Method for producing hair follicle mesenchymal stem or progenitor cells, and application of same

Abstract

The present invention addresses the problem of providing: a method for reprogramming differentiated hair follicle mesenchymal cells to produce stem or progenitor cells of the hair follicle mesenchymal cells; and a composition for preventing or ameliorating the thinning or graying of hair by using a secretion factor derived from the cells.　A method for producing hair follicle mesenchymal stem or progenitor cells which solves the problem of the present invention is characterized by comprising bringing hair follicle mesenchymal cells into contact with a ROCK inhibitor in vitro. A composition that solves the problem of the present invention is characterized by containing an exosome or microRNA derived from the cells, and is capable of suppressing losing or graying of hair.

Description

Method for producing hair follicle mesenchymal stem cells or precursor cells and their applications

[0001] The present invention relates to a method for producing stem cells or progenitor cells from hair follicle mesenchymal cells using a low-molecular-weight compound.

[0002] Hair follicles (tissues) are skin appendages that produce hair and are primarily composed of epithelial and mesenchymal cells. Dermal papilla cells, a type of mesenchymal cell that constitutes hair follicles, send proliferation and differentiation signals to epithelial cells, causing hair formation. Therefore, dermal papilla cells are thought to play a central role in hair growth and hair production, and are used in the development of hair growth agents and research into hair regenerative medicine. Root sheath cells (including hair bulb root sheath cells), which also belong to the same hair follicle mesenchymal cells, are developmentally very similar to dermal papilla cells (Non-Patent Document 1) and have the same hair growth-inducing ability as dermal papilla cells (Non-Patent Document 2), and are similarly used as cells similar to dermal papilla cells. In recent years, it has been suggested that both dermal papilla cells and root sheath cells are cells supplied by differentiation of hair follicle mesenchymal stem / progenitor cells (Non-Patent Document 3).

[0003] If dermal papilla cells could be widely and commonly used, significant benefits would be expected in the development of hair growth agents, such as drug screening using dermal papilla cells, and in hair regenerative medicine using dermal papilla cells and their secreted factors. However, there is a problem in that prolonged culture of dermal papilla cells reduces their proliferation ability and causes a loss of characteristic properties of dermal papilla cells, such as hair follicle induction ability. To date, a method for culturing dermal papilla cells in vitro while maintaining their properties has been proposed, in which cytokines such as PDGF and FGF2 (bFGF) are added to the culture medium (Non-Patent Document 4). It has also been reported that forming dermal papilla cells into three-dimensional aggregates can restore the characteristic properties lost through differentiation (Non-Patent Document 5). However, the addition of cytokines has not yet resolved the decline in proliferation ability of dermal papilla cells due to long-term culture, and the culture of three-dimensional aggregates poses the problem of being unable to increase the cell number even with continued culture.

[0004] Meanwhile, the development of a method for reprogramming differentiated cells has been reported in recent years. This reprogramming method makes it possible to safely and quickly produce stem or progenitor cells from differentiated cells that possess both self-renewal capacity (proliferation capacity) and specific cell functions (differentiation capacity, regenerative activity, etc.), and is expected to be applied to drug evaluation systems and regenerative medicine. Previously, methods for reprogramming cells derived from endoderm (particularly liver and pancreas) without in vitro gene transfer using specific low-molecular-weight compounds have been reported (Patent Documents 1 to 3). However, there have been no reports of such methods for meso-ectodermal cells, from which hair follicle mesenchymal cells are derived.

[0005] In addition, pigment cells are also present among the epithelial cells that make up hair follicles. Pigment cells produce melanosomes in a way that emphasizes hair formation by epithelial cells, causing hair to turn black. Hair follicle epithelial stem cells and melanocyte stem cells, which are the ancestors (sources) of epithelial cells, are both present in the bulge region of hair follicles. It is known that the number of these stem cells decreases with aging and DNA damage. Depletion of hair follicle epithelial stem cells leads to hair follicle miniaturization, thinning, and vellus hair, ultimately resulting in age-related alopecia (Non-Patent Document 6), and depletion of melanocyte stem cells contributes to hair graying (Non-Patent Document 7). Hair follicle epithelial stem cells secrete TGF-β, maintaining melanocyte stem cells. When the ability of hair follicle epithelial stem cells to secrete TGF-β is reduced, melanocyte stem cells are depleted through ectopic differentiation, resulting in subsequent hair graying (Non-Patent Document 8). Therefore, the decline in hair follicle epithelial stem cells and their functional impairment lead to general hair follicle aging traits, such as hair follicle miniaturization (vellus hair), hair follicle loss, and hair graying. Meanwhile, COL17A1 is a crucial factor for the maintenance and survival of hair follicle epithelial stem cells, and overexpression of this gene can prevent the decline and depletion of hair follicle epithelial stem cells and the resulting hair follicle aging traits (Non-Patent Document 6). Biological drugs utilize the cell-specific functions described above, and are expected to produce highly effective drugs with relatively few side effects compared to the small molecule drugs that have been the mainstream until now.

[0006] International Publication No. 2017 / 119512 International Publication No. 2018 / 079714 International Publication No. 2020 / 080550

[0007] Cell. 2021 Jul 22;184(15):3852-3872. doi: 10.1016 / j.cell.2021.06.024.J Invest Dermatol. 2003 Dec;121(6):1267-75. doi: 10.1111 / j.1523-1747.2003.12568.x.Dev Cell. 2020 Apr 20;53(2):185-198.e7. doi: 10.1016 / j.devcel.2020.03.019.J Dermatol Sci. 2015 Aug;79(2):110-8. doi: 10.1016 / j.jdermsci.2015.04.007. Epub 2015 Apr 27.Proc Natl Acad Sci US A. 2013 Dec 3;110(49):19679-88. doi: 10.1073 / pnas.1309970110. Epub 2013 Oct 21.Science. 2016 Feb 5;351(6273):aad4395. doi: 10.1126 / science.aad4395.Cell. 2009 Jun 12;137(6):1088-99. doi: 10.1016 / j.cell.2009.03.037.Cell Stem Cell. 2011 Feb 4;8(2):177-87. doi: 10.1016 / j.stem.2010.11.029.

[0008] The object of the present invention is to provide a method for reprogramming differentiated hair follicle mesenchymal cells to produce stem cells or progenitor cells, and to provide a biological preparation for preventing or ameliorating the occurrence of thinning hair and graying hair, utilizing secretory factors derived from the reprogrammed hair follicle mesenchymal cells.

[0009] As a result of extensive research, the present inventors discovered that contacting differentiated hair follicle mesenchymal cells with a ROCK inhibitor can reprogram them to a state with properties characteristic of stem cells or progenitor cells, such as cell proliferation and expression of specific markers, and that exosomes and microRNAs derived from these cells have the effect of suppressing hair loss or graying, leading to the completion of the present invention.

[0010] That is, the present invention provides the following: (1) A method for producing hair follicular mesenchymal stem / progenitor cells from hair follicular mesenchymal cells, comprising contacting hair follicular mesenchymal cells with a ROCK inhibitor in vitro. (2) The method for producing hair follicular mesenchymal stem / progenitor cells according to (1), wherein the contacting of hair follicular mesenchymal cells with a ROCK inhibitor is carried out by culturing the hair follicular mesenchymal cells in a medium containing the ROCK inhibitor. (3) The method for producing hair follicular mesenchymal stem / progenitor cells according to (1) or (2), wherein the ROCK inhibitor is Y-27632. (4) The method for producing hair follicular mesenchymal stem or progenitor cells according to any of (1) to (3), wherein the hair follicular mesenchymal cells are dermal papilla cells or root sheath cells. (5) The method for producing hair follicular mesenchymal stem / progenitor cells according to any of (1) to (4), wherein the hair follicular mesenchymal cells are derived from a human, rat, or mouse. (6) A method for maintaining or expanding hair follicle mesenchymal stem / progenitor cells, comprising subculturing hair follicle mesenchymal stem / progenitor cells prepared by the preparation method described in any one of (1) to (5) in the presence of a ROCK inhibitor. (7) A hair growth agent containing hair follicle mesenchymal stem / progenitor cells prepared by the preparation method described in any one of (1) to (5). (8) A method for screening for growth agents, comprising the following steps (i) to (ii): (i) preparing hair follicle mesenchymal stem / progenitor cells by the method described in any one of (1) to (5), and (ii) contacting the hair follicle mesenchymal stem / progenitor cells with a test compound. (9) A hair follicle mesenchymal stem / progenitor cell exosome secretion amplifier containing Y-27632. (10) A hair anti-aging agent containing exosomes derived from hair follicle mesenchymal stem / progenitor cells. (11) The hair anti-aging agent according to (10), which is for inhibiting or improving hair loss, graying, miniaturization of hair follicles, thinning, or vellus hair. (12) A COL17A1 production promoter containing exosomes derived from hair follicle mesenchymal stem / progenitor cells. (13) A TGFB2 production promoter containing exosomes derived from hair follicle mesenchymal stem / progenitor cells.(14) A hair growth agent containing one or more microRNAs selected from the group consisting of miR-16-5p, miR-199a-3p, let-7b-5p, miR-21-5p, miR-191-5p, miR-93-5p, miR-30a-5p, miR-99b-5p, miR-143-3p, miR-10b-5p, miR-127-3p, miR-27b-3p, and miR-151a-3p. (15) A WNT7B production promoter containing one or more microRNAs selected from the group consisting of miR-16-5p, miR-199a-3p, let-7b-5p, miR-21-5p, miR-191-5p, miR-93-5p, miR-30a-5p, miR-99b-5p, miR-143-3p, miR-10b-5p, miR-127-3p, miR-27b-3p, and miR-151a-3p. (16) A hair anti-aging agent containing one or more microRNAs selected from the group consisting of miR-16-5p, let-7b-5p, miR-221-3p, miR-191-5p, miR-4655-5p, miR-125b-5p, miR-30a-5p, miR-26b-5p, miR-143-3p, and miR-10b-5p. (17) A COL17A1 production promoter containing one or more microRNAs selected from the group consisting of miR-16-5p, let-7b-5p, miR-221-3p, miR-191-5p, and miR-4655-5p. (18) A TGFB2 production promoter containing one or more microRNAs selected from the group consisting of miR-125b-5p, miR-16-5p, let-7b-5p, miR-30a-5p, miR-26b-5p, miR-143-3p, and miR-10b-5p. (19) A hair growth agent containing exosomes derived from hair follicle mesenchymal stem / progenitor cells produced by the production method described in any one of (1) to (5). (20) An NFATC1 production inhibitor containing exosomes derived from hair follicle mesenchymal stem / progenitor cells produced by the production method described in any one of (1) to (5).

[0011] According to the present invention, differentiated hair follicle mesenchymal cells can be induced to become hair follicle mesenchymal stem cells or progenitor cells with proliferation and hair follicle induction capabilities. Therefore, it is possible to increase the number of hair follicle mesenchymal cells, such as dermal papilla cells, by repeated passage while maintaining their characteristics. Furthermore, by applying the induced hair follicle mesenchymal stem cells or progenitor cells and their secreted exosomes, it is possible to achieve excellent hair growth effects or hair aging inhibitory effects, such as anti-graying effects, through the promotion of COL17A1 and TGFB2 production or the inhibition of NFATC1 production.

[0012] 1 shows the growth curves of dermal papilla cells in Test Example 1. (N: drug-free medium, Y: Y-27632-added medium, A: A-83-01-added medium, C: CHIR99021-added medium) Images of cell culture in Test Example 1 (Y-27632-added medium and drug-free medium are images of cell culture at passage 7 (P7), and 10% FBS-containing DMEM is an image of cell culture at passage 5 (P5)). This figure shows the average cell proliferation rate per passage (4 days) at passages 8 to 11 in Y-27632-added medium (Y) or non-added medium (N) in Test Example 1. This figure shows the expression level of SOX2 in dermal papilla cells in Test Example 2. (Non: Y-27632-free medium, Y: Y-27632-added medium, DMEM: DMEM medium) Images of exosomes derived from dermal papilla cells in Test Example 3. FIG. 1 is a diagram showing the expression level of WNT7B in Test Example 4 (48 hours after addition of each exosome). FIG. 2 is a diagram showing the expression level of NFATC1 in Test Example 4 (24 hours after addition of each exosome). FIG. 3 is a diagram showing the expression level of COL17A1 in Test Example 5. FIG. 4 is a diagram showing the expression level of TGFB2 in Test Example 5. FIG. 5 is a diagram showing the expression level of WNT7B in Test Example 6. FIG. 6 is a diagram showing the expression level of COL17A1 in Test Example 7. FIG. 7 is a diagram showing the expression level of TGFB2 in Test Example 7.

[0013] As used herein, "hair follicle mesenchymal cells" refers to mesenchymal cells of hair follicles, and includes hair follicle root sheath cells such as dermal papilla cells and hair bulb root sheath cells. A "stem cell" of a hair follicle mesenchymal cell refers to a cell that has the ability to self-renew and the ability to induce hair follicles, and a "progenitor cell" of a hair follicle mesenchymal cell refers to a cell that arises from a stem cell and is at an intermediate stage before differentiating into terminally differentiated cells such as dermal papilla cells and root sheath cells. Furthermore, as used herein, stem cells or progenitor cells reprogrammed from these differentiated hair follicle mesenchymal cells are collectively referred to as "hair follicle mesenchymal stem / progenitor cells," and include "dermal papilla stem / progenitor cells."

[0014] In the present invention, "ectoderm" and "mesoderm" refer to one of the three germ layers that arise during the development of metazoans; during development, the ectoderm forms nervous tissue, epithelial tissue, etc., while the mesoderm forms mesenchymal tissue, etc. "Neural crest" is a part of the ectoderm that transforms into mesenchymal tissue, and is considered the fourth germ layer due to its developmental importance. Hair follicles are a type of skin accessory organ with a structure that envelops the hair shaft. Hair follicle mesenchyme is mesenchymal tissue; while the developmental origin of hair follicle mesenchyme in the body is mainly mesoderm, the origin of some hair follicle mesenchyme on the head is neural crest, so hair follicle mesenchyme is derived from either the ectoderm or mesoderm.

[0015] The dermal papilla cells and root sheath cells (including bulb sheath cells) that make up the hair follicle mesenchyme are functional cells involved in hair growth, but because they are differentiated (mature) cells, it is difficult to maintain their function in vitro while culturing them for long periods of time. However, it has been reported that SOX2-positive cells are contained within each population of dermal papilla cells and root sheath cells, and that these SOX2-positive cells serve as stem / progenitor cells for the hair follicle mesenchyme and serve as the source of dermal papilla cells and root sheath cells. Therefore, maintaining SOX2 expression throughout the entire cell population is considered important for culturing hair follicle mesenchymal cells in vitro while maintaining their function.

[0016] Hair follicles have the characteristic of alternating cycles of anagen and telogen. Hair is produced during anagen and stops during telogen. When hair follicle epithelial cells, which are quiescent during telogen, are activated, the hair follicle transitions to anagen. The activated hair follicle epithelial cells then actively divide and differentiate, producing hair. NFATC1 is known as a transcription factor involved in the quiescent state of hair follicle epithelial cells. When NFATC1 is inhibited, hair follicle epithelial cells are activated, and the hair follicle transitions to anagen. Meanwhile, WNT signaling is known to contribute to hair follicle epithelial activation and hair growth. Activated hair follicle epithelial cells undergo WNT autocrine and paracrine regulation, increasing the intensity of WNT signaling throughout the hair follicle epithelium and activating the entire hair follicle epithelium. WNT7B is known to be the main WNT involved in hair follicle growth.

[0017] According to the present invention, hair follicle mesenchymal cells other than endodermal tissue can also be reprogrammed from differentiated cells to stem cells or progenitor cells, and these stem / progenitor cells can be produced from differentiated dermal papilla cells or root sheath cells by contacting them with a ROCK inhibitor. Furthermore, this reprogramming amplifies the secretion of exosomes from hair follicle mesenchymal stem / progenitor cells, and exosomes derived from hair follicle mesenchymal stem / progenitor cells have a stronger inhibitory effect on NFATC1 production than exosomes derived from cells that have not been reprogrammed, thereby activating hair follicle epithelial cells and achieving a more effective hair growth effect.

[0018] The hair follicle mesenchymal cells used as a starting material in the present invention may be any mesenchymal cells that constitute hair follicles, such as dermal papilla cells and root sheath cells (including hair bulb root sheath cells). The hair follicle mesenchymal cells are not particularly limited, but are preferably derived from humans, rats, or mice, with humans being particularly preferred. Hair follicle mesenchymal cells isolated from hair follicles of these mammals and passaged appropriately can be used, but the passage number is preferably four or less, and it is particularly preferred to use primary cultured hair follicle mesenchymal cells. For example, in the case of human dermal papilla cells, dermal papilla cells collected from hair follicles obtained by biopsy or surgery and then cultured and frozen (frozen dermal papilla cells) can be used. To obtain purified primary cultures of dermal papilla cells and root sheath cells, the hair bulb can be excised from the collected hair follicle tissue, the epithelial tissue can be removed from the hair bulb, and the dermal papilla and root sheath can be separated and primary cultured, respectively.

[0019] Hair follicle mesenchymal cells prepared as described above can be reprogrammed (induced) into hair follicle mesenchymal stem / progenitor cells by contacting them with a ROCK inhibitor. The ROCK inhibitor used in the present invention is not particularly limited as long as it inhibits the function of Rho-associated kinase, and examples include GSK269962A (Axon Medchem), Fasudil hydrochloride (Tocris Bioscience), Y-27632, and H-1152 (all Wako Pure Chemical Industries, Ltd.). Of these, Y-27632 is preferred from the viewpoint of the efficiency of reprogramming hair follicle mesenchymal cells into hair follicle mesenchymal stem / progenitor cells. These ROCK inhibitors may be used alone or in combination of two or more compounds.

[0020] In the present invention, it is possible to use a ROCK inhibitor in combination with a small molecule signaling pathway inhibitor, such as an inhibitor of Rho, which is located upstream of ROCK signaling (NSC23766, zoledronic acid, etc.); however, since there is a possibility that the induction of hair follicle mesenchymal cells into hair follicle mesenchymal stem / progenitor cells may be inhibited, it is preferable to avoid using the inhibitor in combination with a TGF-β inhibitor, a GSK3 inhibitor, etc.

[0021] In the present invention, the method for contacting hair follicle mesenchymal cells with a ROCK inhibitor is not particularly limited, but a preferred embodiment is a method of culturing hair follicle mesenchymal cells in a medium supplemented with a ROCK inhibitor. As the induction medium, a medium widely used for culturing animal cells can be used as the basal medium, and commercially available basal media can also be used. Examples include minimal essential medium (MEM), Dulbecco's modified minimal essential medium (DMEM), RPMI1641 medium, and Follicle Dermal Papilla Cell Growth Medium (supplemented with FCS, pituitary extract, FGF2 (bFGF), and insulin), and these can be used alone or in combination of two or more. Furthermore, when culturing hair follicle mesenchymal cells, it is preferable to use Follicle Dermal Papilla Cell Growth Medium among the above media.

[0022] The concentration of the ROCK inhibitor added to the induction medium is not particularly limited, but from the viewpoint of the effect of inducing hair follicle mesenchymal cells to hair follicle mesenchymal stem / progenitor cells, the concentration is preferably, for example, 0.0001 to 500 μM, more preferably 1 to 15 μM, and particularly preferably 10 μM. When the ROCK inhibitor is a water-insoluble or poorly water-soluble compound, it can be dissolved in a small amount of a low-toxicity organic solvent (e.g., DMSO) and then added to the induction medium to achieve the above-mentioned final concentration.

[0023] Known additives can be used in the induction medium as long as they do not impair the effects of the present invention. Examples of such additives include FGF2 (bFGF), insulin, pituitary extract, serum, etc. The medium can also be supplemented with 1 to 20% by mass of serum (e.g., FBS), but it can also be a serum-free medium. In the case of a serum-free medium, a serum substitute (e.g., BSA, HAS, KSR) can also be added. Furthermore, factors such as growth factors, cytokines, and hormones can also be added. Specific examples include platelet-derived growth factor (PDGF), insulin, transferrin, hydrocortisone 21-hemisuccinate or a salt thereof, and dexamethasone.

[0024] The culture vessel used for the culture is not particularly limited as long as it is suitable for adherent culture, and examples include dishes, Petri dishes, tissue culture dishes, cell culture plates, cell culture flasks, etc. When performing suspension culture, it is also possible to use culture vessels whose surfaces have been treated to prevent cell adhesion. For adherent culture, it is possible to use vessels whose inner surfaces are coated with a cell support substrate to improve cell adhesion, and examples of such cell support substrates include collagen, gelatin, Matrigel, poly-L-lysine, laminin, and fibronectin.

[0025] Hair follicle mesenchymal cells are seeded into the induction medium prepared as described above. The seeding cell density is not particularly limited, but for example, 1,000 to 10,000 cells / cm ２ , preferably 2,000 to 2,500 cells / cm ２ At such a seeding cell density, CO ２ In the incubator, CO is preferably 1 to 10% v / v, more preferably 2 to 8% v / v, and even more preferably 4 to 6% v / v. ２ The cells can be cultured in an atmosphere of a high concentration. The culture temperature is preferably 30 to 40°C, more preferably 35 to 37.5°C, and even more preferably 36.5 to 37.5°C. The culture period is not particularly limited, but in the case of induction into stem / progenitor cells, the cells may be cultured for, for example, 1 to 25 days, preferably 10 to 20 days. In the case of maintenance and expansion of stem / progenitor cells after stem / progenitor cell induction, the cells may be cultured for, for example, 26 to 50 days, preferably 26 to 40 days.

[0026] As described above, contacting hair follicle mesenchymal cells with a ROCK inhibitor allows them to be reprogrammed (induced) into hair follicle mesenchymal stem / progenitor cells, thereby increasing the proportion of hair follicle mesenchymal stem / progenitor cells within the cell population. Herein, a cell population with an increased proportion of hair follicle mesenchymal stem / progenitor cells as described above is referred to as a hair follicle mesenchymal stem / progenitor cell population. Furthermore, herein, "differentiated hair follicle mesenchymal cells" refer to hair follicle mesenchymal cells that are larger and flatter than hair follicle mesenchymal stem / progenitor cells and have low cell proliferation ability. For example, the proportion of differentiated hair follicle mesenchymal cells increases by passage for 15 or more generations or by culturing in a general-purpose medium such as DMEM medium containing 10% FBS. A cell population with an increased proportion of differentiated hair follicle mesenchymal cells is referred to as a "differentiated hair follicle mesenchymal cell population."

[0027] The hair follicle mesenchymal stem / progenitor cells and hair follicle mesenchymal stem / progenitor cell populations obtained as described above have the following properties (a) and (b): (a) high SOX2 expression; and (b) high cell proliferation potential. SOX2 is a stemness marker for hair follicle mesenchymal cells. For a hair follicle mesenchymal stem / progenitor cell population, (a) high SOX2 expression means, for example, that the expression level of SOX2 mRNA, measured by the method described in Test Example 2 herein, is higher than that of a differentiated hair follicle mesenchymal cell population, preferably at least 5-fold, more preferably at least 8-fold, relative to that of a differentiated hair follicle mesenchymal cell population. Even if the cell population already has high SOX2 expression, reprogramming (induction) can further increase SOX2 expression, preferably by at least 1.2-fold, compared to the cell population before contact with a ROCK inhibitor. For a hair follicle mesenchymal stem / progenitor cell population, (b) high cell proliferation potential means, for example, that the cell proliferation rate over four days, measured by the method described in Test Example 1 herein, is preferably at least 5-fold, more preferably at least 6-fold. By reprogramming (induction), a state of high cell proliferation ability due to passage can be maintained preferably from the time of addition of a ROCK inhibitor up to passage 8. Furthermore, the period of high SOX2 expression and the period of high cell proliferation ability generally coincide.

[0028] Furthermore, the hair follicle mesenchymal stem / progenitor cells and hair follicle mesenchymal stem / progenitor cell population of the present invention preferably further have one or more of the following properties (c) to (e): (c) The cells have a bipolar or multipolar, elongated morphology; (d) The apparent proliferation rate does not decrease for at least four passages, preferably eight or more passages, after the addition of a ROCK inhibitor; and (e) SOX2 expression is maintained for at least four passages, preferably eight or more passages, after the addition of a ROCK inhibitor. The above property (c) can be confirmed by microscopic observation, etc., and while differentiated hair follicle mesenchymal cells are flat, hair follicle mesenchymal stem / progenitor cells have a bipolar or multipolar, elongated morphology and are smaller than differentiated hair follicle mesenchymal cells. For the hair follicle mesenchymal stem / progenitor cell population, the above property (d) means, for example, that when hair follicle mesenchymal cells are passage-cultured by the method described in Test Example 1 herein, the cell proliferation rate is 4.5-fold or more for at least four passages, preferably eight or more passages. As for the hair follicle mesenchymal stem / progenitor cell population, (e) above means that hair follicle mesenchymal cells are passage-cultured by the method described in Test Example 1 herein, and for cells cultured for at least four passages, preferably eight passages or more, the expression level of SOX2 mRNA measured by the method described in Test Example 2 is two-fold or more higher than that of a differentiated hair follicle mesenchymal cell population. The above-mentioned passage number can also be expressed as the cumulative number of days in culture after seeding the cells, with the number of days in culture for four passages being 14 to 18 days and for eight passages being 29 to 35 days.

[0029] The hair follicle mesenchymal stem / progenitor cells and hair follicle mesenchymal stem / progenitor cell population of the present invention obtained as described above can be maintained and expanded in an undifferentiated state with properties such as hair follicle inducibility by further subculturing in a medium supplemented with a ROCK inhibitor. Examples of media used for subculturing include Follicle Dermal Papilla Cell Growth Medium (supplemented with FCS, pituitary extract, FGF2 (bFGF), and insulin).

[0030] Specifically, when the hair follicle mesenchymal stem / progenitor cells obtained as described above reach 70 to 100% confluence, they are cultured in Follicle Dermal Papilla Cell Growth Medium at a density of 2,000 to 2,500 cells / cm. ２ The medium used in the induction culture of hair follicle mesenchymal stem / progenitor cells from hair follicle mesenchymal cells can also be used as the medium. The concentration of the ROCK inhibitor added can be in the same range as in the induction culture of hair follicle mesenchymal stem / progenitor cells from hair follicle mesenchymal cells, and the culture temperature, CO ２ The same applies to the concentration and culture vessel. When the cells reach 70 to 100% confluence, they are dissociated by trypsin treatment and then passaged. The dermal papilla stem / progenitor cells of the present invention can be purified by conventionally known methods, as needed.

[0031] Hair follicles have the characteristic of alternating cycles of anagen and telogen. Hair is produced during anagen and stops during telogen. When hair follicle epithelial cells, which are quiescent during telogen, are activated, the hair follicle transitions to anagen. The activated hair follicle epithelial cells then actively divide and differentiate, producing hair. NFATC1 is known as a transcription factor involved in the quiescent state of hair follicle epithelial cells. When NFATC1 is inhibited, hair follicle epithelial cells are activated, and the hair follicle transitions to anagen. Meanwhile, WNT signaling is known to contribute to hair follicle epithelial activation and hair growth. Activated hair follicle epithelial cells undergo WNT autocrine and paracrine regulation, increasing the intensity of WNT signaling throughout the hair follicle epithelium and activating the entire hair follicle epithelium. WNT7B is known to be the main WNT involved in hair follicle growth. Hair follicle epithelial stem cells maintain melanocyte stem cells through TGF-β secretion. When the ability of hair follicle epithelial stem cells to secrete TGF-β is reduced, melanocyte stem cells become depleted through ectopic differentiation, resulting in hair graying. Therefore, activating TGF-β signaling pathways such as TGFB2 can suppress the depletion and depletion of melanocyte stem cells and the associated graying of hair. Meanwhile, depletion of hair follicle epithelial stem cells leads to hair follicle miniaturization, thinning, and vellus hair, leading to hair loss. COL17A1 is a crucial factor for the maintenance and survival of hair follicle epithelial stem cells, and expression of this gene can prevent the depletion and depletion of hair follicle epithelial stem cells and the associated development of the aging traits described above in hair follicles.

[0032] The hair follicle mesenchymal stem / progenitor cells of the present invention obtained as described above maintain excellent hair follicle induction ability, and the exosomes produced therefrom have hair follicle epithelium activation effects, such as the inhibitory effect on NFATC1 production and the promotion of WNT7B production, as well as the inhibitory effect on the reduction and depletion of hair follicle epithelial stem cells and melanocyte stem cells due to the promotion of TGFB2 production and COL17A1 production. Therefore, by administering the cells as a cell preparation, or a culture supernatant preparation or exosome preparation utilizing the secreted exosomes, it is possible to obtain hair follicle neogenesis induction effects, hair growth effects, and effects to inhibit hair aging such as graying.

[0033] Furthermore, the microRNAs (miRNAs) of the hair follicle mesenchymal stem / progenitor cells of the present invention obtained as described above can induce hair follicle neogenesis, promote hair growth, and inhibit hair anti-aging by promoting the production of one or more of WNT7B, TGFB2, and COL17A1. For example, miR-16-5p, miR-199a-3p, let-7b-5p, miR-21-5p, miR-191-5p, miR-93-5p, miR-30a-5p, miR-99b-5p, miR-143-3p, miR-10b-5p, miR-127-3p, miR-27b-3p, and miR-151a-3p have hair follicle neogenesis and hair growth effects due to their promotion of WNT7B production. Furthermore, miR-16-5p, let-7b-5p, miR-221-3p, miR-191-5p, and miR-4655-5p promote COL17A1 production, while miR-125b-5p, miR-16-5p, let-7b-5p, miR-30a-5p, miR-26b-5p, miR-143-3p, and miR-10b-5p promote TGFB2 production, demonstrating anti-aging effects on hair. miRNAs are a type of non-coding small RNA consisting of approximately 22 nucleotides. In recent years, it has been reported that miRNAs are secreted extracellularly within exosomes, and that exosome-encapsulated miRNAs are secretory cell-specific. These findings have attracted attention as diagnostic markers for various diseases and novel therapeutic modalities. The origin of the miRNAs is not particularly limited, but examples include humans, monkeys, pigs, dogs, and cats, with human origin being preferred. Sequence information for these miRNAs is available from databases such as miRBase. The nucleotide sequences of each miRNA are represented by the SEQ ID NOs: 1 to 14. The accession numbers for each miRNA are also shown in Table 1. The miRNAs used in the present invention include polynucleotides in which one or several nucleotides have been deleted, added, or substituted in the nucleotide sequence set forth in any of SEQ ID NOs: 1 to 14, or polynucleotides that have 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more identity to the nucleotide sequence set forth in any of SEQ ID NOs: 1 to 14.

[0034]

[0035] The NFATC1 production inhibitors, WNT7B production promoters, TGFB2 production promoters, COL17A1 production promoters, hair follicle neogenesis inducers, hair growth agents, and hair anti-aging agents (hereinafter referred to as "hair growth agents, etc.") of the present invention can be formulated by suspending the above-mentioned hair follicle mesenchymal stem / progenitor cells in an appropriate isotonic buffer such as PBS, and can be prepared according to known manufacturing methods using pharmaceutically acceptable additives as needed. Furthermore, epithelial stem cells, epithelial stem cell-derived culture supernatant, secreted exosomes, and miRNA can also be incorporated. In the present invention, exosomes are lipid particles constantly secreted from cells themselves and are synonymous with extracellular vesicles. Exosomes are microparticles (a type of membrane vesicle) with a diameter of approximately 30 to 120 nm composed of a lipid bilayer membrane, and contain RNA (mRNA, miRNA), DNA, in addition to proteins and lipids. The dosage of the hair growth agent of the present invention, in the case of a cell preparation, is appropriately determined depending on the severity of symptoms, etc. In the case of an adult, for example, the number of hair follicle mesenchymal stem / progenitor cells per administration site is preferably 1 to 9 × 10 ４ pieces, more preferably 1 to 9 × 10 ５ pcs, more preferably 1 to 9 × 10 ６ It is sufficient to administer the amount of the culture supernatant per administration site so that the number of cells reaches approximately 100 μg. In the case of a hair follicle neogenesis inducer, in addition to hair follicle mesenchymal stem / progenitor cells, it is possible to simultaneously administer approximately the same amount of epithelial stem cells to the same site. In the case of a culture supernatant preparation of hair follicle mesenchymal stem / progenitor cells, for example, the amount of culture supernatant components per administration site is preferably 0.1 to 100 μg, more preferably 1 to 10 μg. In the case of an exosome preparation of hair follicle mesenchymal stem / progenitor cells, for example, the amount of exosome components per administration site is preferably 0.1 to 100 μg, more preferably 1 to 10 μg. In the case of an miRNA preparation, for example, the amount of miRNA per administration site is preferably 0.01 to 10 μg, more preferably 0.1 to 1 μg.

[0036] By administering the hair growth agent etc. of the present invention to the dermis or subcutaneous tissue of the site where hair regeneration is required, it can promote hair follicle formation and hair growth at the site, and can regenerate hair.The hair growth agent etc. of the present invention can prevent and improve symptoms such as hair loss and thinning hair caused by diffuse alopecia, androgenetic alopecia, seborrheic alopecia, senile alopecia, telogen effluvium, female pattern alopecia, etc.It can also suppress hair aging such as graying, miniaturization of hair follicles, thinning, and softening.

[0037] The method of administering the hair growth agent of the present invention is not particularly limited, and any conventionally known administration method can be used, such as topical application, electrical introduction (iontophoresis, etc.), injection using a syringe or a microneedle, etc.

[0038] The screening method of the present invention is a method for screening a hair follicle neogenesis inducer or a hair growth agent, comprising: step (1) preparing hair follicle mesenchymal stem / progenitor cells by the method described above; and step (2) contacting the hair follicle mesenchymal stem / progenitor cells with a test compound. For example, after performing steps (1) and (2), the expression level of a gene that promotes or suppresses hair follicle induction or hair growth in the hair follicle mesenchymal stem / progenitor cells can be measured, and the presence or absence and degree of a hair follicle neogenesis inducer effect or hair growth effect can be evaluated using this as an index.

[0039] Examples of genes that have the effect of promoting hair follicle neogenesis induction or hair growth include FGF7, NOG, RSPO1, VEGFA, LEF1, etc. Examples of genes that suppress hair follicle induction or hair growth include BMP4, TGFB2, WNT5A, etc.

[0040] In this specification, the expression level of a gene used as an indicator includes transcription products such as mRNA transcribed from the gene, and translation products such as polypeptides and proteins translated using the transcription products as templates. The expression level of a gene can be measured by measuring these using known measurement methods. For example, when measuring a transcription product of a gene, methods such as quantitative PCR, in situ hybridization, Northern blotting, and DNA microarray can be used using a probe corresponding to the sequence of the target gene. When measuring a translation product of a gene, an antibody that detects the protein translated by the target gene can be used, and measurement can be performed using methods such as Western blotting, flow cytometry, and ELISA.

[0041] The present invention will be explained in more detail below by way of test examples, but the present invention is not limited to these examples in any way.

[0042] Test Example 1: Evaluation of cell proliferation rate of dermal papilla cells 8,000 human dermal papilla cells (PromoCell) at passage 3 were seeded in a 12-well plate and incubated with CO ２ Incubator (CO ２The cells were cultured in a 5% HCl solution (5% HCl, 37°C) and passaged every four days. If cell growth slowed, the number of seeded cells (8,000) was kept the same, but the number of culture days before passage was increased. The medium used was Follicle Dermal Papilla Cell Growth Medium (PromoCell). The compounds added were the ROCK inhibitor Y-27632 (Fujifilm Wako Pure Chemical Industries), the TGF-β inhibitor A-83-01 (Fujifilm Wako Pure Chemical Industries), and the GSK3 inhibitor CHIR99021 (Axon Medchem). Compounds were added one day after seeding at passage 4, with Y-27632 at 10 μM, A-83-01 at 0.5 μM, and CHIR99021 at 3 μM. For subculture, cells were detached using 0.05% trypsin / EDTA solution (Thermo Fisher Scientific), neutralized with Defined Trypsin Inhibitor (Thermo Fisher Scientific), and then harvested. The cell number was counted using trypan blue staining. The harvested cell suspension was diluted with medium to the desired seeding density (8,000 cells per well) and seeded.

[0043] The cell proliferation rate from seeding to passage was calculated by dividing the number of cells recovered at each passage by the number of cells seeded (8,000). Starting from the seeding of 8,000 cells at passage 3, this was multiplied by the cell proliferation rate at each passage to calculate the maximum cell number calculated from the initial seeding number, and a proliferation curve was created. Figure 1 shows the proliferation curves of dermal papilla cells in various compound-containing media, and Figure 2 shows cell culture images. Similarly, Figure 2 shows cell culture images of human dermal papilla cells passaged repeatedly using 10% FBS-containing DMEM (Dulbecco's Modified Eagle Medium). Figure 3 shows the average cell proliferation rate (cell proliferation rate per passage) over each passage period (4 days) at passages 8 to 11, with or without Y-27632. Cells maintained a high proliferation rate for a long period in Y-27632-supplemented media, with the difference compared to unsupplemented media being particularly pronounced from passages 8 to 11. For statistical analysis, Student's t-test was used after confirming homogeneity of variance.

[0044] Test Example 2: Evaluation of SOX2 expression level in dermal papilla cells Human dermal papilla cells (PromoCell) were passaged in the same manner as in Test Example 1 (except that a 100 mm culture dish was used and the seeding cell density was the same) using Follicle Dermal Papilla Cell Growth Medium with or without 10 μM Y-27632, or DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS. At the 7th or 11th passage, CO ２ Incubator (CO ２After culturing the cells in a medium containing 5% ATP and 37°C until they reached 60-70% confluence, mRNA was isolated from the cells using the miRNeasy Mini Kit (Qiagen) according to the kit's protocol. As a control, mRNA was isolated from dermal papilla cells (PromoCell) at passage 3 cultured in Follicle Dermal Papilla Cell Growth Medium using a similar method. Using this RNA as a template, cDNA was synthesized by reverse transcription using SuperScript IV VILO MasterMix (Thermo Fisher Scientific). The synthesized cDNA was then analyzed using a real-time PCR system (Step One Plus, Thermo Fisher Scientific) to measure the expression level of the target gene, SOX2, using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) and the TaqMan method. The SOX2 expression level was then normalized by the expression level of the internal control gene, RPLP0, which was measured in the same way. For statistical analysis, Dunnett's test was used after confirming homogeneity of variance. The information on the primers used in Test Example 2 is as shown in Table 2 below.

[0045]

[0046] SOX2 expression normalized by RPLP0 is shown in Figure 4. In detail, the SOX2 expression levels in dermal papilla cells cultured under the following conditions (i) to (vi) are shown. (i) P3: Dermal papilla cells of passage 3 cultured in Follicle Dermal Papilla Cell Growth Medium (ii) Non P7: Dermal papilla cells of passage 7 (after 15 days of culture) passaged in a medium not supplemented with Y-27632 (iii) Non P11: Dermal papilla cells of passage 11 (after 29 to 35 days of culture) passaged in a medium not supplemented with Y-27632 (iv) Y P7: Dermal papilla cells of passage 7 (after 15 days of culture) passaged in a medium supplemented with Y-27632 (v) Y P11: Dermal papilla cells of passage 11 (after 29 to 35 days of culture) passaged in a medium supplemented with Y-27632 (vi) DMEM P7: Dermal papilla cells of passage 7 (after 15 days of culture) passaged in DMEM containing 10% FBS Regarding the expression of SOX2, a marker characteristic of hair follicle mesenchymal stem / progenitor cells, when cultured in Y-27632-supplemented medium, SOX2 expression was significantly higher at passage 7 (after 15 days of culture) than before the addition of Y-27632 (passage 3), confirming the reprogramming phenomenon (Student's t-test). Furthermore, the decline in SOX2 expression during long-term culture in Y-27632-supplemented medium (passage 11; after 29 to 35 days of culture) was more gradual than in groups cultured in Y-27632-free medium or DMEM, confirming that stem / progenitor cell status is maintained over the long term. These results demonstrate that dermal papilla stem / progenitor cells can be induced and expanded under Y-27632-supplemented conditions.

[0047] Test Example 3: Measurement of exosome amount in culture supernatant of dermal papilla cells Test Example 3-1 Human dermal papilla cells (PromoCell) that had been passaged (passage number 7) using Follicle Dermal Papilla Cell Growth Medium (PromoCell) with or without Y-27632 were seeded on a 15 cm dish and incubated with CO ２ Incubator (CO ２The cells were cultured in a 5% PBS buffer (5% PBS, 37°C) until they reached 80-90% confluence. The medium was then changed to Advanced DMEM (Thermo Fisher Scientific) containing a specified amount of Antibiotic-Antimycotic (Thermo Fisher Scientific) for exosome recovery. After two more days of culture, the culture supernatant was collected and ultracentrifuged at 210,000 x G for 70 min at 4°C using an ultracentrifuge (Optima XE-90, Beckman Coulter) to recover the precipitate containing dermal papilla cell-derived exosomes. The precipitate was diluted and washed with DPBS, no calcium, no magnesium (Thermo Fisher Scientific), and then again ultracentrifuged at 210,000 x G for 70 min at 4°C to recover dermal papilla cell-derived exosomes.

[0048] Test Example 3-2 After exosome collection, the volume of the exosome solution was measured using a pipette, and the protein concentration in the exosome solution was then measured. Protein concentration was measured using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and a Qubit Protein Assay Kit (Thermo Fisher Scientific) according to the kit's protocol. The total protein amount of exosomes was calculated from the volume and protein concentration of the exosome solution, and the exosome protein concentration in the culture supernatant was calculated by dividing this total protein amount by the volume of the centrifuged culture supernatant. Furthermore, the exosome protein concentration in the culture supernatant was divided by the volume of the medium in the 15-cm dish from which the exosomes were collected to calculate the exosome amount per 15-cm dish. The average exosome amount per 15-cm dish for each dermal papilla cell type is shown in Figure 5. These results confirmed that the addition of Y-27632 amplifies exosome secretion from dermal papilla stem / progenitor cells.

[0049] Test Example 4: Evaluation of hair growth effect of exosomes derived from dermal papilla cells Test Example 4-1 Human hair follicle keratinocytes (ScienCell Research Laboratories) were placed in a 48-well plate (AGC Technoglass) coated with type I collagen at 1.25 × 10４ The cells were seeded at a cell density of 1000 cells / well and incubated under CO ２ Incubator (CO ２ The cells were cultured in a 5% soluble lecithin (5%) at 37°C. The medium used was Humedia-KB2 (Kurabo HuMedia-KG2 basal medium) supplemented with a specified amount of gentamicin / amphotericin B.

[0050] Test Example 4-2 On the day after seeding, exosomes derived from dermal papilla cells or adipose stem cells under the following conditions (i) to (ii) were added to the medium to a concentration of 10 μg / mL, and cultured for a further 24 or 48 hours (NFATC1 expression was evaluated for 24 hours, and WNT7B expression was evaluated for 48 hours). PBS containing no exosomes was used as a control. Exosomes derived from adipose stem cells were prepared from human adipose-derived mesenchymal stem cells (StemPro TM Human adipose-derived mesenchymal stem cells were collected from adipose tissue culture medium (Human Adipose-Derived Stem Cells, Thermo Fisher Scientific). Human adipose-derived mesenchymal stem cells were cultured in MesenPRO RS medium supplemented with 2 mM L-glutamine (Thermo Fisher Scientific) and an antibiotic / antimycotic (Thermo Fisher Scientific). TMExosomes were extracted using the same method as for dermal papilla cells. (i) Exosomes derived from dermal papilla cells at passage 7 cultured in Y-27632-supplemented medium (Y condition P7 DP exosomes). (ii) Exosomes derived from adipose stem cells (ASCs). mRNA was then extracted from the cells using a Maxwell RSC48 (Promega) and the simplyRNA Cells Kit (Promega) according to the protocol. Using this RNA as a template, cDNA was synthesized by reverse transcription using SuperScript IV VILO MasterMix (Thermo Fisher Scientific). The mRNA expression levels of WNT7B and NFATC1 were measured using a real-time PCR system (QuantStudio, Thermo Fisher Scientific) with TB Green Premix Ex Taq II (Takara Bio) using the SYBR Green method. The expression levels of WNT7B and NFATC1 were then corrected using the expression level of RPLP2, an internal standard gene, measured in the same manner. For statistical analysis, Student's t-test or Dunnett's test was used after confirming homogeneity of variance. The primer information used in Test Example 4 is shown in Table 3 below.

[0051]

[0052] Figure 6 shows the results of evaluating the effect of each dermal papilla cell-derived exosome on WNT7B expression. An increase in WNT7B expression was confirmed with the addition of dermal papilla cell-derived exosomes. This confirmed that dermal papilla cell-derived exosomes have the effect of promoting WNT7B production. Figure 7 shows the results of evaluating the effect of each dermal papilla cell-derived exosome on NFATC1 expression. A decrease in NFATC1 expression was confirmed with the addition of dermal papilla cell-derived exosomes in Y-27632-supplemented medium. This confirmed that dermal papilla cell-derived exosomes in Y-27632-supplemented medium have the effect of suppressing NFATC1 production.

[0053] Test Example 5: Evaluation of the Anti-Aging Effect of Dermal Papilla Cell-Derived Exosomes Human dermal papilla cell-derived exosomes were collected using the same method as in Test Example 3-1. Human hair follicle keratinocytes were cultured using the same method as in Test Example 4-1. As in Test Example 4-2, the day after culture, exosomes derived from dermal papilla cells (passage 7) in Y-27632-supplemented medium were added to the medium at a concentration of 10 μg / mL and cultured for an additional 48 hours. PBS containing no exosomes was used as a control. Subsequently, mRNA was isolated from the cells using Maxwell RSC48 and the simplyRNA Cells Kit according to the protocol. Using this RNA as a template, cDNA was synthesized by reverse transcription using SuperScript IV VILO MasterMix. From the synthesized cDNA, the expression levels of TGFB2 and COL17A1 mRNA were measured using a real-time PCR system with TB Green Premix Ex Taq II and the SYBR Green method. The expression levels of TGFB2 and COL17A1 were then corrected using the expression level of RPLP2, an internal standard gene, measured in the same manner. For statistical analysis, Student's t-test was used after confirming homogeneity of variance. The primer information used in Test Example 5 is shown in Table 4 below.

[0054]

[0055] Figure 8 shows the results of evaluating the effect of exosomes derived from each type of dermal papilla cell on COL17A1 expression. An increase in COL17A1 expression was confirmed by the addition of exosomes derived from dermal papilla cells cultured in Y-27632-supplemented medium. This confirms that exosomes derived from dermal papilla cells cultured in Y-27632-supplemented medium have the effect of promoting COL17A1 production. Figure 9 shows the results of evaluating the effect of exosomes derived from each type of dermal papilla cell on TGFB2 expression. An increase in TGFB2 expression was confirmed by the addition of exosomes derived from dermal papilla cells cultured in Y-27632-supplemented medium. This confirms that exosomes derived from dermal papilla cells cultured in Y-27632-supplemented medium have the effect of promoting TGFB2 production.

[0056] Test Example 6: Evaluation of hair growth effect by miRNA Lipofectamine was used to introduce miRNA into cells. TM A reaction solution was prepared using RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer's protocol, with the miRNA concentration at 100 nM. The sequences of the miRNAs used are shown in Table 5. Human hair follicle keratinocytes were seeded and cultured using the same procedure as in Example 4-1. After 1 day of culture, the miRNA was introduced into the cells by adding the pre-prepared miRNA solution to the medium, and the cells were cultured for an additional 48 hours. mRNA was then isolated from the cells using a Maxwell RSC48 and the simplyRNA Cells Kit. Using this RNA as a template, cDNA was synthesized by reverse transcription using SuperScript IV VILO MasterMix. The mRNA expression levels of the target genes were measured using a real-time PCR system with TB Green Premix Ex Taq II and the SYBR Green method. The expression levels of the target genes were then corrected for the expression levels of the internal control gene, RPLP2, measured in the same way. Statistical analysis was performed using Student's t-test or Dunnett's test after confirming homogeneity of variance. The primers used in Test Example 6 are as shown in Table 3. The miRNAs used in Test Example 6 are as shown in Table 5.

[0057]

[0058] The effects of each miRNA sequence on WNT7B expression were evaluated and the results are shown in Figure 10. The introduction of miR-16-5p, miR-199a-3p, let-7b-5p, miR-21-5p, miR-191-5p, miR-93-5p, miR-30a-5p, miR-99b-5p, miR-143-3p, miR-10b-5p, miR-127-3p, miR-27b-3p, and miR-151a-3p resulted in increased WNT7B expression. This confirms that these miRNAs promote WNT7B production.

[0059] Test Example 7: Evaluation of the anti-aging effect of miRNAs. Using the same method as in Test Example 6, the miRNAs listed in Table 5 were introduced into human hair follicle keratinocytes, and the effects on COL17A1 and TGFB2 expression were evaluated. The results of evaluating the effects of each miRNA sequence on COL17A1 expression are shown in Figure 11 , and the results of evaluating the effects on TGFB2 expression are shown in Figure 12 . Introduction of miR-16-5p, let-7b-5p, miR-221-3p, miR-191-5p, and miR-4655-5p confirmed increased COL17A1 expression. This confirmed that these miRNAs promote COL17A1 production. Furthermore, introduction of miR-125b-5p, miR-16-5p, let-7b-5p, miR-30a-5p, miR-26b-5p, miR-143-3p, and miR-10b-5p confirmed increased TGFB2 expression. This confirmed that these miRNAs have the effect of promoting TGFB2 production.

[0060] The present invention enables the proliferation of hair follicle mesenchymal stem / progenitor cells, such as dermal papilla cells, through repeated passage while maintaining characteristics such as hair follicle inducibility. Therefore, the present invention is useful for drug screening and as a hair regenerative medical material using dermal papilla cells and their secreted factors. Furthermore, dermal papilla cell-derived exosomes have anti-hair-graying effects in addition to hair growth effects, making them useful as anti-hair-graying materials. Furthermore, miRNAs with specific sequences are useful as hair growth and anti-hair-graying materials.

Claims

1. A method for producing hair follicle mesenchymal stem / progenitor cells from hair follicle mesenchymal cells, which comprises contacting the hair follicle mesenchymal cells in vitro with a ROCK inhibitor.

2. A method for producing hair follicle mesenchymal stem / progenitor cells according to claim 1, wherein the contact of hair follicle mesenchymal cells with a ROCK inhibitor is achieved by culturing the hair follicle mesenchymal cells in a medium containing a ROCK inhibitor.

3. The method for producing hair follicle mesenchymal stem / progenitor cells according to claim 1, wherein the ROCK inhibitor is Y-27632.

4. A method for producing stem or precursor cells of hair follicle mesenchymal cells according to claim 1, wherein the hair follicle mesenchymal cells are dermal papilla cells or root sheath cells.

5. A method for producing hair follicle mesenchymal stem / progenitor cells according to claim 1, wherein the hair follicle mesenchymal cells are derived from a human, rat or mouse.

6. A method for maintaining or expanding hair follicle mesenchymal stem / progenitor cells, comprising subculturing hair follicle mesenchymal stem / progenitor cells prepared by the method of any one of claims 1 to 5 in the presence of a ROCK inhibitor.

7. A hair growth agent containing hair follicle mesenchymal stem / progenitor cells prepared by the method according to any one of claims 1 to 5.

8. A method for screening hair growth agents, comprising the following steps (1) and (2): (1) preparing hair follicle mesenchymal stem / progenitor cells by the method according to any one of claims 1 to 5; and (2) contacting the hair follicle mesenchymal stem / progenitor cells with a test compound.

9. A hair follicle mesenchymal stem / progenitor cell exosome secretion enhancer containing Y-27632.

10. An anti-aging agent for hair containing exosomes derived from hair follicle mesenchymal stem / progenitor cells.

11. The hair anti-aging agent according to claim 10, which is used to inhibit or improve hair loss, graying, miniaturization of hair follicles, thinning or softening of hair.

12. A COL17A1 production promoter containing exosomes derived from hair follicle mesenchymal stem / progenitor cells.

13. A TGFB2 production promoter containing exosomes derived from hair follicle mesenchymal stem / progenitor cells.

14. A hair growth agent containing one or more microRNAs selected from the group consisting of miR-16-5p, miR-199a-3p, let-7b-5p, miR-21-5p, miR-191-5p, miR-93-5p, miR-30a-5p, miR-99b-5p, miR-143-3p, miR-10b-5p, miR-127-3p, miR-27b-3p, and miR-151a-3p.

15. A WNT7B production promoter containing one or more microRNAs selected from the group consisting of miR-16-5p, miR-199a-3p, let-7b-5p, miR-21-5p, miR-191-5p, miR-93-5p, miR-30a-5p, miR-99b-5p, miR-143-3p, miR-10b-5p, miR-127-3p, miR-27b-3p, and miR-151a-3p.

16. An anti-aging agent for hair containing one or more microRNAs selected from the group consisting of miR-16-5p, let-7b-5p, miR-221-3p, miR-191-5p, miR-4655-5p, miR-125b-5p, miR-30a-5p, miR-26b-5p, miR-143-3p, and miR-10b-5p.

17. A COL17A1 production promoter comprising one or more microRNAs selected from the group consisting of miR-16-5p, let-7b-5p, miR-221-3p, miR-191-5p, and miR-4655-5p.

18. A TGFB2 production promoter containing one or more microRNAs selected from the group consisting of miR-125b-5p, miR-16-5p, let-7b-5p, miR-30a-5p, miR-26b-5p, miR-143-3p, and miR-10b-5p.

19. A hair growth agent containing exosomes derived from hair follicle mesenchymal stem / progenitor cells produced by the production method described in any one of claims 1 to 5.

20. An NFATC1 production inhibitor containing exosomes derived from hair follicle mesenchymal stem / progenitor cells produced by the production method described in any one of claims 1 to 5.

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