Method for producing marginal cells of the inner ear vascular stria, method for evaluating drugs, and cell culture for drug evaluation.

Abstract

The present invention provides a technology for creating functional inner ear stria vascularis marginal cells that are potentially applicable for screening of chemical agents and pathological analysis of hearing impairment. This method for producing inner ear stria vascularis marginal cells comprises a step for culturing a cell population including inner ear precursor cells, in an insulin-free culture medium that does not contain insulin or contains insulin only in a trace amount.

Description

[Technical Field]

[0001] This invention relates to a method for producing marginal cells of the inner ear's stria vascularis, a method for evaluating drugs, and a drug evaluation cell culture. [Background technology]

[0002] Hearing loss is a disease that significantly reduces quality of life, yet there is still no fundamental cure. Furthermore, hearing loss has recently attracted attention as a risk factor for dementia and other conditions, making the understanding of the mechanisms of hearing loss and the establishment of treatment methods important and urgent issues.

[0003] Until now, genetically modified mice and rodents such as drug-treated mice and rats have been mainly used as model animals for analyzing the mechanisms of hearing loss. However, there have been many reports of cases where the phenotype of rodent model animals does not necessarily match the pathology of humans, and in recent years, it has become particularly important to perform pathological analysis using human cells and tissues. However, it is difficult to anatomically and non-invasively collect the stria vascularis from humans, as with other inner ear tissues, and furthermore, since hearing loss itself is not a fatal disease, it is difficult to obtain human pathological tissue. For these reasons, it is not easy to analyze the mechanisms of hearing loss using human cells and tissues.

[0004] In recent years, differentiated cells derived from pluripotent stem cells, such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), have attracted attention as research tools to solve these problems. The inventors' research laboratory has also developed a method to induce differentiation of inner ear cell-like cells from human iPS cells (Patent Document 1), and is using this to construct a pathological analysis system for Pendred syndrome (PDS), a type of hearing loss, and to search for therapeutic drugs (Non-Patent Document 1). Furthermore, the efficacy of some of the candidate drugs obtained as a result of this search has been confirmed in physician-initiated clinical trials (Clinical Trial Registration ID: UMIN000033083).

[0005] On the other hand, the stria vascularis of the inner ear cochlea is an essential tissue for maintaining homeostasis of the ionic environment in the inner ear cochlea (see Figure 21, left panel), and dysfunction of the stria vascularis is thought to be one of the causes of many types of hearing loss, including hereditary, drug-induced, age-related, noise-induced, and viral infection-induced hearing loss. Of the three types of cells that make up the stria vascularis, marginal cells are cells that have essential functions in unidirectional ion transport and barrier formation between tissue and endolymph (see Figure 21, right panel). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Makoto Hosoya, Masato Fujioka, Takefumi Sone, Satoshi Okamoto, Wado Akamatsu, Hideki Ukai, Hiroki R. Ueda, Kaoru Ogawa, Tatsuo Matsunaga, and Hideyuki Okano, "Cochlear Cell Modeling Using Disease-Specific iPSCs Unveils a Degenerative Phenotype and Suggests Treatments for Congenital Progressive Hearing Loss," Cell Reports 18, 68-81, January 3, 2017. [Patent Documents]

[0007] [Patent Document 1] Patent No. 6218152 [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] While marginal cell-like cells differentiated using conventional techniques exhibit some marker expression, they do not form the cobblestone-like planar structure created by tight junction proteins between cells necessary for maintaining potassium ion homeostasis. Therefore, they cannot be considered to exhibit the same physiological functions as marginal cells of the stria vascularis inherent in the inner ear organs. Consequently, they have not been applied to the pathological analysis of hearing loss involving inner ear stria vascularis marginal cells or to drug screening.

[0009] Therefore, the object of the present invention is to provide a technique for producing functional inner ear stria vascularis marginal cells that have potential applications in the pathophysiological analysis of hearing loss and drug screening. [Means for solving the problem]

[0010] The inventors of this invention have diligently conducted research to achieve the above objectives and have now completed this invention.

[0011] In other words, in its first aspect, the present invention provides a method for producing marginal cells of the inner ear stria vascularis, comprising the step of culturing a cell population including inner ear progenitor cells differentiated from pluripotent stem cells in an insulin-free medium that contains no insulin or only trace amounts of insulin.

[0012] According to the method for producing marginal cells of the inner ear stria vascularis according to the present invention, a cell population including inner ear progenitor cells differentiated from pluripotent stem cells is cultured in an insulin-free medium that contains no insulin or only trace amounts of insulin, thereby obtaining functional marginal cells of the inner ear stria vascularis that have potential applications in the pathophysiological analysis of hearing loss and drug screening.

[0013] In the method for producing marginal cells of the inner ear stria vascularis according to the present invention, it is preferable that the insulin-free medium has an insulin concentration of 0 nM or more and 100 nM or less. This makes it possible to more reliably induce differentiation into high-quality marginal cells of the inner ear stria vascularis.

[0014] In the method for producing marginal cells of the inner ear stria vascularis according to the present invention, the insulin-free medium preferably contains EGF and further contains at least one or more selected from the group consisting of bFGF, FGF3, and BMP4. In particular, in suspension culture, it is preferable that the initial insulin-free medium during the suspension culture period contains EGF, FGF3, and BMP4. This makes it possible to more reliably induce differentiation into high-quality marginal cells of the inner ear stria vascularis.

[0015] The method for producing marginal cells of the inner ear vascular stria according to the present invention preferably includes the following steps (1) and (2). (1) A process of detaching and dispersing a cell population, including inner ear progenitor cells differentiated from pluripotent stem cells. (2) A step of culturing the cells or cell population obtained in step (1) in suspension in the insulin-free medium in the presence of an extracellular matrix material.

[0016] The extracellular matrix material is preferably at least one or more selected from the group consisting of Matrigel, pronectin, collagen, laminin, and fibronectin.

[0017] According to the above manufacturing method, the cell population, including inner ear progenitor cells differentiated from pluripotent stem cells, is subjected to cell detachment and dispersion treatment. This eliminates cells unsuitable for culture, ensuring that the cells are of good quality. Furthermore, suspension culture in the presence of extracellular matrix material provides a scaffold for differentiation into stria vascularis marginal cells. Consequently, high-quality inner ear stria vascularis marginal cells can be obtained with greater reproducibility.

[0018] The method for producing marginal cells of the inner ear vascular stria according to the present invention preferably includes the following steps (1) to (3). (1) A process of detaching and dispersing a cell population, including inner ear progenitor cells differentiated from pluripotent stem cells. (2) The step of culturing the cells or cell population obtained in step (1) in suspension in the insulin-free medium in the presence of an extracellular matrix material (3) The step of seeding the cells or cell population after the suspension culture in step (2) on feeder cells that have been separately adherently cultured in advance and culturing them in the insulin-free medium

[0019] Preferably, the feeder cells are melanocytes or melanocyte-like cells.

[0020] According to the above production method, since a cell population containing inner ear progenitor cells induced to differentiate from pluripotent stem cells is subjected to cell detachment and dispersion treatment, cells that are not suitable for culture are eliminated, making it more certain that the inner ear progenitor cells have good quality. In addition, since suspension culture is performed in an insulin-free medium in the presence of an extracellular matrix material, the presence of the extracellular matrix material has an effect of serving as a scaffold for differentiation into stria vascularis marginal cells. Furthermore, since the cells or cell population after suspension culture are seeded on feeder cells that have been separately adherently cultured in advance and cultured in an insulin-free medium, for the stria vascularis marginal cells co-cultured with the adherently cultured feeder cells, the formation of a structure (layered) in which the stria vascularis marginal cells are two-dimensionally cultured on the surface of the feeder cells is promoted. As a result, two-dimensional culture of stria vascularis marginal cells with good quality can be performed with better reproducibility.

[0021] In the method for producing stria vascularis marginal cells according to the present invention, it is preferable that the cell population containing inner ear progenitor cells obtained by inducing differentiation from the pluripotent stem cells is obtained by a method including the following steps (1) to (4).

[0022] (1) The step of culturing pluripotent stem cells in the absence of growth factors and in the presence of a ROCK inhibitor (2) The step of culturing the cell population obtained in step (1) in the absence of growth factors and in the absence of a ROCK inhibitor (3) The step of culturing the cell population obtained in step (2) in the presence of at least one growth factor selected from the group consisting of bFGF, FGF3, FGF10, and FGF19, and BMP4 (4) A step of culturing the cell population obtained in step (3) in the presence of at least one growth factor selected from the group consisting of bFGF, FGF3, FGF10, and FGF19, but in the absence of BMP4.

[0023] According to the above manufacturing method, it is possible to more reliably induce differentiation of inner ear progenitor cells from pluripotent stem cells, and consequently, to obtain high-quality inner ear stria vascularis marginal cells with greater reproducibility.

[0024] In the method for producing marginal cells of the inner ear vascular striae according to the present invention, it is preferable to obtain the marginal cells of the inner ear vascular striae under serum-free conditions. This reduces the risk of differentiation into unintended cells due to serum factors.

[0025] In the method for producing marginal cells of the Stria vascularis of the inner ear according to the present invention, it is preferable that the marginal cells of the Stria vascularis of the inner ear express potassium channel proteins and tight junction proteins. This ensures that the obtained cells are functional marginal cells of the Stria vascularis of the inner ear, and consequently, allows for the more reproducible acquisition of high-quality marginal cells of the Stria vascularis of the inner ear.

[0026] In a second aspect of the present invention, the present invention provides a method for evaluating a drug, comprising the steps of treating marginal cells of the inner ear striata vascularis, which are differentiated from inner ear progenitor cells, with a test drug, and evaluating the state of the marginal cells of the inner ear striata vascularis treated with the test drug.

[0027] According to the drug evaluation method of the present invention, it is possible to effectively and efficiently evaluate drugs that affect marginal cells of the inner ear vascular stria.

[0028] In its third aspect, the present invention provides a cell culture for drug evaluation containing marginal cells of the inner ear vascular stria, which are differentiated from inner ear progenitor cells.

[0029] The cell culture for drug evaluation according to the present invention contains marginal cells of the inner ear's stria vascularis, which are differentiated from inner ear progenitor cells. Therefore, drugs that affect marginal cells of the inner ear's stria vascularis can be effectively and efficiently evaluated using this culture.

[0030] In its fourth aspect, the present invention provides a method for producing marginal cells of the inner ear stria vascularis, comprising the step of culturing a cell population including inner ear progenitor cells in an insulin-free medium that contains no insulin or only trace amounts of insulin.

[0031] According to the method for producing marginal cells of the inner ear stria vascularis according to the present invention, a cell population including inner ear progenitor cells is cultured in an insulin-free medium that contains no insulin or only trace amounts of insulin, thereby obtaining functional marginal cells of the inner ear stria vascularis that have potential applications in the pathophysiological analysis of hearing loss and drug screening. [Brief explanation of the drawing]

[0032] [Figure 1] This is a flowchart illustrating one embodiment of a method for producing marginal cells of the inner ear vascular stria according to the present invention. [Figure 2] This is a flowchart illustrating another embodiment of the method for producing marginal cells of the inner ear vascular stria according to the present invention. [Figure 3] This flowchart illustrates yet another embodiment of the method for producing marginal cells of the inner ear vascular stria according to the present invention. [Figure 4] This figure shows the results of inducing differentiation from pluripotent stem cells into inner ear progenitor cells in Test Example 1, and detecting the expression of PAX2, PAX8, and SOX2, known marker molecules for inner ear progenitor cells, by immunostaining with their respective specific antibodies. [Figure 5] In Test Example 2, differentiation induction was performed from inner ear progenitor cells to inner ear stria vascularis marginal cells, and the expression of KCNQ1, KCNE1, and LRP2, known functional proteins, in inner ear stria vascularis marginal cells was detected by immunostaining using their respective specific antibodies. This figure shows the results. [Figure 6]In Test Example 2, differentiation induction was performed from inner ear progenitor cells to inner ear stria vascularis marginal cells, and the expression of NKCC1 and KCNQ1, known functional proteins, in inner ear stria vascularis marginal cells was detected by immunostaining using their respective specific antibodies. This figure shows the results. [Figure 7] In Test Example 2, differentiation induction was performed from inner ear progenitor cells to inner ear stria vascularis marginal cells, and the expression of Na / K ATPase, a known functional protein, in inner ear stria vascularis marginal cells was detected by immunostaining with its specific antibody. This figure shows the results. [Figure 8] In Test Example 2, differentiation induction was performed from inner ear progenitor cells to inner ear stria vascularis marginal cells, and the expression of LMX1 and ESRRB, known functional proteins, in inner ear stria vascularis marginal cells was detected by immunostaining using their respective specific antibodies. This figure shows the results. [Figure 9] In Test Example 2, differentiation induction was performed from inner ear progenitor cells to inner ear stria vascularis marginal cells, and the expression of occludin, a known intercellular tight junction protein in inner ear stria vascularis marginal cells, was detected by immunostaining with its specific antibody. This figure shows the results. [Figure 10] In Test Example 2, differentiation induction was performed from inner ear progenitor cells to inner ear stria vascularis marginal cells, and the expression of ZO-1, known as an intercellular tight junction protein, in inner ear stria vascularis marginal cells was detected by immunostaining with its specific antibody. This figure shows the results. [Figure 11] In Test Example 3, pluripotent stem cells were differentiated into inner ear progenitor cells, and further differentiated into inner ear stria vascularis marginal cells. This figure shows the results of detecting the expression of NKCC1, a known functional protein, in inner ear stria vascularis marginal cells by immunostaining with its specific antibody. [Figure 12] In Test Example 3, pluripotent stem cells were differentiated into inner ear progenitor cells, and further differentiated into inner ear stria vascularis marginal cells. The figure shows the results of detecting the expression of ESRRB and KCNQ1, known functional proteins, in inner ear stria vascularis marginal cells by immunostaining with their specific antibodies. [Figure 13]In Test Example 3, pluripotent stem cells were differentiated into inner ear progenitor cells, and then further differentiated into inner ear stria vascularis marginal cells. The figure shows the results of detecting the expression of Occludin, Claudin-1, and ZO-1, known as intercellular tight junction proteins in inner ear stria vascularis marginal cells, by immunostaining with their specific antibodies. [Figure 14] In Test Example 4, differentiation induction from inner ear progenitor cells to inner ear stria vascularis marginal cells was performed by co-culturing with feeder cells (melanocytes) that had been previously cultured with adhesion. The figure shows the results of detecting the expression of ZO-1 and Claudin-1, known as intercellular tight junction proteins, in inner ear stria vascularis marginal cells by immunostaining with their respective specific antibodies. [Figure 15] In Test Example 4, differentiation induction from inner ear progenitor cells to inner ear stria vascularis marginal cells was performed by co-culturing with feeder cells (melanocytes) that had been previously cultured with adhesion. The figure shows the results of detecting the expression of Occludin, a known intercellular tight junction protein in inner ear stria vascularis marginal cells, by immunostaining with its specific antibody. [Figure 16] In Test Example 4, differentiation induction from inner ear progenitor cells to inner ear stria vascularis marginal cells was performed by co-culturing with feeder cells (melanocytes) that had been previously cultured with adhesion. The figure shows the results of detecting the expression of NKCC1, KCNQ1, and LRP2, known functional proteins, in inner ear stria vascularis marginal cells by immunostaining with their respective specific antibodies. [Figure 17] This figure shows the results of quantifying OTX2 gene expression by qPCR in the process of differentiating inner ear progenitor cells into inner ear stria vascularis marginal cells, using a medium containing an insulin-containing serum substitute, an insulin-free medium, or an insulin-free medium to which insulin was added, in Test Example 5. [Figure 18] This figure shows the results of the barrier function assay performed in Test Example 6. The left column shows a photograph of the fluorescence microscope image, and the right column shows a photograph of the bright-field microscope image. [Figure 19]In Test Example 7, after drug treatment of marginal cells of the inner ear's stria vascularis, which were differentiated from pluripotent stem cells, the expression of ZO-1, known as an intercellular tight junction protein, and cleaved caspase-3, known as a marker of apoptosis, was detected by immunostaining with their specific antibodies. This figure shows the results. [Figure 20] This figure shows the results of calculating the percentage of cleaved caspase-3 expressing cells relative to the total number of cells after drug treatment of marginal cells of the inner ear stria vascularis, which were differentiated from pluripotent stem cells, in Test Example 7. [Figure 21] This is a schematic diagram illustrating a magnified view of the stria vascularis region in a cross-section of the cochlea of ​​the inner ear. [Modes for carrying out the invention]

[0033] The present invention relates to a method for inducing differentiation of inner ear cells that constitute the inner ear organs, and more specifically, to a method for producing inner ear stria vascularis marginal cells that has been improved to promote differentiation induction from stem cells or progenitor cells such as pluripotent stem cells into inner ear stria vascularis marginal cells.

[0034] In this specification, "pluripotent stem cells" have the same meaning as generally understood by those skilled in the art, and refer to stem cells that have the ability to differentiate into almost all cells that make up the body. Examples of pluripotent stem cells include induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells). They may be of human origin or of non-human origin. Furthermore, they may be prepared by reprogramming somatic cells from healthy individuals or from somatic cells from individuals with diseases. Diseases that are particularly relevant include those related to the inner ear organs, hearing, and hearing ability, such as Pendred syndrome and Usher syndrome.

[0035] In this specification, "marginal cells of the inner ear stria vascularis" refers to cells that exhibit physiological functions similar to those of marginal cells of the stria vascularis inherent in the inner ear organs. However, "physiological functions similar to those of marginal cells of the stria vascularis inherent in the inner ear organs" includes not only cases where the cells possess all of their inherent functions, but also cases where they partially exhibit some of those physiological functions. Therefore, for example, it includes cells that resemble marginal cells of the inner ear stria vascularis.

[0036] In this specification, "adherent culture" means culturing target cells or cell populations by adhering them to the bottom surface of a culture vessel, while "suspension culture" means culturing target cells or cell populations without adhering them to the bottom surface of a culture vessel. In this case, when cells or cell populations are said to be adhering to the bottom surface of a culture vessel during culture, it means that the cells or cell populations are adhering to the bottom surface of the culture vessel through cell-substrate adhesion molecules contained in the extracellular matrix (ECM), and that the cells or cell populations do not float in the culture medium even when the culture medium is gently agitated. On the other hand, when cells or cell populations are said not to be adhering to the bottom surface of a culture vessel during culture, it means that the cells or cell populations are not adhering to the bottom surface of the culture vessel through cell-substrate adhesion molecules contained in the extracellular matrix (ECM), and that even if they are touching the bottom surface, the cells or cell populations will float in the culture medium when the culture medium is gently agitated. In adherent culture, it is preferable to chemically treat the bottom surface of the plastic dish or coat it with an adhesion-promoting coating agent (such as gelatin, polylysine, or agar) to promote cell adhesion to the substrate. In suspension culture, it is preferable not to treat the bottom surface of the plastic dish or to coat it with an adhesion-inhibiting coating agent (such as poly(2-hydroxyethyl methacrylate)) to prevent cell adhesion to the substrate. Generally, adherent culture makes it easy to promote or suppress differentiation induction, and to perform operations such as changing the culture medium to regulate these processes. On the other hand, suspension culture makes it easier to promote the formation of three-dimensional organoids that mimic tissues in living organisms. Even in adherent culture, it may take time for the target cells to adhere, and they may remain in a suspension state for a certain period of time. However, if the cells eventually adhere even after some time, they will be included in adherent culture.

[0037] [1] Differentiation induction from pluripotent stem cells to inner ear progenitor cells Differentiation induction from pluripotent stem cells into inner ear progenitor cells can be achieved, for example, by following steps (1A) to (4A) as described below. However, the technical scope of the present invention is not limited to using inner ear progenitor cells prepared by the method described below, but can be used with inner ear progenitor cells prepared by any other differentiation induction method. For example, inner ear progenitor cells used in the present invention can also be prepared by the method described in the above-mentioned Japanese Patent Publication No. 6218152, the method described by Makoto Hosoya et al. (Cell Reports 18, 68-81, January 3, 2017), or the method described by Sho Kurihara et al. (Stem Cells Transl Med. 2022 Mar 31;11(3):282-296.).

[0038] (1A) A process of culturing pluripotent stem cells without the addition of growth factors and in the presence of a ROCK inhibitor. (2A) A step in which the cell population obtained in step (1A) is cultured without the addition of growth factors and in the absence of ROCK inhibitors. (3A) A step of culturing the cell population obtained in step (2A) in the presence of at least one growth factor selected from the group consisting of bFGF, FGF3, FGF10, and FGF19, and BMP4. (4A) A step in which the cell population obtained in step (3A) is cultured in the presence of at least one growth factor selected from the group consisting of bFGF, FGF3, FGF10, and FGF19, and in the absence of BMP4.

[0039] The culture medium used in step (1A) above is not particularly limited, as long as it can maintain pluripotent stem cells or cells that are in the process of differentiating from pluripotent stem cells. For example, "mTeSR1" (STEMCELL Technologies), a serum-free medium that does not require feeder cells for maintaining pluripotent stem cells, is a preferred example. However, in this step (1A), the culture is carried out in the absence of growth factors and in the presence of a ROCK (Rho-associated coiled-coil forming kinase / Rho-binding kinase) inhibitor. The ROCK inhibitor has a cell death suppression effect on pluripotent stem cells. Step (1A) is preferably carried out for 1 to 3 days, and more preferably for 1 to 2 days.

[0040] Examples of ROCK inhibitors used in the above step (1A) include Y-27632 ((R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide), Fasudil hydrochloride, K-115 (ripasudil hydrochloride hydrate), and DE-104. The concentration of the ROCK inhibitor can be appropriately determined according to the type of ROCK inhibitor, but for example, in the case of Y-27632, 1 to 100 μM is preferred, and 10 to 20 μM is more preferred.

[0041] The culture medium used in step (2A) described above can be any medium capable of maintaining pluripotent stem cells or cells evolving from pluripotent stem cells, and is not particularly limited. For example, "mTeSR1" (STEMCELL Technologies), a serum-free medium that does not require feeder cells for maintaining pluripotent stem cells, is a preferred example, similar to the medium preferably used for culture in step (1A). However, in this step (2A), the culture is carried out in the absence of growth factors and ROCK inhibitors. Furthermore, in a preferred embodiment, after culturing the cells in the above-mentioned mTeSR1 medium for about one day, it is preferable to replace the culture medium with the basal medium to be used in the subsequent step (for example, the serum-free medium "DMEM / F12" (product name "D-MEM / Ham's F-12", Fujifilm Wako Pure Chemical Corporation), and maintain the cells by culturing them while replacing the medium with fresh medium every day. This allows the cells to adapt well to the basal medium used for the action of growth factors. In addition, supplementary nutrients may be added to the basal medium as appropriate. For example, serum-free supplement "B27" (product name "Gibco B-27 Supplement", Thermo Fisher Scientific), serum-free supplement "N2" (product name "Gibco N-2 Supplement", Thermo Fisher Scientific), serum-free supplement "Gibco GlutaMAX", serum-free supplement "Nonessential" Examples include "aminoacid" (Nacalai Tesque Co., Ltd.). Process (2A) is preferably carried out for a total of 1 to 10 days, more preferably for 2 to 8 days, and even more preferably for 3 to 6 days.

[0042] In addition, in steps (1A) and (2A) above, the meaning of "absence of growth factors and / or ROCK inhibitors" is that the growth factors and / or ROCK inhibitors are substantially absent, and they may be present at concentrations that do not have any effect.

[0043] The culture medium used in step (3A) above is not particularly limited, as long as it can maintain cells that are moving towards differentiation from pluripotent stem cells. For example, similar to the medium preferably used in the later stages of culture in step (2A), a serum-free medium such as "DMEM / F12" (product name "D-MEM / Ham's F-12", Fujifilm Wako Pure Chemical Corporation) can be used, along with serum-free supplements such as "B27" (product name "Gibco B-27 Supplement", Thermo Fisher Scientific), "N2" (product name "Gibco N-2 Supplement", Thermo Fisher Scientific), "Gibco GlutaMAX", and "Nonessential" A culture medium supplemented with "aminoacid" (Nacalai Tesque Co., Ltd.) is a preferred example. However, in this step (3A), the cells are cultured in the presence of at least one growth factor selected from the group consisting of bFGF, FGF3, FGF10, and FGF19, and BMP4. In this case, it is preferable to have all of bFGF, FGF3, FGF10, and FGF19 present. The concentration range of the growth factors bFGF, FGF3, FGF10, FGF19, and BMP4 in the culture medium is preferably 10 to 50 ng / mL, and more preferably 10 to 25 ng / mL, respectively. Furthermore, it is preferable to maintain the cells by changing the culture medium to fresh, for example, approximately every day. This further enhances the effect of the above growth factors in promoting the desired differentiation. Step (3A) is preferably carried out for a total of 1 to 6 days, more preferably 2 to 5 days, and even more preferably 3 to 4 days.

[0044] The culture medium used in step (4A) above is not particularly limited, as long as it can maintain cells that are moving towards differentiation from pluripotent stem cells. For example, similar to the culture medium preferably used in step (3A), a serum-free medium such as "DMEM / F12" (product name "D-MEM / Ham's F-12", Fujifilm Wako Pure Chemical Corporation) can be used, along with serum-free supplements such as "B27" (product name "Gibco B-27 Supplement", Thermo Fisher Scientific), "N2" (product name "Gibco N-2 Supplement", Thermo Fisher Scientific), "Gibco GlutaMAX", and "Nonessential". A culture medium supplemented with "aminoacid" (Nacalai Tesque Co., Ltd.) is a preferred example. However, in this step (4A), the cells are cultured in the presence of at least one growth factor selected from the group consisting of bFGF, FGF3, FGF10, and FGF19, and in the absence of BMP4. In this case, it is preferable to have all of bFGF, FGF3, FGF10, and FGF19 present. The concentration range of the growth factors bFGF, FGF3, FGF10, and FGF19 in the culture medium is preferably 10 to 50 ng / mL, and more preferably 25 ng / mL. Furthermore, it is preferable to maintain the cells by changing the culture medium to fresh, for example, approximately every day. This further enhances the effect of the above growth factors in promoting the desired differentiation. Step (4A) is preferably carried out for a total of 1 to 6 days, more preferably 2 to 5 days, and even more preferably 3 to 4 days.

[0045] In step (4A), the meaning of "absence of BMP4" is that BMP4 is substantially absent, and it may be present at a concentration that has no effect.

[0046] The series of cultures in steps (1A) to (4A) are preferably carried out using adherent culture, where the cells are cultured while adhering to the bottom surface of the culture dish. This allows for efficient cell culture. Furthermore, it facilitates operations such as changing the culture medium to promote, suppress, or regulate differentiation induction. It is also preferable to culture in serum-free medium. This reduces the risk of differentiation into unintended cells due to serum factors.

[0047] Generally, to evaluate the degree of differentiation into inner ear cells, the expression of inner ear progenitor cell markers such as PAX2 and PAX8 is used as an indicator. In other words, when immature progenitor cells undergo a prescribed culture process, they differentiate into inner ear cells, and consequently, the expression of PAX2 and PAX8 increases (Reference 1: Ealy M, Ellwanger DC, Kosaric N, Stapper AP, Heller S. "Single-cell analysis delineates a trajectory toward the human early otic lineage." Proc Natl Acad Sci US A. 2016 Jul 26;113(30):8508-13.; Reference 2: Koehler KR, Nie J, Longworth-Mills E, Liu XP, Lee J, Holt JR, Hashino E. "Generation of inner ear organoids containing functional hair cells from human pluripotent stem cells." Nat Biotechnol. 2017 Jun;35(6):583-589.). Therefore, the degree of differentiation into inner ear cells can be evaluated by examining the expression of PAX2 and PAX8 at the protein expression level or mRNA expression level in the inner ear progenitor cells obtained through the method described above. In other words, the cell population has reached a level at which at least the expression of PAX2 and PAX8 can be detected when examined at the protein expression level or mRNA expression level. In addition to PAX2 and PAX8, other well-known markers for inner ear progenitor cells, such as FoxG1, GATA3, TFAP2A, ECAD, SOX10, and JAG1, may be used as indicators to evaluate the degree of differentiation into inner ear cells, and one or more of these markers may be used for evaluation. In this specification, "inner ear progenitor cells" refers to cells that have the ability to differentiate into inner ear cells that constitute the inner ear organs before differentiation.Therefore, it includes, for example, what are called inner ear stem cells, ectodermal placode cells, cochlear stem cells, cochlear progenitor cells, and tissue stem cells contained in the tissues of inner ear organs.

[0048] [2] Differentiation induction from inner ear progenitor cells to inner ear stria vascular margin cells Figure 1 shows a flowchart illustrating one embodiment of the method for producing marginal cells of the inner ear striata vascularis according to the present invention. As shown in Figure 1, in the present invention, inner ear progenitor cells are subjected to specific treatment to induce differentiation from inner ear progenitor cells into more mature inner ear cells. Specifically, a cell population containing inner ear progenitor cells is cultured in an insulin-free medium that contains no insulin or only trace amounts of insulin to induce differentiation into marginal cells of the inner ear striata vascularis.

[0049] As the basal medium used for culturing in insulin-free medium, as in the preparation of inner ear progenitor cells described above, for example, "DMEM / F12" (product name "D-MEM / Ham's F-12", Fujifilm Wako Pure Chemical Corporation) can be used. In addition, supplemental nutrients may be added to the basal medium as appropriate. Examples include the serum-free supplement "Gibco GlutaMAX" (Thermo Fisher Scientific) and the serum-free supplement "Nonessential aminoacid" (Nacalai Tesque Co., Ltd.). However, the insulin-free medium used in the present invention needs to have a limited insulin concentration. As shown in the examples described later, if the medium has a normal insulin concentration, differentiation induction into the target marginal cells will be hindered. The lower limit of the insulin concentration is preferably 0 nM or higher, and the upper limit is preferably, for example, 100 nM or lower. The upper limit for insulin concentration may be 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1 nM or less.

[0050] In the preparation of inner ear progenitor cells described above, serum-free supplements that can be added to and supplemented in the basal medium were exemplified, such as the serum-free supplement "B27" (product name "Gibco B-27 Supplement," Thermo Fisher Scientific) and the serum-free supplement "N2" (product name "Gibco N-2 Supplement," Thermo Fisher Scientific). However, these contain insulin at a certain concentration or higher. Therefore, it is not advisable to add or supplement the above-mentioned insulin-free medium in the usual amounts according to the product protocol. On the other hand, there are serum-free supplements containing similar components, such as the insulin-free serum-free supplement "N21-Ins" (product name "N21-MAX Insulin-Free Media Supplement," R&D Systems). Such insulin-free culture supplement products can be added or supplemented in the above-mentioned insulin-free medium used in the present invention in the usual amounts according to the product protocol.

[0051] Generally, growth factors that contribute to the differentiation induction of inner ear progenitor cells into more mature inner ear cells include EGF, bFGF, FGF3, and BMP4. Therefore, even in the above-mentioned insulin-free culture medium, one or more of these growth factors can be included in the culture medium. In this case, the preferred concentration range of these growth factors in the culture medium is as follows: For EGF, it is preferably 10-50 ng / mL, and more preferably 20-30 ng / mL. For bFGF, it is preferably 10-50 ng / mL, and more preferably 10-30 ng / mL. For FGF3, it is preferably 10-100 ng / mL, and more preferably 20-80 ng / mL. For BMP4, it is preferably 10-50 ng / mL, and more preferably 20-30 ng / mL.

[0052] The above-mentioned culture using insulin-free medium is preferably carried out in an environment in which at least EGF is present as a growth factor, and at least one or more selected from the group consisting of bFGF, FGF3, and BMP4. In particular, in suspension culture, it is important that the medium initially changed as an insulin-free medium contains EGF, as well as FGF3 and BMP4. More specifically, in suspension culture using insulin-free medium, it is preferable to culture in the presence of at least EGF as a growth factor, and it is even more preferable to change the medium so that each growth factor is present at each stage of the suspension culture period, such as culturing in the presence of EGF, FGF3, and BMP4 in addition to EGF in the first half, culturing in the presence of EGF, FGF3 in addition to EGF in the middle, and culturing in the presence of EGF, bFGF in addition to EGF in the latter half.

[0053] On the other hand, in culturing using the insulin-free medium described above, it is more preferable not to include IGF-1 in the medium. This makes it possible to avoid the provision of insulin-like signals by IGF-1.

[0054] By using the above-mentioned insulin-free medium, and by the presence or replacement of these preferred growth factors, differentiation induction into marginal cells of the inner ear stria vascularis can be made more reliable. As for the culture method, adherent culture or suspension culture may be used, but from the viewpoint of promoting the formation of three-dimensional organoids that mimic in vivo tissues, suspension culture is more preferable. Furthermore, it is preferable to culture in serum-free medium. This reduces the risk of differentiation into unintended cells due to serum factors. The culture period is preferably 50 to 70 days in total from the start of differentiation induction of pluripotent stem cells, more preferably 55 to 65 days, and even more preferably 60 to 65 days. Furthermore, the culture period in insulin-free medium is preferably 30 to 50 days in total, more preferably 35 to 45 days, and even more preferably 40 to 45 days.

[0055] Figure 2 shows a flowchart illustrating another embodiment of the method for producing marginal cells of the inner ear stria vascularis according to the present invention. In this embodiment, the differentiation induction into marginal cells of the inner ear stria vascularis is made more reliable by subjecting the inner ear progenitor cells differentiated from pluripotent stem cells to the following steps (1B) and (2B).

[0056] (1B) A process of detaching and dispersing a cell population, including inner ear progenitor cells differentiated from pluripotent stem cells. (2B) A step of suspension culture of the cells or cell population obtained in step (1B) in the insulin-free medium in the presence of an extracellular matrix material.

[0057] Specifically, as shown in Figure 2, in this embodiment, a cell population including inner ear progenitor cells differentiated from pluripotent stem cells is first subjected to cell detachment and dispersion. In the process of differentiating pluripotent stem cells into inner ear cells through culture, the proliferated cells adhere to each other. However, if the individual cells are separated and then transferred to the next culture, they dissociate into single cells or small clusters of 2 to 10 cells, making the individual cells more susceptible to direct exposure to the culture medium and incubator. At this time, if the cells are undifferentiated or have weak growth activity, they will not survive and will die. This eliminates cells that are unnecessary for differentiation into inner ear cells, ensuring the maintenance of expression levels such as PAX2 and PAX8. The cell detachment and dispersion process can be any method that can detach adhered cells from the culture substrate and disperse them into individual cells; there are no particular restrictions on the method. Examples include enzymatic treatment using trypsin-like enzyme preparations (product name "TrypLE Select," Thermo Fisher Scientific Inc.) or actase (cell detachment enzyme preparation: product name "Accutase," Nacalai Tesque Co., Ltd.). After enzymatic treatment, cell separation can be ensured by pipetting in a liquid culture medium. Alternatively, remaining cell clumps may be removed by passing the cells through a mesh with a predetermined pore size. For this purpose, cell strainers equipped with nylon meshes having predetermined pore sizes in stages, such as 1 to 1000 μm, are commercially available, and a mesh with an appropriate pore size may be selected and used from among such commercially available cell strainers.

[0058] Furthermore, as shown in Figure 2, in this embodiment, cells or cell populations that have undergone cell detachment and dispersion are cultured in suspension in an insulin-free medium that contains no insulin or only trace amounts of insulin in the presence of an extracellular matrix material. For suspension culture, it is preferable to suspend the cells or cell populations that have undergone cell detachment and dispersion in a suitable liquid medium and then culture them in a suspension culture incubator that allows for non-adherent culture. Specifically, as a culture incubator for non-adherent culture, for example, a plastic dish for non-adherent cell culture can be used.

[0059] For the suspension culture described above, the insulin-free medium mentioned above may be used. However, an extracellular matrix material is added to it. The presence of this extracellular matrix material facilitates the formation of cell polarity and provides a scaffold for differentiation into Striae vascularis marginal cells. The extracellular matrix material can be any material that functions as a scaffold when cells grow three-dimensionally, and there are no particular restrictions, but examples include Matrigel, pronectin, collagen, laminin, and fibronectin. These may be used individually or in combination of two or more. The amount of extracellular matrix material in the insulin-free medium may be within the range commonly used by those skilled in the art and is not particularly limited, but typically it may be, for example, 0.01 to 10 mg / mL in terms of unlimited protein amount, 0.05 to 5 mg / mL, or 0.1 to 1 mg / mL.

[0060] This suspension culture is preferably carried out for a total of 30 to 50 days from the start of suspension culture in insulin-free medium, more preferably for 35 to 45 days, and even more preferably for 40 to 45 days. Furthermore, during this suspension culture, the spheres (cell aggregates) formed by centrifugation or the like may be collected without damaging them, and the suspension culture may be continued by replacing the medium with fresh medium or adding fresh medium. In this case, it is preferable to change the medium or add fresh medium every 3 to 4 days during the additional suspension culture.

[0061] The three-dimensional cultured stria vascularis marginal cells that can be prepared in this way are cells that exhibit intrinsic physiological functions similar to those of stria vascularis marginal cells inherent in the inner ear organs, such as expressing intercellular tight junction proteins and ion transporters.

[0062] [3] Two-dimensional culture of marginal cells of the inner ear striata Figure 3 shows a flowchart illustrating yet another embodiment of the method for producing marginal cells of the inner ear striae vessels according to the present invention. In this embodiment, inner ear progenitor cells differentiated from pluripotent stem cells are subjected to the following steps (1C) to (3C) to more reliably induce differentiation into marginal cells of the inner ear striae vessels, and a two-dimensional structure (layered) is formed by the marginal cells of the inner ear striae vessels. In other words, two-dimensional culture (2D culture) of marginal cells of the inner ear striae vessels is made possible.

[0063] (1C) A process of detaching and dispersing a cell population, including inner ear progenitor cells differentiated from pluripotent stem cells. (2C) A step in which the cells or cell population obtained in step (1C) are cultured in suspension in the insulin-free medium in the presence of an extracellular matrix material. (3C) A step in which the cells or cell population after suspension culture in step (2C) are seeded onto feeder cells that have been previously cultured with adherent cells and cultured in the insulin-free medium.

[0064] Specifically, as shown in Figure 3, in this embodiment, steps (1C) and (2C) for suspension culture in insulin-free medium described above are the same as steps (1B) and (2B) described in Figure 2. However, during this process, the cell population in suspension culture is appropriately harvested, dispersed by pipetting in a solution to which enzymes are added as needed, and then seeded onto feeder cells that have been previously cultured with adhesion. Then, the culture in insulin-free medium described above is continued. Melanocytes or melanocyte-like cells can be used as feeder cells. By culturing feeder cells such as melanocytes with adhesion, a two-dimensional structure (layered) is formed along the surface of a two-dimensional structure such as the inner bottom surface of the culture vessel. By seeding and co-culturing on this, the formation of a two-dimensional structure (layered) is also promoted for the marginal cells of the inner ear stria vascularis.

[0065] This co-culture with feeder cells is preferably carried out for a total of 30 to 50 days from the start, more preferably for 35 to 45 days, and even more preferably for 40 to 45 days. Furthermore, the co-culture with feeder cells may be continued by replacing the culture medium with fresh medium or adding fresh medium. In this case, it is preferable to replace the culture medium or add fresh medium every 3 to 4 days during the additional co-culture.

[0066] As shown in the examples below, the two-dimensionally cultured marginal cells of the inner ear's stria vascularis have a layered structure with barrier function. Therefore, they can be more suitably used as functional tool cells that exhibit the same intrinsic physiological functions as marginal cells of the stria vascularis inherent in the inner ear organs, such as tight junctions between cells necessary for maintaining potassium ion concentration homeostasis and unidirectional ion transport, cultured planarly with a layered structure.

[0067] [4] Methods for evaluating drugs In another aspect of the present invention, the present invention can provide a method for evaluating drugs. Specifically, the present invention provides a method for evaluating drugs that includes the steps of treating marginal cells of the inner ear's stria vascularis, differentiated from pluripotent stem cells, with a test drug, and evaluating the state of the marginal cells of the inner ear's stria vascularis treated with the test drug.

[0068] In the drug evaluation method according to the present invention, an arbitrary test drug can be applied to marginal cells of the inner ear stria vascularis differentiated from pluripotent stem cells, and the effect of the test drug on the marginal cells of the inner ear stria vascularis can be evaluated. Therefore, it is useful as a tool for effectively and efficiently screening substances related to the functionality of inner ear organs, for example. In the step of treating with the test drug, although not limited, for example, the marginal cells of the inner ear stria vascularis obtained as described above may be suspended in a buffer solution, the test drug may be added to the solution at a predetermined concentration, and the state of the cells may be observed after a predetermined time has elapsed. Alternatively, the marginal cells of the inner ear stria vascularis obtained as described above may be cultured, a predetermined concentration may be added to the culture medium, cultured for a predetermined time, and the state of the cells thereafter may be observed. Furthermore, in the step of evaluating the state of the marginal cells of the inner ear stria vascularis after treatment, although not limited, for example, the ion permeability, barrier function, apoptosis, oxidative stress, etc. of cells related to the functionality of inner ear organs may be examined.

[0069] [5] Cell cultures for drug evaluation In yet another aspect of the present invention, the present invention provides a cell culture for drug evaluation. Specifically, it provides a cell culture for drug evaluation containing marginal cells of the inner ear vascular striae differentiated from pluripotent stem cells.

[0070] The cell culture for drug evaluation according to the present invention allows for the evaluation of how an arbitrary test drug affects marginal cells of the inner ear stria vascularis by applying it to the cell culture. Therefore, it is useful as a tool for effectively and efficiently screening substances related to the functionality of inner ear organs, for example. The cell culture can be in the form of cells or cell populations containing at least the marginal cells of the inner ear stria vascularis, and is usually in the form of cell aggregates obtained by suspension culture. In this case, it is preferable to store the cell aggregates in a preservation solution such as a medium or culture medium to protect and preserve them. When using the cell culture, the preservation solution is replaced with a test solution containing the test drug, and the state of the cells is observed after a predetermined time has elapsed, allowing the cell culture to be used for evaluation of the test drug.

[0071] Alternatively, as described above, by co-culturing with feeder cells such as melanocytes, the cells can be formed in a two-dimensional structure (layered) along the surface of a two-dimensional structure such as the inner bottom surface of the culture vessel. Therefore, the form of the cell culture can also be that of a two-dimensional culture (2D culture). In this case, it is preferable to store the cell culture in a culture vessel with a preservation solution such as a medium or culture medium to protect and preserve the two-dimensional structure (layered) form. When using the cell culture, the preservation solution is replaced with a test solution containing the test drug, and the state of the cells is observed after a predetermined time has elapsed, allowing the cell culture to be used for evaluation of the test drug. The evaluation is not limited to these, but for example, it may involve examining the ion permeability, barrier function, apoptosis, oxidative stress, etc., of cells involved in the functionality of inner ear organs. [Examples]

[0072] The present invention will be described in more detail below with reference to examples. However, these examples are not intended to limit the scope of the present invention.

[0073] (reagent) (1) Matrigel: Coating agent (product name "Corning Matrigel Basement Membrane Matrix", Corning Corporation) (2) Actase: Enzyme preparation for cell detachment (product name "Accutase", Nacalai Tesque Co., Ltd.) (3) Trypsin-like enzyme preparation (product name "TrypLE Select", Thermo Fisher Scientific Co., Ltd.) (4) Y-27632: ROCK inhibitor (specific inhibitor of Rho-associated coiled-coil forming kinase / Rho-binding kinase) (Trade name "Y-27632", Fujifilm Wako Pure Chemical Corporation) (5) mTeSR1: Maintenance medium for iPS cells (product name "mTeSR1", STEMCELL Technologies) (6) DMEM / F12: Serum-free medium (product name "D-MEM / Ham's F-12", Fujifilm Wako Pure Chemical Industries, Ltd.) (7) B27: Serum-free supplement (50X) (Product name "Gibco B-27 Supplement", Thermo Fisher Scientific) (8) N2: Serum-free supplement (100X) (Product name "Gibco N-2 Supplement", Thermo Fisher Scientific) (9) N21-Ins: Serum-free supplement (50X) (Product name "N21-MAX Insulin Free Media Supplement", R&D Systems) (10) GlutaMAX: Serum-free supplement (100X) (Product name "Gibco GlutaMAX", Thermo Fisher Scientific) (11) Nonessential amino acid (100X): Non-essential amino acid supplement (product name "MEM Non-essential Amino Acid Solution", Nakalai Tesque Co., Ltd.) (12) poly-L-ornithine / fibronectin: coating agent (product name "Poly-L-ornithine", Sigma-Aldrich), (product name "Fibronectin", Sigma-Aldrich) (13) bFGF (product name "Recombinant Human FGF-basic", Peprotech) (14) FGF3 (product name "Recombinant Human FGF-3 protein", R&D Systems) (15) FGF10 (product name "Recombinant Human FGF-10 protein", Peprotech) (16) FGF19 (product name "Recombinant Human FGF-19 protein", Peprotech) (17) BMP4 (product name "Recombinant Human BMP-4 protein", Peprotech) (18) IGF-1 (trade name “Recombinant Human IGF-1 Protein”, R&D Systems) (19) SB431542: TGF-β receptor inhibitor (trade name "SB431542", ReproCELL) (20) Heparin (trade name "heparan sulfate sodium salt", Sigma-Aldrich) (21) L-Glutamine (Trade name "L-Glutamine", Nacalai Tesque Co., Ltd.) (22) iMatrix-511 silk: Coating agent (product name "iMatrix-511 silk", NIPPI Corporation) (23) StemFit AK02N: Maintenance medium for iPS cells (product name "StemFit AK02N", Ajinomoto Co., Inc.) (24) CHIR99021: GSK-3 inhibitor (trade name "CHIR-99021", Focus biomolecules) (25) The primer sequences used in qPCR are shown in Table 1.

[0074] [Table 1]

[0075] In the following test examples, ampicillin at a concentration of 100 μg / mL was used as an antibacterial agent in the cell culture medium as needed. Unless otherwise specified, culture was performed under normal oxygen conditions (O2 20%, CO2 5%).

[0076] [Test Example 1] In this study, human iPS cells were differentiated into inner ear progenitor cells.

[0077] [Differentiation induction method] (Before differentiation induction:(-)Day2) 1) A 6-well plate was coated with Matrigel. 2) Confluent, feeder-free human iPS cells were treated with actase, incubated at 37°C for 2-3 minutes, and then detached from the dish. 3) After dilution with PBS, the cells were centrifuged and collected. 4) The supernatant was discarded, and the cells were suspended in mTeSR1 medium to which the ROCK inhibitor (Y-27632) (10 μM) had been added. 5) The cells were passed through a nylon mesh (pore size 40 μm) and the number of cells was counted using a hemocytometer. 6) mTeSR1 medium containing Y-27632 was added to the wells coated with Matrigel as described in 1) above. 7) 2.5 × 10 per well 4 cells / cm 2 The cell suspension was seeded in this manner.

[0078] (Before differentiation induction:(-)Day1) The culture medium was replaced with mTeSR1 medium that does not contain ROCK inhibitors.

[0079] (Day 0) The culture medium was changed to serum-free medium (DMEM / F12 + 2%B27 + 1%N2 + 1%GlutaMAX + 1%Nonessential aminoacid). The medium was then changed daily until Day 2.

[0080] (Day 3) The culture medium was replaced with a serum-free medium (DMEM / F12 + 2%B27 + 1%N2 + 1%GlutaMAX + 1%Nonessential aminoacid) to which growth factors bFGF, FGF3, FGF10, FGF19, and BMP4 were added at concentrations of 25 ng / mL, 25 ng / mL, 25 ng / mL, 25 ng / mL, and 10 ng / mL, respectively. The medium was changed daily thereafter until Day 5.

[0081] (Day 6) The serum-free medium (DMEM / F12 + 2%B27 + 1%N2 + 1%GlutaMAX + 1%Nonessential aminoacid) was replaced with a medium supplemented with growth factors bFGF, FGF3, FGF10, and FGF19 (all growth factors at a concentration of 25 ng / mL), and the cells were cultured until Day 8.

[0082] (Day 9) Cells were treated with actase and incubated at 37°C for 2-3 minutes. They were then detached from the dish and diluted in PBS. The cells were collected by centrifugation and suspended in a serum-free medium (DMEM / F12+2%B27+1%N2) to which L-glutamine was added to a concentration of 2 mM. Growth factors bFGF, FGF3, FGF10, and FGF19 were then added to concentrations of 25 ng / mL each. This cell suspension was seeded into wells coated with poly-L-ornithine / fibronectin to achieve a cell concentration of approximately one-third of that before cell detachment, and adherent culture was performed under hypoxic conditions (O24%, CO25%).

[0083] (Day 10) The culture medium was prepared by adding L-glutamine to serum-free medium (DMEM / F12+2%B27+1%N2) to a concentration of 2 mM, and then adding growth factors bFGF, EGF, and IGF-1 to concentrations of 20 ng / mL, 20 ng / mL, and 50 ng / mL, respectively, and then performing a medium exchange with this prepared medium.

[0084] (Day 12) For cells on day 12 after the start of differentiation induction, total RNA was extracted using the RNeasy spin column kit (Qiagen). cDNA was synthesized from 1 μg of total RNA from each sample using reverse transcriptase (product name "Invitrogen SuperScript IV Reverse Transcriptase" (Thermo Fisher Scientific)), and the expression levels of marker genes were quantified by qPCR.

[0085] As a result, the expression of genes specific to inner ear progenitor cells, such as PAX2 and PAX8, which are known to be marker molecules for inner ear progenitor cells, was confirmed.

[0086] Furthermore, the expression status of various marker proteins in cells 12 days after the start of differentiation induction (Day 12) was examined by immunostaining.

[0087] [1] PAX2, PAX8, SOX2 (Day 12) Cells on day 12 of differentiation induction were fixed by treating with 4% paraformaldehyde at room temperature for 20 minutes, treated with 0.3% PBST for 30 minutes, and then blocked at room temperature for 1 hour with 10% normal donkey serum / 0.3% PBST. Mouse anti-PAX2 antibody, rabbit anti-PAX8 antibody, and goat anti-SOX2 antibody were used as primary antibodies at concentrations of 1:500, 1:500, and 1:500, respectively, and reacted at room temperature for 2 hours. Fluorescently labeled secondary antibodies specific to the IgG animal species of each primary antibody were reacted at room temperature for 1 hour. The nuclei were also stained with Hoechst 33258. Figure 4 shows microscopic images obtained by confocal microscopy (scale bar: 50 μm).

[0088] As a result, as shown in Figure 4, the expression of PAX2, PAX8, and SOX2, which are known as marker proteins for inner ear progenitor cells, was confirmed. Therefore, it was confirmed that inner ear progenitor cells can be differentiated from human iPS cells using the method described above.

[0089] [Test Example 2] In this study, we induced differentiation of inner ear progenitor cells into inner ear stria vascularis marginal cells.

[0090] [Differentiation induction method] (Day 12) Human iPS cells were differentiated into inner ear progenitor cells using the same method as in Test Example 1. On day 12 after the start of differentiation induction, the cells were detached with actase, an equal volume of PBS was added, and the cells were passed through a nylon mesh (pore size 40 μm) and counted using a hemocytometer.

[0091] 5 x 10 5 Cells were suspended to a concentration of cells / 10mL, seeded into low-adhesion 6-well plates (product name "Corning Ultra-Low Attachment Plate," Corning), and suspension culture was started under hypoxic conditions (O24%, CO25%). At this time, the culture medium prepared was serum-free medium (DMEM / F12+2%B27+1%N2) to which growth factors bFGF, EGF, IGF-1, FGF3, and FGF10 were added at concentrations of 10 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, and 50 ng / mL, respectively. Furthermore, L-glutamine was added to a concentration of 2 mM, Y-27632 to a concentration of 10 μM, Matrigel to a concentration of 1%, SB431542 to a concentration of 2 μM, and heparin to a concentration of 50 ng / mL. Using this medium, cells were suspended and suspension culture was started.

[0092] (Day 16) On the fourth day after the start of suspension culture (Day 16), the entire culture medium was replaced with a medium composition that was the same as the one used at the start of suspension culture but with Matrigel and Y-27632 removed. Suspension culture was then continued under hypoxic conditions (O24%, CO25%).

[0093] (Day 20) On day 8 (Day 20) after the start of suspension culture, a serum-free medium (DMEM / F12 + 2%N21-Ins) was prepared by adding growth factors EGF, FGF3, and BMP4 at concentrations of 10 ng / mL, 50 ng / mL, and 10 ng / mL, respectively. Additionally, L-glutamine was added to a concentration of 2 mM, and heparin was added to a concentration of 50 ng / mL. The entire medium was then replaced, and suspension culture was continued under normal oxygen conditions (O2 20%, CO2 5%).

[0094] (Day 24) On day 12 (Day 24) after the start of suspension culture, a serum-free medium (DMEM / F12 + 2%N21-Ins) was prepared by adding growth factors EGF and FGF3 at concentrations of 10 ng / mL and 50 ng / mL, respectively, and then adding L-glutamine to a concentration of 2 mM, and SB431542 to a concentration of 2 μM. The entire medium was then replaced, and suspension culture was continued under normal oxygen conditions (O2 20%, CO2 5%).

[0095] (Day 32) On day 20 (Day 32) after the start of suspension culture, a culture medium prepared by adding growth factors bFGF and EGF to serum-free medium (DMEM / F12 + 2%N21-Ins) at concentrations of 2 ng / mL and 10 ng / mL, respectively, and further adding L-glutamine to a concentration of 2 mM, and SB431542 to a concentration of 2 μM was used. The entire medium was then replaced, and suspension culture was continued under normal oxygen conditions (O2 20%, CO2 5%).

[0096] (Day 36) On day 24 after the start of suspension culture (Day 36), the culture medium was completely replaced using the same composition as the medium used for the medium change on Day 32, and suspension culture was continued under normal oxygen conditions (O2 20%, CO2 5%).

[0097] (Day 40) On day 28 (Day 40) after the start of suspension culture, the culture medium was completely replaced with a serum-free medium (DMEM / F12 + 2%N21-Ins) to which growth factor EGF was added to a concentration of 10 ng / mL, L-glutamine was added to a concentration of 2 mM, and SB431542 was added to a concentration of 2 μM. Thereafter, the medium was changed every three days until Day 63, and suspension culture was continued under normal oxygen conditions (O2 20%, CO2 5%).

[0098] The expression status of various marker proteins was examined by immunostaining in cells undergoing differentiation induction using the method described above, as well as in cells after differentiation induction.

[0099] [2-1]KCNQ1, KCNE1, LRP2(Day63) Cells cultured in suspension on day 51 (Day 63) were fixed with 4% paraformaldehyde at 4°C for 3 hours, and 7 μm frozen sections were prepared. These frozen sections were treated with 0.3% PBST for 10 minutes, followed by blocking with 10% normal donkey serum / 0.1% PBST at room temperature for 1 hour. Mouse anti-LRP antibody, rabbit anti-KCNE1 antibody, and goat anti-KCNQ1 antibody were used as primary antibodies at concentrations of 1:200, 1:200, and 1:100, respectively, and reacted overnight at 4°C. Fluorescently labeled secondary antibodies specific to the IgG animal species of each primary antibody were reacted at room temperature for 1 hour. The nuclei were also stained with Hoechst 33258. Figure 5 shows microscopic images obtained by confocal microscopy (scale bar: left panel 20 μm, right panel 20 μm).

[0100] As a result, as shown in Figure 5, the expression of KCNQ1, KCNE1, and LRP2, known as functional proteins, was detected in marginal cells of the inner ear stria vascularis. Furthermore, they were co-localized on the cell membrane, suggesting that the expression pattern in endogenous marginal cells was reproduced.

[0101] [2-2]NKCC1, KCNQ1 (Day 63) Cells cultured in suspension on day 51 (Day 63) were fixed with 4% paraformaldehyde at 4°C for 3 hours, and 7 μm frozen sections were prepared. These frozen sections were treated with 0.3% PBST for 10 minutes, followed by blocking with 10% normal donkey serum / 0.1% PBST at room temperature for 1 hour. Rabbit anti-KCNQ1 antibody and goat anti-NKCC1 antibody were used as primary antibodies at a concentration of 1:200, and reacted overnight at 4°C. Fluorescently labeled secondary antibodies specific to the IgG animal species of each primary antibody were reacted at room temperature for 1 hour. The nuclei were also stained with Hoechst 33258. Figure 6 shows microscopic images obtained by confocal microscopy (scale bar: 50 μm).

[0102] As a result, as shown in Figure 6, the expression of NKCC1 and KCNQ1, known as functional proteins, was detected in marginal cells of the inner ear stria vascularis. Furthermore, they were not co-localized on the cell membrane, suggesting that the expression pattern in endogenous marginal cells was successfully reproduced.

[0103] [2-3]Na / K ATPase (Day 24) Cells on day 12 (Day 24) after the start of suspension culture were fixed by treatment with 10% TCA at 4°C for 15 minutes, and blocked at room temperature for 1 hour with 10% normal donkey serum / 0.1% PBST. Rabbit anti-Na / K ATPase antibody was used as the primary antibody at a concentration of 1:100 and reacted overnight at 4°C. A fluorescently labeled secondary antibody specific to the IgG of the primary antibody was reacted at room temperature for 1 hour, and the cells were observed using a confocal microscope. Figure 7 shows the microscopic images obtained by confocal microscopy (scale bar: 20 μm).

[0104] As a result, as shown in Figure 7, the expression of Na / K ATPase, a functional protein known as Na / K ATPase, was detected in marginal cells of the inner ear stria vascularis. Furthermore, it was localized to the cell membrane, suggesting that the expression pattern in endogenous marginal cells was being reproduced.

[0105] [2-4] LMX1, ESRRB (Day 40) Cells on day 28 (Day 40) after the start of suspension culture were fixed by treatment with 4% paraformaldehyde at room temperature for 20 minutes, treated with 0.3% PBST for 20 minutes, and then blocked with 10% normal donkey serum / 0.3% PBST at room temperature for 1 hour. Mouse anti-ESRRB antibody and rabbit anti-LMX1 antibody were used as primary antibodies at concentrations of 1:100, and reacted overnight at 4°C. Fluorescently labeled secondary antibodies specific to the IgG animal species of each primary antibody were reacted at room temperature for 2 hours. The nuclei were also stained with Hoechst 33258. Figure 8 shows microscopic images obtained by confocal microscopy (scale bar: 20 μm).

[0106] As a result, as shown in Figure 8, the expression of LMX1 and ESRRB, known as functional proteins, was detected in marginal cells of the inner ear stria vascularis. Furthermore, they were localized in the nucleus, suggesting that the expression pattern in endogenous marginal cells was being reproduced.

[0107] [2-5] Occludin (Day 52) Cells cultured in suspension forty days (Day 52) were fixed by treatment with 10% TCA at 4°C for 15 minutes, treated with 0.3% PBST for 10 minutes, and then blocked at room temperature for 1 hour with 10% normal donkey serum / 0.1% PBST. Rabbit anti-Occludin antibody was used as the primary antibody at a concentration of 1:100 and reacted overnight at 4°C. A fluorescently labeled secondary antibody specific to the IgG of the primary antibody was reacted at room temperature for 1 hour, and observed under a confocal microscope. Figure 9 shows the microscopic images obtained under a confocal microscope (scale bar: 20 μm).

[0108] As a result, as shown in Figure 9, the expression of Occludin, a known tight junction protein between cells in the marginal cells of the inner ear's stria vascularis, was detected. Furthermore, it exhibited a characteristic cobblestone-like signaling pattern, suggesting that the differentiated cells were epithelial cell-like cells, specifically the marginal cells of the inner ear's stria vascularis.

[0109] [2-6]ZO-1 (Day 43) Cells on day 31 (Day 43) after the start of suspension culture were fixed by treatment with 10% TCA at 4°C for 15 minutes, and blocked at room temperature for 1 hour with 10% normal donkey serum / 0.1% PBST. As the primary antibody, goat anti-ZO-1 antibody was used at a concentration of 1:100 and reacted overnight at 4°C. A fluorescently labeled secondary antibody specific to the IgG of the primary antibody was reacted at room temperature for 2 hours, and observed with a confocal microscope. Figure 10 shows the microscopic images obtained by observation with a confocal microscope (scale bar: 20 μm).

[0110] As a result, as shown in Figure 10, the expression of ZO-1, a functional protein known to be present in marginal cells of the inner ear's stria vascularis, was detected. Furthermore, it exhibited a cobblestone-like signal characteristic of epithelial cells, suggesting that the differentiated cells were epithelial cell-like cells, specifically marginal cells of the stria vascularis.

[0111] From these results, it became clear that by culturing cells using this induction method, inner ear progenitor cells obtained by differentiation induction from pluripotent stem cells can be further differentiated into inner ear stria vascularis marginal cells.

[0112] [Test Example 3] In this study, human iPS cells were differentiated into inner ear progenitor cells using a method different from that used in Study Example 1, and these inner ear progenitor cells were then differentiated into inner ear stria vascularis marginal cells.

[0113] [Method for inducing differentiation of inner ear progenitor cells] (Before differentiation induction:(-)Day2) 1) Six-well plates were coated with iMatrix-511 silk. 2) Confluent, feeder-free human iPS cells were treated with actase, incubated at 37°C for 2-3 minutes, and then detached from the dish. 3) After dilution with PBS, the cells were centrifuged and collected. 4) The supernatant was discarded, and the cells were suspended in StemFit AK02N to which the ROCK inhibitor (Y-27632) (10 μM) had been added. 5) The cells were passed through a nylon mesh (pore size 40 μm) and the number of cells was counted using a hemocytometer. 6) StemFit AK02N containing Y-27632 was added to the wells coated with iMatrix-511 silk as described in 1) above. 7) 2.5 × 10 per well 4 cells / cm 2 The cell suspension was seeded in this manner.

[0114] (Before differentiation induction:(-)Day1) The culture medium was replaced with StemFit AK02N, which does not contain ROCK inhibitors.

[0115] (Day 0) The culture medium was changed to serum-free medium (DMEM / F12 + 2%B27 + 1%N2 + 1%GlutaMAX + 1%Nonessential aminoacid). The medium was then changed daily until Day 2.

[0116] (Day 3) The culture medium was replaced with a serum-free medium (DMEM / F12 + 2%B27 + 1%N2 + 1%GlutaMAX + 1%Nonessential aminoacid) to which growth factors bFGF, FGF3, FGF10, FGF19, and BMP4 were added at concentrations of 25 ng / mL, 25 ng / mL, 25 ng / mL, 25 ng / mL, and 10 ng / mL, respectively. The medium was changed daily thereafter until Day 5.

[0117] (Day 6) The serum-free medium (DMEM / F12 + 2%B27 + 1%N2 + 1%GlutaMAX + 1%Nonessential aminoacid) was replaced with a medium supplemented with growth factors bFGF, FGF3, FGF10, FGF19, and CHIR99021 (all growth factors were at a concentration of 25 ng / mL, and the concentration of CHIR99021, a GSK-3 inhibitor, was 8 μM), and the cells were cultured until Day 8.

[0118] (Day 9) Cells were treated with actase and incubated at 37°C for 2-3 minutes. They were then detached from the dish and diluted in PBS. The cells were collected by centrifugation and suspended in a serum-free medium (DMEM / F12+2%B27+1%N2) to which L-glutamine was added to a concentration of 2 mM. Growth factors bFGF, FGF3, FGF10, and FGF19 were then added to concentrations of 25 ng / mL each. This cell suspension was seeded into wells coated with poly-L-ornithine / fibronectin to achieve a cell concentration of approximately one-third of that before cell detachment, and adherent culture was performed under hypoxic conditions (O24%, CO25%).

[0119] (Day 10) The culture medium was prepared by adding L-glutamine to serum-free medium (DMEM / F12+2%B27+1%N2) to a concentration of 2 mM, and then adding growth factors bFGF, EGF, and IGF-1 to concentrations of 20 ng / mL, 20 ng / mL, and 50 ng / mL, respectively, and then performing a medium exchange with this prepared medium.

[0120] [Method for inducing differentiation of marginal cells in the inner ear's vascular striatum] (Day 11) In the same method as in Test Example 2 (from Day 12 onwards), marginal cells of the inner ear vascular stria were differentiated.

[0121] The expression status of various marker proteins in cells differentiated using the method described above was examined by immunostaining.

[0122] [3-1]NKCC1 (Day 60) Cells cultured in suspension for 49 days (Day 60) were fixed with 4% paraformaldehyde at room temperature for 15 minutes, and 7 μm frozen sections were prepared. These frozen sections were treated with 0.3% PBST for 10 minutes, followed by blocking with 10% normal goat serum / 0.1% PBST at room temperature for 1 hour. Rabbit anti-NKCC1 antibody was used as the primary antibody at a concentration of 1:100 and reacted overnight at 4°C. A fluorescently labeled secondary antibody specific to the IgG of the primary antibody was reacted at room temperature for 1 hour. The nuclei were also stained with Hoechst 33258. Figure 11 shows microscopic images obtained by confocal microscopy (scale bar: 10 μm).

[0123] As a result, as shown in Figure 11, the expression of NKCC1, a known functional protein, was confirmed in the marginal cells of the inner ear stria vascularis. Therefore, it was confirmed that marginal cells of the inner ear stria vascularis can be differentiated from inner ear progenitor cells differentiated from human iPS cells using a method different from that of Test Example 1.

[0124] [3-2] ESRRB, KCNQ1 (Day 60) Cells cultured in suspension for 49 days (Day 60) were fixed with 4% paraformaldehyde at room temperature for 15 minutes, and 7 μm frozen sections were prepared. These frozen sections were treated with 0.3% PBST for 10 minutes, followed by blocking with 10% normal donkey serum / 0.1% PBST at room temperature for 1 hour. Mouse anti-ESRRB antibody and rabbit anti-KCNQ1 antibody were used as primary antibodies at concentrations of 1:100, and reacted overnight at 4°C. Secondary antibodies, which were IgG species-specific and fluorescently labeled, were reacted with the primary antibodies at room temperature for 1 hour. The nuclei were also stained with Hoechst 33258. Figure 12 shows microscopic images obtained by confocal microscopy (scale bar: 10 μm).

[0125] As a result, as shown in Figure 12, the expression of ESRRB and KCNQ1, known functional proteins, was detected in marginal cells of the inner ear stria vascularis. Furthermore, ESRRB was localized in the nucleus, suggesting that its expression pattern in endogenous marginal cells was successfully reproduced.

[0126] [3-3]ZO-1, Claudin-1, Occludin (Day60) Cells on day 49 (Day 60) after the start of suspension culture were fixed by treatment with 10% TCA at 4°C for 15 minutes, and 7 μm frozen sections were prepared. These frozen sections were treated with 0.3% PBST for 10 minutes, and then blocked at room temperature for 1 hour with 10% normal goat serum / 0.1% PBST (ZO-1 antibody was not blocked). As primary antibodies, goat anti-ZO-1 antibody, rabbit anti-Claudin-1 antibody, and rabbit anti-Occludin antibody were used at concentrations of 1:100, and reacted overnight at 4°C. Secondary antibodies specific to the IgG animal species of the primary antibodies were reacted at room temperature for 1 hour. The nuclei were also stained with Hoechst 33258. Figure 13 shows microscopic images obtained by observation with a Keyence BZ-X810 microscope (scale bar: 10 μm).

[0127] As a result, as shown in Figure 13, the expression of ZO-1, Claudin-1, and Occludin, known as intercellular tight junction proteins in marginal cells of the inner ear's stria vascularis, was detected. Furthermore, these cells exhibited characteristic cobblestone-like signaling, suggesting that the differentiated cells were epithelial cell-like cells, specifically marginal cells of the inner ear's stria vascularis.

[0128] [Test Example 4] In this study, we attempted to induce differentiation of inner ear progenitor cells into inner ear stria vascularis marginal cells by attempting two-dimensional culture of these cells.

[0129] [Differentiation Induction Methods (Part 1)] (Day 20) In the same method as in Test Example 1, inner ear progenitor cells were obtained from human iPS cells, and these inner ear progenitor cells were cultured until Day 20 using the same method as in Test Example 2.

[0130] Cells were collected and treated with a trypsin-like enzyme preparation (product name "TrypLE Select," Thermo Fisher Scientific Inc.) with EDTA added to a concentration of 1 mM for 20 minutes at 37°C. Then, 3 to 5 times the volume of DMEM / F12 was added, the cells were passed through a nylon mesh (pore size 40 μm), and the number of cells was counted using a hemocytometer.

[0131] The cells after detachment are 1.2 × 10 6 The cells were suspended in a culture medium with the same composition as the Day 20 medium to a concentration of cells / 10mL, and seeded into feeder cells that had been cultured separately. Melanocytes (product name "Normal Human Epidermal Melanocytes", TAKARA Corporation) were used as feeder cells, with 1 x 10⁶ cells per well of an 8-well chamber (product name "Chamber Slide II", IWAKI Corporation). 5 cells / cm 2 The cells were prepared by seeding and culturing them in M2 medium for melanocyte proliferation (TAKARA Corporation) for 6-7 days until confluence was achieved through adherent culture.

[0132] Subsequently, co-culture with feeder cells (melanocytes) was performed using a culture medium with the same composition as when suspension culture was performed, and the timing of medium changes was also the same, until 53 days after the start of suspension culture (Day 65).

[0133] [4-1]ZO-1, Claudin-1 (Day 65) Cells cultured in suspension on day 53 (Day 65) were fixed by treatment with 10% TCA at 4°C for 15 minutes, treated with 0.3% PBST for 10 minutes, and then blocked at room temperature for 1 hour with 10% normal donkey serum / 0.1% PBST. As primary antibodies, goat anti-ZO-1 antibody and rabbit anti-Claudin-1 antibody were used at concentrations of 1:100, and reacted overnight at 4°C. Fluorescently labeled secondary antibodies specific to the IgG animal species of the primary antibodies were reacted at room temperature for 1 hour. The nuclei were also stained with Hoechst 33258. Figure 14 shows microscopic images obtained by confocal microscopy (scale bar: 20 μm).

[0134] As a result, as shown in Figure 14, the expression of ZO-1 and Claudin-1, known as intercellular tight junction proteins, was detected in marginal cells of the inner ear stria vascularis. Furthermore, it exhibited a cobblestone-like signal characteristic of epithelial cells, suggesting that differentiation of epithelial cell-like cells, such as marginal cells of the inner ear stria vascularis, could be induced even in two-dimensional cultures in which the cells proliferated and spread as a monolayer on melanocytes through co-culture with pre-adherent cultured melanocytes.

[0135] [Differentiation Induction Methods (Part 2)] (Day 20) In the same method as in Test Example 1, inner ear progenitor cells were obtained from human iPS cells, and these inner ear progenitor cells were cultured until Day 21 using the same method as in Test Example 2.

[0136] Subsequently, co-culture with feeder cells (melanocytes) was carried out in the same manner as in "Differentiation Induction Method (Part 1)" until Day 60 (48 days after the start of suspension culture).

[0137] [4-2] Occludin (Day 60) Cells on day 48 (Day 60) after the start of suspension culture were fixed by treatment with 4% paraformaldehyde at room temperature for 15 minutes, treated with 0.3% PBST for 10 minutes, and then blocked at room temperature for 1 hour with 10% normal donkey serum / 0.1% PBST. Rabbit anti-Occludin antibody was used as the primary antibody at a concentration of 1:100 and reacted overnight at 4°C. A fluorescently labeled secondary antibody specific to the IgG animal species of the primary antibody was reacted at room temperature for 1 hour. The nuclei were also stained with Hoechst 33258. Figure 15 shows microscopic images obtained by confocal microscopy (scale bar: 10 μm).

[0138] As a result, as shown in Figure 15, the expression of Occludin, a known tight junction protein between cells, was detected in marginal cells of the inner ear stria vascularis. Furthermore, it exhibited a cobblestone-like signal characteristic of epithelial cells, suggesting that differentiation of epithelial cell-like cells, such as marginal cells of the inner ear stria vascularis, could be induced even in a two-dimensional culture in which the cells proliferated and spread as a monolayer on melanocytes through co-culture with pre-adherent cultured melanocytes.

[0139] [Differentiation Induction Methods (Part 3)] (Day 20) In the same method as in Test Example 1, inner ear progenitor cells were obtained from human iPS cells, and these inner ear progenitor cells were cultured until Day 20 using the same method as in Test Example 2.

[0140] Subsequently, co-culture with feeder cells (melanocytes) was carried out in the same manner as in "Differentiation Induction Method (Part 1)" until day 53 (Day 65) after the start of suspension culture.

[0141] [4-3]NKCC1, KCNQ1, LRP2(Day65) On the 48th day after the start of suspension culture (Day60), the cells were fixed by treating overnight at 4°C with 4% paraformaldehyde, treated with 0.3% PBST for 10 minutes, and then blocked at room temperature for 1 hour with 10% normal donkey serum / 0.1% PBST. As primary antibodies, goat anti-NKCC1 antibody, rabbit anti-KCNQ1 antibody, and mouse anti-LRP2 antibody were used at a concentration of 1:100 each and reacted overnight at 4°C. A fluorescent-labeled secondary antibody specific to the IgG animal species of the primary antibody was reacted at room temperature for 1 hour. Also, the nucleus was stained with Hoechst33258. Figure 16 shows the microscopic images obtained by observation with a confocal microscope (scale bar: 10 μm in the upper row, 5 μm in the lower row).

[0142] As a result, as shown in Figure 16, the expression of NKCC1, KCNQ1, and LRP2, known as functional proteins in the marginal cells of the stria vascularis of the inner ear, was detected. Also, while KCNQ1 and LRP2 co-localize in the cell membrane, NKCC1 and KCNQ1 do not co-localize in the cell membrane, and it was considered that in two-dimensional culture as well, cells expressing the marginal cell marker with the same polarity as the inner marginal cells could be induced to differentiate.

[0143] [Test Example 5] In this test example, in the process of inducing differentiation from inner ear progenitor cells to marginal cells of the stria vascularis of the inner ear, the effect of using an insulin-free medium as the culture medium was examined.

[0144] [Differentiation Induction Method] (Day12) Differentiation of human iPS cells into inner ear progenitor cells was induced in the same manner as in Test Example 1. On the 12th day (Day12) from the start of differentiation induction, the cells were detached with trypsin, an equal amount of PBS was added, and the cells were passed through a nylon mesh (pore size 40 μm), and the cell count was performed using a hemocytometer.

[0145] 5×10 5Cells were suspended to a concentration of cells / 10mL, seeded into low-adhesion 6-well plates (product name "Corning Ultra-Low Attachment Plate," Corning), and suspension culture was started under hypoxic conditions (O24%, CO25%). At this time, the culture medium prepared was serum-free medium (DMEM / F12+2%B27+1%N2) to which growth factors bFGF, EGF, IGF-1, FGF3, and FGF10 were added at concentrations of 10 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, and 50 ng / mL, respectively. Furthermore, L-glutamine was added to a concentration of 2 mM, Y-27632 to a concentration of 10 μM, Matrigel to a concentration of 1%, SB431542 to a concentration of 1 μM, and heparin to a concentration of 50 ng / mL. Using this medium, cells were suspended and suspension culture was started.

[0146] From day 8 (Day 20) after the start of suspension culture, the cells were cultured under the following three different culture conditions.

[0147] Condition 1) Culture medium using insulin-containing serum substitute: Except for using serum-free medium (DMEM / F12+2%B27+1%N2) as the culture medium, suspension culture was continued until Day 59 using the same method as in Test Example 2 - Day 20 onwards.

[0148] Condition 2) Insulin-free medium: Except for using serum-free medium (DMEM / F12 + 2%N21-Ins) as the culture medium, suspension culture was continued until Day 59 using the same method as in Test Example 2 - from Day 20 onwards.

[0149] Condition 3) Medium to which insulin has been added to insulin-free medium: Except for using serum-free medium (DMEM / F12 + 2%N21-Ins) to which insulin was added to a concentration of 1.5 μM, suspension culture was continued until Day 59 using the same method as in Test Example 2 from Day 20 onwards.

[0150] (Day 60) In all of the above conditions 1) to 3), cells were collected on day 48 (Day 60) after the start of suspension culture, and the expression level of the OTX2 gene, which is known to be expressed in the Reissner membrane, an organ of the inner ear, but is poorly expressed in the marginal cells of the inner ear stria vascularis, was quantified by qPCR in the same manner as in Test Example 1.

[0151] As a result, as shown in Figure 17, it was found that OTX2 expression levels were lower when insulin was not present in the culture medium compared to when insulin was included (using insulin-containing serum substitute medium and insulin-free medium with added insulin). In other words, OTX2 expression levels could be reduced by not adding insulin to the culture medium. Therefore, it was concluded that using insulin-free medium is necessary to obtain cells that exhibit intrinsic physiological functions similar to endogenous marginal cells.

[0152] [Test Example 6] Inner ear progenitor cells were obtained from human iPS cells using the same method as in Test Example 1, and these inner ear progenitor cells were used in suspension culture until day 49 (Day 61) after the start of suspension culture using the same method as in Test Example 2.

[0153] The obtained cells were subjected to a barrier function assay. Specifically, the cultures, including the cells, were placed in 1.5 mL microcentrifuge tubes after suspension culture, centrifuged, the medium was discarded, and 0.5 mL of HBSS containing 2 mM EEDTA was added. As a control, a sample was prepared under the same conditions with HBBS without EDTA added. Each sample was placed on ice for 15 minutes, and then 4 kDa FITC-Dextran was added to each to a concentration of 2 mg / mL, the cells were resuspended, and immediately observed under a confocal microscope.

[0154] As a result, as shown in the upper panel of Figure 18, no fluorescence image indicating the migration of FITC-Dextran (fluorescence) into the cell aggregate was observed in the untreated cells, whereas as shown in the lower panel of Figure 18, a fluorescence image indicating the migration of FITC-Dextran (fluorescence) into the cell aggregate was observed with EDTA treatment. This suggests that, in the untreated cells, the intercellular barrier function was maintained, preventing migration into the cell aggregate, whereas with EDTA treatment, the calcium-dependent barrier function was impaired, leading to migration into the cell aggregate.

[0155] From the above, it has become clear that the method of the present invention can produce cells that exhibit intrinsic physiological functions similar to those of marginal cells of the stria vascularis inherent in the inner ear organs.

[0156] [Test Example 7] In this study, drug evaluation was performed using marginal cells of the inner ear vascular stria vascularis, which were differentiated using the same method as in Study Example 3.

[0157] Specifically, in Test Example 3, on day 47 (Day 58) after the start of suspension culture, 10 μM cisplatin (an anticancer drug) or 100 μM neomycin (an aminoglycoside antibiotic) was added to the culture medium, and suspension culture was continued for 48 hours until Day 60 under normal oxygen conditions (O2 20%, CO2 5%).

[0158] The expression status of various marker proteins in cells treated with the above-described method was examined by immunohistochemical staining.

[0159] [7-1]cleaved caspase-3, ZO-1(Day60) Cells were fixed by treating them with 4% paraformaldehyde at room temperature for 30 minutes on day 49 of suspension culture (Day 60) and 2 days after the start of drug treatment, and 7 μm frozen sections were prepared. As a control, frozen sections were similarly prepared from cells that had not undergone drug treatment, also on day 49 of suspension culture (Day 60). Each frozen section was treated with 0.3% PBST for 10 minutes, followed by blocking with 10% normal donkey serum / 0.1% PBST at room temperature for 1 hour. Rabbit anti-cleaved caspase-3 antibody and goat anti-ZO-1 antibody were used as primary antibodies at a concentration of 1:200, and reacted overnight at 4°C. Fluorescently labeled secondary antibodies specific to the IgG of the primary antibodies were reacted at room temperature for 1 hour. The nuclei were stained with Hoechst 33258. Figure 19 shows microscopic images obtained by confocal microscopy (scale bar: 20 μm).

[0160] Furthermore, the total number of cells was counted using Hoechst33258 in each microscopic image, and the number of cells expressing cleaved caspase-3 was also counted. The percentage of cells expressing cleaved caspase-3 relative to the total number of cells was then calculated. The results are shown in Figure 20.

[0161] As a result, as shown in Figures 19 and 20, it was confirmed that treatment with each drug increased the number of cells expressing cleaved caspase-3, a known marker of apoptosis, compared to the control group. From these findings, it became clear that ototoxicity caused by various drugs can be evaluated using marginal cells of the inner ear stria vascularis, which are differentiated from human iPS cells.

Claims

1. The process includes culturing a cell population containing inner ear progenitor cells differentiated from pluripotent stem cells in an insulin-free medium that contains no insulin or only trace amounts of insulin. The aforementioned insulin-free medium has an insulin concentration of 0 nM or more and 100 nM or less. A method for producing marginal cells of the inner ear stria vascularis, wherein the insulin-free medium contains EGF and further contains at least one or more selected from the group consisting of bFGF, FGF3, and BMP4.

2. The method for producing marginal cells of the Stria vascularis according to claim 1, wherein the insulin-free medium is selected to contain FGF3 and BMP4.

3. A method for producing marginal cells of the inner ear striae vascularis according to claim 1, comprising the following steps (1) and (2). (1) A process of detaching and dispersing a cell population, including inner ear progenitor cells differentiated from pluripotent stem cells. (2) A step of suspension culture of the cells or cell population obtained in step (1) in the insulin-free medium in the presence of an extracellular matrix material.

4. The method for producing marginal cells of the inner ear vascular stria according to claim 3, wherein the extracellular matrix material comprises at least one or more selected from the group consisting of Matrigel, pronectin, collagen, laminin, and fibronectin.

5. A method for producing marginal cells of the inner ear striae vascularis according to claim 1, comprising the following steps (1) to (3). (1) A process of detaching and dispersing a cell population, including inner ear progenitor cells differentiated from pluripotent stem cells. (2) A step of suspension culture of the cells or cell population obtained in step (1) in the insulin-free medium in the presence of an extracellular matrix material. (3) A step in which the cells or cell population after suspension culture in step (2) are seeded onto feeder cells that have been previously cultured with adherent cells and cultured in the insulin-free medium.

6. The method for producing marginal cells of the inner ear vascular stria according to claim 5, wherein the feeder cells are melanocytes or melanocyte-like cells.

7. A method for producing marginal cells of the inner ear vascular stria of the inner ear according to claim 1, wherein the cell population including inner ear progenitor cells differentiated from the pluripotent stem cells is obtained by a method comprising the following steps (1) to (4). (1) A process of culturing pluripotent stem cells without the addition of growth factors and in the presence of a ROCK inhibitor. (2) A step of culturing the cell population obtained in step (1) in the absence of growth factors and ROCK inhibitors. (3) A step of culturing the cell population obtained in step (2) in the presence of at least one growth factor selected from the group consisting of bFGF, FGF3, FGF10, and FGF19, and BMP4. (4) A step of culturing the cell population obtained in step (3) in the presence of at least one growth factor selected from the group consisting of bFGF, FGF3, FGF10, and FGF19, and in the absence of BMP4.

8. A method for producing marginal cells of the inner ear striae under serum-free conditions, according to claim 1.

9. The method for producing marginal cells of the inner ear stria vascularis according to claim 1, wherein the marginal cells of the inner ear stria vascularis express potassium channel proteins and tight junction proteins.

10. A method for evaluating a drug, comprising the steps of: obtaining marginal cells of the inner ear vascular stria by the manufacturing method described in claim 1; treating the marginal cells of the inner ear vascular stria with a test drug; and evaluating the state of the marginal cells of the inner ear vascular stria treated with the test drug.

11. The process includes culturing a cell population containing inner ear progenitor cells in an insulin-free medium that contains no insulin or only trace amounts of insulin. The aforementioned insulin-free medium has an insulin concentration of 0 nM or more and 100 nM or less. A method for producing marginal cells of the inner ear stria vascularis, wherein the insulin-free medium contains EGF and further contains at least one or more selected from the group consisting of bFGF, FGF3, and BMP4.

12. A method for evaluating a drug, comprising the steps of: obtaining marginal cells of the inner ear vascular stria by the manufacturing method described in claim 11; treating the marginal cells of the inner ear vascular stria with a test drug; and evaluating the state of the marginal cells of the inner ear vascular stria treated with the test drug.

Images