferation basal medium hESF-GRO, primate ES cell differentiation induction basal medium hESF-DIF, primate ES cell proliferation medium CSTI-7, etc. can be used. Specifically, culture can be performed using the media exemplified in Patent Document 2.

[0022] In this specification, "closed culture vessel" refers to a sealed culture vessel that allows cell culture without opening or closing the vessel, and has a structure that allows for the exchange of culture medium via a tube or other device connected to the vessel through an opening (see Figure 1). The use of closed culture vessels is particularly advantageous in terms of hygiene, as it reduces the risk of biological contamination during culture or culture medium exchange, for example, and is superior in that it reduces the burden on the culture environment.

[0023] The "closed culture vessel" used in the present invention is superior in that it allows for suspension culture within the closed culture vessel followed by adherent culture without changing the culture vessel. The closed culture vessel is a culture vessel having both a suspension culture surface and an adherent culture surface, and is equipped with a culture surface on which suspension cells can be cultured in suspension (hereinafter also referred to as the "non-adherent culture surface") and a culture surface on which adherent cells can adhere (hereinafter also referred to as the "adherent culture surface"). The non-adherent culture surface and the adherent culture surface inside the vessel are arranged so that cells can be cultured on either the culture surface when the vessel is standing still. For example, as shown in Figure 1, the closed culture vessel has a bag-like shape, with the non-adherent culture surface located at the bottom when the vessel is standing still and the adherent culture surface located at the top. When a cell suspension is introduced and cultured using a vessel of this shape, cells can be cultured in suspension with the non-adherent culture surface located at the bottom, and then by inverting the top and bottom surfaces, the cell suspension of the suspension-cultured cells comes into contact with the adherent culture surface, allowing for adherent culture. It should be noted that the closed culture vessel shown in Figure 1 is only one embodiment, and it is clear that it should not be limited to the form shown in Figure 1.

[0024] In the method for producing Leydig-like cells of the present invention, human pluripotent stem cells can be cultured in suspension until embryoid bodies (EBs) begin to form, and then adherent culture can be performed. 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 EBs begin to form, and then adherent culture can be performed by inverting the closed culture vessel without transferring the cell suspension to another culture vessel. More specifically, human pluripotent stem cells can be cultured in suspension for 10 days, 8 days, more preferably 6 days from the start of culture. EBs are formed during the suspension culture process of human pluripotent stem cells 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.

[0025] For suspension culture of human pluripotent stem cells until EB formation begins, it is preferable to culture them on a culture surface having well-like depressions that allow for the formation of spherical small cell aggregates (small spheres) and spherical large cell aggregates (large spheres). Therefore, the non-adherent culture surface placed in the closed culture vessel used in the present invention is preferably a well-like shape that allows for the culture of cell aggregates in a suspended state. For example, in a suspension culture system, single-cell suspension cells can be seeded to form EBs of uniform size. A well-like shape that allows for the formation of approximately 50 to 20,000 cells, preferably approximately 50 to 5,000 cells, and more preferably approximately 100 to 200 cells per EB is preferable.

[0026] The closed-system culture vessel used in the present invention allows for the supply and exchange of culture medium necessary for cultivation through an opening (culture medium exchange port) connected to the vessel via a tube or the like. Furthermore, to maintain the required concentration of gaseous state during cultivation, gas containing, for example, carbon dioxide may be supplied through the opening. The placement of the opening is not particularly limited and should be positioned where suspension culture and / or adherent culture are possible.

[0027] The material of the closed-system culture vessel is not particularly limited, as long as it allows for suspension culture and adhesive culture. A suitable material for suspension culture or adhesive culture in a closed system is a material that allows gas exchange so that gases such as oxygen and carbon dioxide can be maintained at appropriate concentrations during culture, or a material that is designed to allow gas exchange. For example, it may be a flexible packaging material or a rigid packaging material. Preferably, it is a packaging material, and olefin resins such as polyethylene and polypropylene can be suitably used. Other materials that can be used include polystyrene, polymethylpentene, cyclic olefin polymer, cyclic olefin copolymer, 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, polyethylene chloride, and the like. Furthermore, 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, and nylon-based thermoplastic elastomers can also be used. In addition, thermosetting elastomers such as urethane rubber, silicone rubber, and fluororubber, as well as thermosetting resins such as phenolic resin, epoxy resin, melamine resin, urea resin, unsaturated polyester resin, alkyd resin, urethane resin, and thermosetting polyimide can also be used.

[0028] For suspension culture and / or adherent culture, the culture surface of the culture vessel may be coated with a known or future-developed coating agent and / or surface-treated. The coating agent is not particularly limited, but a non-adherent surface can be created by using, for example, an untreated polyethylene film, maintaining a high static water contact angle of 80° to 100°, thereby preventing adherent cells from adhering. Alternatively, a non-adherent surface can be created by, 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. Examples of cell adhesion inhibitors include phospholipid polymers, polyvinyl alcohol derivatives, phospholipid polymer complexes, polyhydroxyethyl methacrylate, polyvinyl alcohol, agarose, chitosan, polyethylene glycol, albumin, etc., and these may 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 culture, but examples include Matrigel. TM Collagen, gelatin, laminin, heparan sulfate proteoglycan, and entactin (manufactured by BD), or combinations thereof, may also be used. For surface treatment of the substrate that will be the adhesion surface of the culture vessel, for example, plasma treatment, excimer treatment, corona treatment, etc., can be applied.

[0029] The method for producing human pluripotent stem cell-derived Leydig-like cells according to the present invention can be adapted to the method described in Patent Document 3. Specifically, the process from human pluripotent stem cells to the initiation of EB formation may include a step of forcibly expressing NR5A1 in human pluripotent stem cells. More specifically, the method may include a step of forcibly expressing NR5A1 in human pluripotent stem cells in the presence of one or more 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 used in the present invention, and then adherent culture. In the adherent culture step, the method may include a step of removing the present WNT canonical pathway activator, BMP4, and VEGF, and adding cAMP.

[0030] In the preparation of human pluripotent stem cell-derived Leydig-like cells according to the present invention, the NR5A1 gene is introduced into human pluripotent stem cells, and embryoid bodies (EBs) are formed. To form EBs, a cytokine cocktail, specifically one or more cytokines selected from the group consisting of WNT canonical pathway activators, BMP4 (Bone Morphogenetic Protein 4), and VEGF (vascular endothelial growth factor), can be added to the culture system of human pluripotent stem cells. WNT canonical pathway activation refers to classical Wnt pathway activation, also known as activation of the β-catenin pathway, in which β-catenin regulates gene expression. Examples of WNT canonical pathway activators include glycogen synthase kinase 3 (GSK-3) inhibitors, and more specifically, CHIR99021. The concentration of the WNT canonical pathway activator is not particularly limited but can be appropriately selected within a range 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 is not particularly limited but can be appropriately selected within a range 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 is not particularly limited but can be appropriately selected within a range 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.

[0031] Human pluripotent stem cells are preferably cultured in suspension for 10 days, 8 days, more preferably 6 days, from the start of culture. EBs are formed during the suspension culture process of human pluripotent stem cells 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.

[0032] In this specification, the "NR5A1 gene" can be identified, for example, by GenBank Accession No. NM_004959 (SEQ ID NO: 1). As a method for introducing the NR5A1 gene into human pluripotent stem cells, any method known per se or any method to be developed in the future can be applied. The cDNA of NR5A1 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.

[0033] The present invention includes a step of forcibly expressing a transgene in human pluripotent stem cells into which the above NR5A1 gene has been introduced. The gene can be expressed using any gene expression system known per se or to be developed in the future. The timing of gene expression can be controlled using any method known per se or to be developed in the future. As an experimental system capable of reversibly regulating the expression of a target gene in cells or the like, there is a Tet-On / Tet-Off system that expresses a gene dependently on tetracycline. TM / Off TM According to such an expression system, the expression of the target gene can be regulated by the presence or absence of addition of an antibiotic tetracycline or a tetracycline derivative doxycycline (Dox) to the culture medium. According to the Tet-Off system, for example, a Tet repressor (TetR) that functions in the Escherichia coli tetracycline resistance operon and a Tet operator sequence (tetO sequence) are used. TetR binds to the tetO sequence in the absence of tetracycline, and NR5A1 can be forcibly expressed. TM In the method for producing human pluripotent stem cell-derived Lydig-like cells of the present invention, either the Tet-Off system or the Tet-On system may be used to control the expression of the NR5A1 gene. However, it is preferable to use the Tet-Off system that forcibly expresses NR5A1 in the absence of tetracycline or doxycycline.

[0034] In the method for producing human pluripotent stem cell-derived Lydig-like cells of the present invention, either the Tet-Off system or the Tet-On system may be used to control the expression of the NR5A1 gene. However, it is preferable to use the Tet-Off system that forcibly expresses NR5A1 in the absence of tetracycline or doxycycline. TM system and Tet-On TM system may be used to control the expression of the NR5A1 gene, but it is preferable to use the Tet-Off system that forcibly expresses NR5A1 in the absence of tetracycline or doxycycline. TM system. TMBy using this system, differentiation into human pluripotent stem cells (HPS) that have been pre-transmitted with the NR5A1 gene can be suppressed by culturing them in the presence of tetracycline or doxycycline. Then, by removing tetracycline or doxycycline at the time when differentiation into HPS cells is necessary, NR5A1 can be continuously forced to be expressed. Furthermore, since tetracycline or doxycycline is removed during the continuous forced expression of NR5A1, undesirable effects from these drugs can be avoided, making this method preferable. As a result, differentiation into human pluripotent stem cells (HPS) can be more effectively controlled and promoted.

[0035] Although Patent Document 2 (International Publication WO2018 / 088240) also describes Tet-on / off in its examples, Patent Document 2 describes Tet-On as the forced expression of NR5A1 in the presence of tetracycline or doxycycline. TM The description is based on the system. The Tet-off system in the example of Patent Document 2 describes a case where NR5A1 is not forced to be expressed in the absence of tetracycline or doxycycline, which is the basis of the Tet-off of the present invention. TM This differs from the system.

[0036] The present invention provides a method for producing human pluripotent stem cell-derived Leydig-like cells, which includes a step of performing suspension culture in a closed culture vessel and then adhering culture of the cells without changing the culture vessel. Adhering culture refers to culturing in a monolayer state. Human mesoderm cells can be differentiated into Leydig-like cells during the adhering culture step. In the adhering culture step, it is preferable to remove the cytokine cocktail (one or more cytokines selected from the group consisting of WNT canonical pathway activator, BMP4, and VEGF that are present in the culture system) that was added to the human pluripotent stem cell culture system used in the suspension culture step.

[0037] In the above adhesion culture step, it is preferable to further treat with cAMP. When using 8-Br-cAMP as cAMP, it can be added to the culture medium at a concentration of 0.01 to 4 mM, preferably 0.1 to 1 mM. Along with the cAMP treatment, it is preferable to further stimulate with Forskolin. Forskolin can be added to the culture medium at a concentration of 0.1 to 100 μM, preferably 2 to 100 μM, and more preferably 5 to 100 μM. It is preferable to remove cAMP 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 17 days after the start of culture. On the other hand, it is preferable to continue adding Forskolin to the culture medium even after the cAMP has been removed.

[0038] The duration of adherent culture is not particularly limited and can be continued as long as it is culturable. Furthermore, cells can be subcultured during the adherent culture process. Cell subculturing is preferably done using cell-separating enzymes such as TrypLE Select, TrypLE Express, or Accutase. Alternatively, it can be done using ROCK inhibitors such as Y27632. The seeding density of cells during subculturing is not particularly limited as long as it is at a concentration in which the cells can survive, but for example, 1 to 1.0 × 10⁻⁶ is suitable. 6 The number of cells per well is preferably 1000 to 1.0 × 10 5 The number of cells / well is approximately 1.0 × 10⁶. 4 cells / well (9.6cm) 2 )

[0039] Furthermore, adherent cultured cells can be cryopreserved. The methods and conditions for cryopreserving cells are not particularly limited and can be any method practiced by those skilled in the art; any known method or any method developed in the future can be applied. For example, it is necessary to use a culture medium containing a cryoprotective agent such as DMSO (Dimethyl sulfoxide) or glycerol as the freezing medium, and commercially available media can also be used. An example of a commercially available media is STEM-CELLBANKER. TM(Zenogen Pharma), Recovery TM Cell Culture Freezing Medium (ThermoFisher, SCIENTIFIC) or Synth-a-Freeze TM Cryopreservation medium (ThermoFisher, SCIENTIFIC), etc., can be used. Cells can be frozen and stored, for example, at -80°C, and can also be stored at even lower temperatures in liquid nitrogen. Thawing of frozen cells can be done by any method practiced by those skilled in the art, and is not particularly limited; any method already known or any method developed in the future can be applied.

[0040] According to the method for producing human pluripotent stem cell-derived Leydig-like cells of the present invention, differentiation induction efficiency from human pluripotent stem cells to human pluripotent stem cell-derived Leydig-like cells is induced at a high rate 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 method of the present invention. The human pluripotent stem cell-derived Leydig-like cells of the present invention can continuously produce testosterone for at least 14 days, preferably 22 days or more, and more preferably 32 days or more. Furthermore, it is considered possible to continuously produce testosterone for 60 days or more, and even 120 days or more. Moreover, the Leydig-like cell population of the present invention is a human pluripotent stem cell-derived Leydig-like cell population that contains a high rate of 80% or more, preferably 90% or more, and more preferably 95% or more of Leydig-like cells capable of producing testosterone.

[0041] Leydig-like cells derived from human pluripotent stem cells, prepared by adherent culture in the above process, can be harvested and further cultured in suspension. Any culture vessel capable of three-dimensional culture can be used for suspension culture, and there are no particular restrictions; however, the same type of culture vessel used in the process of EB formation from human pluripotent stem cells or the process of forcing NR5A1 expression in human pluripotent stem cells can be used.

[0042] The present invention also extends to pharmaceutical compositions containing human pluripotent stem cell-derived Leydig-like cells or cell populations prepared by the method of the present invention as an active ingredient. Targets of the pharmaceutical compositions of the present invention include diseases associated with low testosterone levels and conditions requiring testosterone supplementation. The present invention also extends to therapeutic agents for diseases associated with low testosterone levels, containing human pluripotent stem cell-derived Leydig-like cells or cell populations prepared by the method of the present invention as an active ingredient. Specifically, examples include LOH syndrome, Klinefelter syndrome, testicular trauma, secondary hypogonadism, and cases where biologically female individuals, such as transgender people, desire male hormone supplementation.

[0043] A pharmaceutical composition containing human pluripotent stem cell-derived Leydig-like cells or cell populations as an active ingredient, or a therapeutic agent for diseases associated with testosterone deficiency, may contain a pharmaceutically acceptable carrier in addition to the produced Leydig-like cells. Examples of carriers include the culture medium used to produce the Leydig-like cells. Furthermore, the human pluripotent stem cell-derived Leydig-like cells may be subjected to immunoisolation.

[0044] The present invention also extends to devices for transplanting human pluripotent stem cell-derived endocrine cells. That is, the present invention also extends to devices used before adhering culture of the human pluripotent stem cell-derived Leydig-like cell population of the present invention. In this specification, "device for transplanting pluripotent stem cell-derived endocrine cells" means a device on which pluripotent stem cell-derived endocrine cells can be transplanted, and which, when pluripotent stem cell-derived endocrine cells are cultured on the device, is capable of effectively secreting substances such as hormones produced by the pluripotent stem cell-derived endocrine cells, specifically testosterone. Materials that can be used as devices for transplanting pluripotent stem cell-derived endocrine cells must have biocompatibility, meaning they do not have adverse effects on living organisms. As biocompatible materials, materials with biocompatibility are preferred, and after the transplanted human pluripotent stem cell-derived Leydig-like cell population has engrafted in the living organism, the material may be bioabsorbable. 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, but sheet-like structures such as biocompatible membranes can be used.

[0045] The present invention also extends to a cell transplantation composition comprising a pluripotent stem cell-derived endocrine cell transplantation device to which a population of human pluripotent stem cells-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 immunoisolated. The cell transplantation composition can be prepared by adhering and culturing the human pluripotent stem cell-derived Leydig-like cell population 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 to which cells can be transplanted, but it can be, for example, a sheet or a capsule.

[0046] In this specification, "immunoisolation treatment" refers to a treatment that allows water, nutrients, and hormones to pass through without hindering angiogenesis, but prevents immune cells and transplanted cells from passing through, thus preventing the transplanted cells from being rejected in the local environment. Immunoisolation treatment can be performed using any method that is already known or will be developed in the future. For example, immunoisolation treatment can be performed 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, hydroxypropyl methylcellulose (HPMC) membranes, etc. Such immunoisolation treatment can avoid problems such as immune rejection, cell diffusion, and infiltration associated with cell transplantation. Even if the human pluripotent stem cell-derived Leydig-like cells produced through immunoisolation are not derived from the organism's own cells (autologous cells), they can be used to treat conditions that require a continuous supply of substances produced by human pluripotent stem cell-derived Leydig-like cells, specifically testosterone, to the organism.

[0047] The present invention also extends to a method for transplanting a population of human pluripotent stem cells (Leydig-like cells) produced by the method of the present invention. The transplantation method can be achieved by transplanting the cell population, along with a pluripotent stem cell-derived endocrine cell transplantation device to which the cell population is attached, to a desired site in the body, preferably subcutaneously. The target patients for transplantation of the human pluripotent stem cell-derived Leydig-like cell population of the present invention are, as with the target patients for the pharmaceutical composition described above, diseases associated with testosterone deficiency and symptoms requiring testosterone replacement. Specifically, these include LOH syndrome, Klinefelter syndrome, testicular trauma, secondary hypogonadism, and cases where biologically female individuals, such as transgender people, desire to supplement male hormones.

[0048] The present invention also relates to a method for treating diseases associated with testosterone deficiency by administering a pharmaceutical composition containing a human pluripotent stem cell-derived Leydig-like cell population as an active ingredient, a method for treating diseases associated with testosterone deficiency by transplanting a cell transplantation composition including a pluripotent stem cell-derived endocrine cell transplantation device to which a human pluripotent stem cell-derived Leydig-like cell population is attached, or a method for treating symptoms requiring testosterone supplementation. [Examples]

[0049] 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.

[0050] (Example 1) Method for preparing Leydig-like cells This example describes a method for producing Leydig-like cells (see Figure 2).

[0051] 1.Cells used In this example, differentiation induction treatment was performed using iPS cells (FFPB3AB4).

[0052] 2. NR5A1 gene Following 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 constructed, and the gene was introduced into iPS cells (FFPB3AB4). Tet-Off TM NR5A1 was forcibly expressed using the Advanced Expression Induction System (Clontech).

[0053] 3. Induction of differentiation into Leydig-like cells Tet-Off TM Human iPS cells (KW107_121-3_NR5A1_Leydig_P12+2(EX564)) in which the NR5A1 gene was forcibly expressed in the system were cultured in a bag-shaped closed culture vessel, with 3.6 × 10⁶ cells per bag on the suspension culture surface (non-adherent surface).6 Cells were seeded. For culture, the cells were cultured for 6 days in a differentiation induction medium (DMEM medium (Invitrogen) containing 15% KSR (ThermoFisher, SCIENTIFIC)) containing a cytokine cocktail of 20 mM CHIR99021, 100 μg / mL BMP4, and 100 μg / mL VEGF. The non-adherent surface of the closed culture vessel contained approximately 18,000 well-like depressions per bag, and after 6 days of culture, one EB was formed from approximately 200 cells in one depression (Figure 3). Figure 4 shows the shape of the EBs when 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 compared to EBs produced by the conventional method.

[0054] The culture medium for adherent culture (1 mM 8-Br-cAMP, 10 μM Y27632, and 100 μM Forskolin, 10% FBS-containing DMEM medium (Invitrogen) without KSR) was replaced through the opening of the closed culture vessel. The closed culture vessel was then inverted so that the culture surface (adhesion surface) suitable for adherent culture was at the bottom, and the cell suspension containing KW107_121-3_NR5A1_Leydig_P12+2, which had formed EBs within the closed culture vessel, was placed on the adhesion surface of the closed culture vessel (Figure 5). The shape of the EBs before and after inversion on day 6 of culture is shown in Figure 6. The cells were cultured for a further 11 days with the cells in contact with the adhesion surface, and 8-Br-cAMP was removed on day 17. Subsequently, the cells were maintained by adherent culture in 10% FBS-containing DMEM medium (Invitrogen) with 100 μM Forskolin.

[0055] The properties of Leydig-like cells produced by the method of the present invention were confirmed in the following experimental examples.

[0056] (Experimental Example 1-1) Cell morphology and testosterone concentration in culture supernatant In this experiment, cells prepared by culturing in a closed culture vessel according to the preparation method of Example 1, and cells prepared by the conventional method (Example 1 of Patent Document 3) using an open culture vessel, with the culture vessels changed for suspension culture and adherent culture respectively, were observed for Leydig-like cells derived from KW111_3AB4_2S6_NR5A1_hiPSC (both were Leydig-like cells) on day 18 of culture. The amount of testosterone in the culture supernatant of each was measured. Testosterone concentration was measured by the ECLIA (Electrochemiluminescence immunoassay) method. As a result, all adherent cells were spindle-shaped with no morphological differences, and the testosterone concentration in the culture supernatant was comparable to that of the conventional method (Figure 7).

[0057] (Experimental Examples 1-2) Expression of Leydig cell markers In this experiment, the expression of the Leydig cell marker HSD17B3 was confirmed by cell immunostaining in cultured cells prepared in Example 1 on day 18. As a result, the expression of HSD17B3 was confirmed (Figure 8).

[0058] (Experimental Examples 1-3) Changes in testosterone secretion levels in cultured cells In this experiment, the testosterone concentration in the culture supernatant was measured over time for cells produced by culturing in a closed culture vessel according to the preparation method of Example 1, and cells produced by the conventional method (Example 1 of Patent Document 3) using an open culture vessel, with the culture vessels changed for suspension culture and adherent culture respectively. (Both were Leydig-like cells derived from KW111_3AB4_2S6_NR5A1_hiPSC). The testosterone concentration was measured using the same method as in Experimental Example 1-1. In the first experiment, measurements were taken from day 8 to day 22 of culture, and in the second experiment, measurements were taken from day 8 to day 32 of culture. As a result, the testosterone concentration of Leydig-like cells produced by the method of the present invention was comparable to that of the conventional method (Figure 9). [Industrial applicability]

[0059] As detailed above, in the process of producing human pluripotent stem cell-derived Leydig-like cells, using a closed culture vessel and performing suspension culture followed by adherent culture makes the medium exchange process easier compared to conventional methods that use open culture vessels and require changing the culture vessel when switching from suspension culture to adherent culture, thus reducing the risk of biological contamination. Furthermore, the produced Leydig-like cells can stably and continuously secrete testosterone, comparable to conventional methods. Moreover, the method of the present invention allows for easy scaling up to large-scale culture, enabling mass production of Leydig-like cells toward practical application.

Claims

1. A method for producing Leydig-like cells derived from human pluripotent stem cells, comprising a culture step in which suspension culture is performed in a closed culture vessel followed by adherent culture.

2. The method for producing human pluripotent stem cell-derived Leydig-like cells according to claim 1, wherein the closed culture vessel is a closed culture vessel that includes a culture surface inside the vessel to which suspension cells can be cultured in suspension and a culture surface to which adherent cells can adhere.

3. The method for producing human pluripotent stem cell-derived Leydig-like cells according to claim 2, characterized in that a closed culture vessel is used in which a culture surface capable of suspension culture and a culture surface capable of adhesion inside the vessel are arranged such that cells can be cultured on either culture surface when the vessel is standing still.

4. The method for producing human pluripotent stem cell-derived Leydig-like cells according to claim 2, characterized in that a closed culture vessel is used in which the culture surface inside the container, which allows for suspension culture of suspended cells, has a well-like shape that allows for culture of cell aggregates in a suspended state.

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

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

7. A method for producing human pluripotent stem cell-derived Leydig-like cells according to claim 1, wherein the culture step, which involves performing suspension culture in a closed culture vessel followed by adherent culture, includes the step of culturing the suspended cells on a culture surface inside the vessel that allows for suspension culture, and then inverting the cell suspension onto a culture surface that allows for adherent culture.

8. A method for producing human pluripotent stem cell-derived Leydig-like cells according to claim 1, comprising the step of performing suspension culture from human pluripotent stem cells until embryoid body formation begins, and then performing adherent culture.

9. A method for producing human pluripotent stem cell-derived Leydig-like cells according to claim 8, comprising the step of forcibly expressing NR5A1 in human pluripotent stem cells in the process from human pluripotent stem cells to the initiation of embryoid body formation.

10. A method for producing human pluripotent stem cell-derived Leydig-like cells according to claim 9, comprising the steps of forcing human pluripotent stem cells to express NR5A1 in the presence of one or more selected from the group consisting of a WNT canonical pathway activator, BMP4, and VEGF, culturing them in suspension for 2 to 10 days, and then performing adherent culture.

11. A method for producing human pluripotent stem cell-derived Leydig-like cells according to claim 10, comprising the step of removing the 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 claim 11, comprising the step of removing cAMP from the culture system between 2 and 40 days after the start of treatment with cAMP.

13. A method for producing human pluripotent stem cell-derived Leydig-like cells according to claim 10, comprising the step of removing the WNT canonical pathway activator, BMP4, and VEGF present in the adhesion culture step, and then treating with forskolin.

14. Leydig-like cells derived from human pluripotent stem cells, prepared by the method described in claim 1.

15. The human pluripotent stem cell-derived Leydig-like cells according to claim 14, wherein the human pluripotent stem cell-derived Leydig-like cells are Leydig-like cells that continuously produce testosterone for at least 60 days or more.

16. Human pluripotent stem cell-derived Leydig-like cells as described in claim 14, after being passaged at least once.

17. A population of human pluripotent stem cell-derived Leydig-like cells, including the human pluripotent stem cell-derived Leydig-like cells described in claim 14.

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