Epithelial-derived cells for use in producing regenerated hair follicle primordia

Abstract

[Problem] To provide a novel epithelium-derived cell that can be used for manufacturing regenerated hair follicle germs. [Solution] Provided are: an epithelium-derived cell expressing integrin β5 (Itgb5) at a detectable level; a cell population including said cell; and a method for producing regenerated hair follicle germs by using the cell population.

Description

[Technical Field]

[0001] The present invention relates to epithelial-derived cells that can be used to produce regenerated hair follicle primordia. [Background technology]

[0002] To realize hair regenerative medicine, it is necessary to obtain epithelial-derived stem cells and mesenchymal stem cells that have the ability to induce hair follicles, which are necessary for reconstructing hair follicle primordia, and to proliferate them in vitro. Regarding mesenchymal stem cells, it is known that dermal papilla cells, which can be obtained from the dermal papilla, have the ability to induce hair follicles, and it has been shown that they can be obtained by culturing them in vitro (Non-Patent Document 1).

[0003] On the other hand, with regard to epithelial-derived stem cells, a culture technique for Lgr5 (leucine-rich reapeat-containing G-protein coupled receptor 5)-positive stem cells from intestinal epithelial tissue has been established, and it has been shown that intestinal villi can be regenerated from cultured Lgr5-positive stem cells (Non-patent Document 2).

[0004] The presence of stem cells capable of inducing hair follicles in the bulge region of hair follicles has been suggested (Non-Patent Document 3), and regarding in vitro proliferation, a method for proliferating stem cells positive for the cell surface markers CD34 (CD34 antigen) and CD49f (integrin α6) by three-dimensional culture of mouse hair follicle-derived cells has been reported (Non-Patent Document 4), but their organ regeneration ability has not been fully demonstrated. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Tissue Eng.13,975-82,2007 [Non-patent document 2] Nature 459, 262-5, 2009 [Non-patent document 3] Exp. Cell Res. 316, 1422-8, 2010 [Non-patent document 4] EMBO J. 36, 151-64, 2017 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide novel epithelium-derived cells that can be used to produce regenerated hair follicle primordia. [Means for solving the problem]

[0007] As a result of extensive research to solve the above-mentioned problems, the inventors discovered that a population of epithelial-derived cells expressing detectable levels of integrin β5 (Itgb5) can induce regenerated hair follicle primordia that enable sustained hair growth, thereby completing the present invention.

[0008] That is, the present invention includes the following features: [1] Epithelial-derived cells, express detectable levels of integrin β5 (Itgb5), characterized in that Epithelial-derived cells.

[0009] [2] The epithelial-derived cells according to [1], further express detectable levels of the cell surface markers CD34 and / or CD49f, characterized in that Epithelial-derived cells.

[0010] [3] The epithelial-derived cell according to [1] or [2], additionally express detectable levels of the cell surface markers CD34 and CD49f, characterized in that Epithelial-derived cells.

[0011] [4] The epithelial-derived cell according to any one of [1] to [3], Expression of PDGFRα (platelet-derived growth factor receptor-α) and the cell surface marker CD86 is not detectable by flow cytometry characterized in that Epithelial-derived cells.

[0012] [5] An epithelial-derived cell population comprising the epithelial-derived cell according to any one of [1] to [4].

[0013] [6] The epithelial-derived cell population according to [5], In vitro cultures, Epithelial-derived cell populations.

[0014] [7] The epithelial-derived cell population according to [5] or [6], containing at least 2% of cells expressing Itgb5, characterized in that Epithelial-derived cell populations.

[0015] [8] The epithelial-derived cell population according to [5] or [6], containing cells expressing Itgb5 in at least 10% of the cells, characterized in that Epithelial-derived cell populations.

[0016] [9] The epithelial-derived cell population according to any one of [5] to [8], It is characterized in that it is used for producing a regenerated hair follicle primordium. Cell population.

[0017]

[10] A method for producing a regenerated hair follicle primordium, comprising: [5] to [8], and a mesenchymal cell-derived cell population are contacted and cultured to obtain a regenerated hair follicle primordium. Manufacturing method.

[0018] Any combination of one or more of the features of the present invention described above is also included within the scope of the present invention. [Effects of the Invention]

[0019] According to the present invention, novel epithelium-derived cells that can be used to produce regenerated hair follicle primordia are provided. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 shows a flow cytometric analysis of mouse Itgb5-positive cells after isolation (right frame) from cultured mouse skin epithelium-derived cells (left frame). [Figure 2] FIG. 2 shows the organ primordium method used to examine the differentiation potential of mouse Itgb5-positive cells in hair follicles (A) and histological analysis demonstrating the differentiation potential of mouse Itgb5-positive cells (B). [Figure 3] Figure 3 shows flow cytometry analysis of cell populations expressing CD34 and CD49f in cultured mouse skin epithelium-derived cells (A) and cell populations expressing CD34 and Itgb5 before and after isolation (B) (left frame: before isolation; center frame: Itgb5-negative / positive cells; right frame: Itgb5-negative cells). [Figure 4] Figure 4 shows the organ primordium method used to examine the functional analysis of Itgb5-positive cells in relation to the persistence of hair growth, a representative example of hair growth after transplantation (white arrowhead: hair growth), and the hair growth rate and the proportion of mouse Itgb5-negative cells and mouse Itgb5-negative / positive cells with three or more hair cycles (three or more hair cycles). [Figure 5] Figure 5 shows the CD34 and CD49f expression cell populations (A), CD34 and Itgb5 expression cell populations (gating within the black box (1) in A, B), and CD86 and Itgb5 expression cell populations (gating within the black box (2) in B, C) of cultured mouse skin epithelium-derived cells. DETAILED DESCRIPTION OF THE INVENTION

[0021] Hereinafter, embodiments of the present invention will be described in detail. The present invention relates to novel epithelial-derived cells, which are prepared from epithelial cells of animals (typically humans) and are characterized by expressing a detectable level of Itgb5.

[0022] As used herein, the term "detectable level" refers to a level at which expression of a cell surface marker can be detected relative to a negative control when the marker is detected by a method known to those skilled in the art. Therefore, the degree of expression is not important, and in the present invention, even a slight expression level can be considered a "detectable level" as long as the expression is detected relative to a negative control.

[0023] In the present invention, techniques used to detect cell surface marker mRNA (messenger RNA), cDNA (complementary DNA), or protein include colorimetric, luminescent, fluorescent, ultraviolet (UV), and radioisotope (RI) techniques. Specific examples include, but are not limited to, flow cytometry, Northern blotting, Western blotting, reverse transcription-polymerase chain reaction (RT-PCR), reverse transcription-quantitative polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent spot assay (ELISA), enzyme-linked immunosorbent spot assay (ELISPOT), competitive enzyme-multiplied immunoassay (ENMA), radioallergen adsorption (RAST) test, radioimmunoassay, radiobinding assay, gel filtration chromatography, agarose gel electrophoresis, and acrylamide gel electrophoresis. In one embodiment of the present invention, the technique used to detect cell surface markers is flow cytometry.

[0024] In one embodiment, the epithelial-derived cells of the present invention further express detectable levels of other epithelial cell markers, including, but not limited to, CD34, CD49f, integrin α5, integrin β1, integrin β6, integrin β8, TGFβ (Transforming growth factor beta) receptor 1, TGFβ receptor 2, TGFβ receptor 3, retinoic acid receptor α, inhibitor of DNA binding 1, inhibitor of DNA binding 2, inhibitor of DNA binding 3, histone deacetylase 9, Kruppel-like factor 4, Kruppel-like factor 5, Kruppel-like factor 7, activating transcription factor 3, nuclear receptor subfamily 4 group A member 1, nuclear receptor subfamily 4 group A member 2, cytokeratin 15, LIM homeobox protein 2, sex determining region Y-box 9, collagen type XVII α1, and cadherin 3. In one embodiment of the invention, the epithelial-derived cells of the invention further express detectable levels of CD34 and CD49f.

[0025] In one embodiment, the epithelial-derived cells of the present invention do not express detectable levels of specific cell surface markers, such as PDGFRα and CD86. In one embodiment, the epithelial-derived cells of the present invention do not express detectable levels of PDGFRα and CD86.

[0026] The method for producing epithelial-derived cells or a cell population containing said cells of the present invention will be described below.

[0027] The epithelial-derived cells of the present invention can be obtained by culturing cells derived from animal epithelial tissue in a basal medium containing a specific expression inducer according to a conventional method.

[0028] Basal media refers to media containing carbon sources, nitrogen sources, inorganic salts, and the like essential for culturing cells, particularly mammalian cells (e.g., human). Basal media that can be used for producing the epithelial-derived cells of the present invention can be media generally used for culturing animal cells, including, but not limited to, minimum essential media (MEM) such as Eagle's medium, Dulbecco's modified Eagle's medium (DMEM), minimum essential medium α (MEM-α), Ham's F-12 and F-10 media, DMEM / F12 medium, Williams' medium E, RPMI-1640 medium, MCDB medium, 199 medium, Fisher's medium, Iscove's modified Dulbecco's medium (IMDM), McCoy's modified medium, DEF-CS medium, CnT-PR medium, Advanced DMEM medium, Advanced MEM medium, Advanced DMEM / F12 medium, and mixtures thereof.

[0029] In one embodiment, the production of epithelial-derived cells of the present invention uses at least the following expression inducers: at least one bone morphogenetic protein (BMP) inhibitor; At least one fibroblast growth factor (FGF); and At least one of Sonic hedgehog (SHH) or an SHH agonist.

[0030] The BMP inhibitor used to produce the epithelial-derived cells of the present invention is not particularly limited as long as it can prevent or inhibit the binding of BMP molecules to BMP receptors. Whether a molecule or compound has BMP inhibitory activity can be determined by measuring the transcriptional activity of BMP using methods known to those skilled in the art (Zilberberg et al., BMC Cell Biol, 8:41, 2007).

[0031] Examples of BMP inhibitors used in the production of epithelial-derived cells of the present invention include, but are not limited to, Noggin, Dorsomorphin, Chordin, Follistatin, and Ectodin. In the present invention, one BMP inhibitor may be used alone, or multiple BMP inhibitors may be used in combination.

[0032] The concentration of the BMP inhibitor used in producing the epithelium-derived cells of the present invention can be in the range of 0.1 ng / mL to 1000 ng / mL, preferably 0.3 ng / mL to 300 ng / mL, and more preferably 1 ng / mL to 100 ng / mL.

[0033] Fibroblast growth factors (FGFs) used in the production of epithelial-derived cells of the present invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, and FGF-10. In the production of epithelial-derived cells of the present invention, one fibroblast growth factor may be used alone, or multiple fibroblast growth factors may be used in combination. In another embodiment of the present invention, at least two fibroblast growth factors are used in combination.

[0034] In one embodiment of the present invention, at least FGF-7 is used as the fibroblast growth factor.

[0035] The concentration of fibroblast growth factor used in producing the epithelium-derived cells of the present invention can be in the range of 0.1 ng / mL to 1000 ng / mL, preferably 0.3 ng / mL to 300 ng / mL, and more preferably 1 ng / mL to 100 ng / mL.

[0036] The SHH agonist used in the production of epithelial-derived cells of the present invention is not particularly limited as long as it can enhance SHH-mediated signal transduction. Whether a molecule or compound has SHH agonist activity can be determined using, for example, the method taught in Japanese Patent Application Laid-Open No. 2009-213442. Examples of SHH agonists used in the production of epithelial-derived cells of the present invention include, but are not limited to, SAG (Smoothened agonist) and purmorphamin.

[0037] The concentration of SHH or SHH agonist used in producing the epithelium-derived cells of the present invention can be in the range of 0.1 ng / mL to 1000 ng / mL, preferably 0.3 ng / mL to 300 ng / mL, and more preferably 1 ng / mL to 100 ng / mL.

[0038] In one embodiment, at least one receptor tyrosine kinase ligand is further used in the production of the epithelium-derived cells of the present invention. The use of a receptor tyrosine kinase ligand in the production of the epithelium-derived cells of the present invention is preferred because it significantly improves the engraftment rate when the produced regenerated hair follicle primordium is transplanted into a living body. Receptor tyrosine kinase ligands used in the production of epithelial-derived cells of the present invention include, but are not limited to, epidermal growth factor (EGF), transforming growth factor α (TGFα), amphiregulin, and heparin-binding EGF-like growth factor (HB-EGF).

[0039] The concentration of the receptor tyrosine kinase ligand used in the production of the epithelium-derived cells of the present invention can be in the range of 0.1 ng / mL to 1000 ng / mL, preferably 0.3 ng / mL to 300 ng / mL, and more preferably 1 ng / mL to 100 ng / mL.

[0040] If necessary, at least one TGFβ receptor / ALK5 (Anaplastic lymphoma kinase 5) inhibitor is further used in the production of the epithelial-derived cells of the present invention. The TGFβ receptor / ALK5 inhibitor used in the production of the epithelial-derived cells of the present invention is not particularly limited as long as it can inhibit the interaction between TGFβ receptor and ALK5, and may include a TGFβ receptor inhibitor or an ALK5 inhibitor, etc. Examples of TGFβ receptor / ALK5 inhibitors used in the production of epithelial-derived cells of the present invention include, but are not limited to, SB431542, A83-01, ALK5 Inhibitor, D4476, LY364947, SB525334, and SD208.

[0041] The concentration of the TGFβ receptor / ALK5 inhibitor used in producing the epithelium-derived cells of the present invention can be in the range of 0.001 ng / mL to 1000 ng / mL, preferably 0.01 ng / mL to 100 ng / mL, and more preferably 0.1 ng / mL to 10 ng / mL.

[0042] In producing the epithelial-derived cells of the present invention, other components for optimizing the cell culture medium, e.g., the cell culture medium for epithelial-derived cells, may be used, including, but not limited to, Glutamax®. TM Examples of suitable anti-inflammatory drugs include serum substitutes such as B27 supplement (Thermo Fisher Scientific) and N2 supplement (Thermo Fisher Scientific), and rock inhibitors such as Y-27632.

[0043] In an exemplary embodiment of the invention, a combination of a basal medium, the following additives, and other components to optimize the cell culture medium are used in the production of the epithelial-derived cells of the invention: Noggin, EGF, FGF-7, FGF-10, SAG.

[0044] In another exemplary embodiment of the invention, a combination of a basal medium, the following additives, and other components to optimize the cell culture medium are used in the production of epithelial-derived cells of the invention: Noggin, FGF-7, FGF-10, SAG.

[0045] In producing the epithelium-derived cells of the present invention, the epithelium-derived cells to be cultured can typically be prepared from hair follicles, and for example, the bulge region (e.g., the outermost layer cells of the outer root sheath of the bulge region), the hair matrix base, or hair follicle epithelium induced from iPS cells (induced pluripotent stem cells) or ES cells (embryonic stem cells) can be used.

[0046] Epithelial tissues that can be used in the present invention can be collected from a variety of animals, including mammalian primates (e.g., humans, monkeys, etc.), ungulates (e.g., pigs, cattle, horses, etc.), small mammalian rodents (e.g., mice, rats, rabbits, etc.), as well as dogs and cats. Epithelial tissues can be collected by applying the same conditions as those normally used for collecting tissues, and can be extracted under sterile conditions and stored in an appropriate preservative solution.

[0047] For example, epithelial tissue-derived cells from hair follicles can be prepared by first isolating the hair follicles from surrounding tissues and separating them into epithelial tissue and mesenchymal tissue according to their shape. Enzymes may be used to facilitate the separation. Examples of enzymes include known enzymes such as dispase, collagenase, and trypsin, and those skilled in the art can use any enzyme they like.

[0048] Furthermore, cells derived from tissues other than hair follicles may also be used as epithelial tissue-derived cells. Examples of cells derived from tissues other than hair follicles include, but are not limited to, epithelial cells derived from the skin or oral mucosa or gingiva, preferably immature epithelial-derived progenitor cells capable of differentiating into differentiated epithelial cells, such as keratinized or parakeratinized epithelial cells, such as skin or mucosa, such as non-keratinized epithelial cells or their stem cells. Specifically, an example of using oral epithelial-derived cells or primary cultured cells thereof as epithelial-derived cells is described in Japanese Patent Application Laid-Open No. 2008-29756, the disclosure of which is incorporated herein by reference in its entirety.

[0049] The origin of ES cells that can be used in the present invention is not particularly limited, and ES cells derived from the inner cell mass of an animal can be used. For example, ES cells derived from the inner cell mass of a human, mouse, rat, dog, cat, rabbit, cow, horse, sheep, goat, pig, or monkey can be used.

[0050] "iPS cells" generally refer to cells that have been given the pluripotency to differentiate into a large number of cells, like ES cells, and the ability to self-replicate, maintaining pluripotency even after division and proliferation, by introducing, for example, several types of genes and / or drugs into somatic cells. However, the present invention is not limited to the above explanation and broadly includes cells that those skilled in the art recognize as "iPS cells."

[0051] The origin of iPS cells that can be used in the present invention is not particularly limited, and iPS cells derived from animals can be used. For example, iPS cells derived from humans, mice, rats, dogs, cats, rabbits, cows, horses, sheep, goats, pigs, or monkeys can be used. The somatic cells from which iPS cells can be derived are also not particularly limited, and iPS cells induced from cells derived from any tissue can be used. Furthermore, the method for deriving iPS cells that can be used in the present invention is also not particularly limited, and iPS cells induced by any method can be used as long as they can be derived from somatic cells.

[0052] When epithelial tissue-derived cells collected from a living organism are used as epithelial tissue-derived cells, it is preferable to perform a single-cell disaggregation process before culturing. In the present invention, "single-cell disaggregation" refers to a process of separating multiple cells (typically from tissues or organs) that are bound or adhered to each other into individual cells. Such single-cell disaggregation can be performed using methods known to those skilled in the art, including, but not limited to, enzyme treatment. Enzymes that can be used in such enzymatic treatment and the conditions for their use are also known to those skilled in the art; for example, enzymes such as dispase, collagenase, and trypsin can be used. Disaggregation into single cells is preferable from the viewpoint of promoting efficient proliferation of the desired epithelial tissue-derived cells.

[0053] In one embodiment, the epithelial tissue-derived cells are cells derived from the epithelial tissue of the bulge region. Such cells can be prepared by collecting tissue from the bulge region epithelium (e.g., the outermost layer of the outer root sheath) and subjecting it to a single-cell treatment.

[0054] The epithelial tissue-derived cells can be cultured under conditions generally used for culturing animal cells, such as culturing for 5 to 8 days in an incubator at a temperature of about 37°C and a 5% CO2 concentration. Antibiotics such as streptomycin may also be added to the culture medium as appropriate.

[0055] For the culture, an extracellular matrix known to those skilled in the art may be used as appropriate, such as Matrigel (registered trademark) (Corning), Type IV collagen, atelocollagen, Type I collagen, or Type III collagen.

[0056] The cell population expanded as described above is an in vitro culture containing a certain proportion of cells expressing Itgb5, and if necessary, positive selection based on Itgb5 may be performed to separate, isolate, or enrich for cells expressing Itgb5. Negative and positive selection techniques based on surface markers are also known to those skilled in the art, and any antibody-based technique can be used, including, for example, fluorescence-activated cell sorting (FACS) and magnetic bead separation.

[0057] The epithelium-derived cells of the present invention can be typically used to produce regenerated hair follicle primordia used in hair follicle regenerative medicine. The hair follicle primordium is the tissue from which hair follicles originate and is composed of epithelial-derived cells and mesenchymal cells. The hair follicle primordium is formed during fetal development by the thickening of a portion of the epidermis and the aggregation of adjacent mesenchymal cells. In the present invention, the term "regenerated hair follicle primordium" refers to a hair follicle primordium that is artificially induced or regenerated from epithelial-derived cells and mesenchymal cells.

[0058] The epithelium-derived cells of the present invention can be typically used to produce regenerated hair follicle primordia used in hair follicle regenerative medicine. When the epithelium-derived cells of the present invention are used to produce regenerated hair follicle primordia, a cell population containing the epithelium-derived cells can be cultured in contact with a cell population derived from mesenchymal cells to produce a regenerated hair follicle primordium.

[0059] The "cell population containing epithelial-derived cells" is preferably a cell population containing cells that express detectable levels of Itgb5 at a rate of at least 2%, for example at least 10%, in order to enable the production of regenerated hair follicle primordia, preferably regenerated hair follicle primordia that have sustained hair growth potential (i.e., have the ability to grow hair for one or more hair cycles, preferably two, three, four or five or more hair cycles).

[0060] The culture conditions for contacting and culturing a cell population containing epithelial-derived cells with a cell population derived from mesenchymal cells can be the conditions used for culturing general animal cells, for example, for at least 12 hours, preferably at least 16 hours, more preferably at least 24 hours, and even more preferably at least 40 hours, and the medium and culture conditions may be changed during the culture.

[0061] The terms used in this specification, unless otherwise defined, are used to describe particular embodiments and are not intended to limit the invention.

[0062] Furthermore, the term "comprise" used in this specification intends that the described items (components, steps, elements, numbers, etc.) are present, unless the context clearly dictates otherwise, and does not exclude the presence of other items (components, steps, elements, numbers, etc.).

[0063] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs. Terms used herein should be interpreted as having a meaning consistent with the meaning in this specification and the related technical field, and should not be interpreted in an idealized or overly formal sense, unless otherwise defined.

[0064] The present invention will be described in more detail below with reference to examples. However, the present invention may be embodied in various forms and should not be construed as being limited to the examples set forth herein. [Example]

[0065] [Example 1] Analysis of the differentiation potential of Itgb5-positive cells by cell lineage tracing 1) Preparation of reagents 1-1) Preparation of dissection medium DMEM (Thermo Fisher Scientific) was mixed with FBS (Biowest, final concentration: 10%), HEPES (Thermo Fisher Scientific, final concentration: 10 mM), and penicillin-streptomycin (Thermo Fisher Scientific, final concentration: 1%).

[0066] 1-2) Preparation of HEPES HEPES (Dojindo Laboratories) was dissolved in Milli-Q water, and the pH was adjusted to 7.4 with NaOH (Fujifilm Wako Pure Chemical Industries) to prepare 1 M HEPES.

[0067] 1-3) Preparation of NaHCO3 1 M NaHCO3 was prepared by dissolving NaHCO3 (Fujifilm Wako Pure Chemical) in Milli-Q water.

[0068] 1-4) Preparation of DMEM 10x DMEM was prepared by dissolving 1 L of DMEM powder (Thermo Fisher Scientific) in 100 mL of Milli-Q water.

[0069] 1-5) Preparation of reconstitution buffer A reconstitution buffer solution was prepared by dissolving 2.2 g of NaHCO3 (Fujifilm Wako Pure Chemicals) and 4.77 g of HEPES (Fujifilm Wako Pure Chemicals) in 100 mL of 0.05 N NaOH solution (prepared by dissolving NaOH (Fujifilm Wako Pure Chemicals) in Milli-Q water).

[0070] 1-6) Preparation of 10x αMEM 10x αMEM was prepared by dissolving 1 L of αMEM (Sigma) in 100 mL of Milli-Q water.

[0071] 1-7) Preparation of gel for reconstitution Cellmatrix Type IA (Nitta Gelatin) and 10x αMEM (mentioned above) were mixed at a mixing ratio of 8:1, and then the same volume of reconstitution buffer solution (mentioned above) as that of 10x αMEM was added and mixed.

[0072] 1-8) 1x PBS 10x PBS was prepared by dissolving KCl (Fujifilm Wako Pure Chemical Industries, final concentration 27 mM), KH2PO4 (Fujifilm Wako Pure Chemical Industries, final concentration 15 mM), Na2HPO4 (Fujifilm Wako Pure Chemical Industries, final concentration 80 mM), and NaCl (Fujifilm Wako Pure Chemical Industries, final concentration 1.37 M) in Milli-Q water. 1x PBS was prepared by diluting 10x PBS 10 times with Milli-Q water.

[0073] 2) Seeding, culture, and recovery of mouse skin epithelial-derived cells Skin samples were collected from the backs of 7-8-week-old CAG-EGFP mice (SLC) and incubated with 0.25% trypsin (Thermo Fisher Scientific) diluted 1:10 in PBS(-) (Nacalai Tesque) at 37°C for 50 minutes. After incubation, the tissue was transferred to dissection medium, and the epidermis (epithelial layer) was separated. The cells were then detached by pipetting. The cell solution containing the detached cells was passed through a 70 μm cell strainer (Corning) and centrifuged at 250 x g for 5 minutes at 4°C in a high-speed microcentrifuge (Hitachi Kogyo). After centrifugation, the supernatant was removed, and mouse skin epithelial cells were collected by adding and mixing Advanced DMEM / F-12 (Thermo Fisher Scientific). The cell concentration of mouse skin epithelial cells was measured, and the cell suspension was then aliquoted into tubes. The tube was centrifuged at 310 x g for 3 minutes at 4°C, the supernatant was removed, and a 1% atelocollagen solution (Koken) containing 1 M HEPES (Dojindo Laboratories, final concentration: 10 mM), NaHCO3 (ibid., final concentration: 10 mM), and DMEM (ibid., final concentration: 1x) was added and mixed to obtain 1x10 5A cell suspension was prepared at a concentration of 100 cells / mL. After removing air bubbles by centrifugation, the cell suspension was seeded into a 6-well plate (Becton Dickinson) at 90 μL / well. After allowing the gel to solidify for approximately 30 minutes in a CO2 incubator (Panasonic) set at 37°C and 5% CO2, the medium was adjusted to Advanced DMEM / F-12 as the basal medium, supplemented with 10 mM HEPES (Thermo Fisher Scientific), 1x Glutamax (Thermo Fisher Scientific), 1x B27 supplement (Thermo Fisher Scientific), 1x N2 supplement (Thermo Fisher Scientific), 10 μM Rock inhibitor (Y-27632, Fujifilm Wako Pure Chemical Industries), 50 ng / mL EGF (Peprotech), 50 ng / mL FGF-7 (R&D), 50 ng / mL FGF-10 (R&D), 50 ng / mL SHH agonist (SAG, cayman), and 50 ng / mL BMP inhibitor (Noggin, Peprotech), and 1% Penicillin-Streptomycin (Thermo Fisher Scientific). Culture medium (NFFSE medium) containing ThermoFisher Scientific) was added at 3 mL / well, and 3D culture was performed for 6 days in a CO2 incubator (ThermoFisher Scientific) set at 37°C and 5% CO2.

[0074] After 6 days of culture, gels with cell colonies were detached from the culture dish using a cell scraper. The detached gels were collected in a 1.5 mL tube (maximum of 3 gels per tube) and 1 mL of culture supernatant was added. Collagenase I (Worthington) was added to a final concentration of 100 U / mL and incubated at 37°C for 60–90 minutes to dissolve the gel. After centrifugation at 590 × g and 4°C for 3 minutes, the supernatant was removed and the cells were washed once with 1 mL of PBS(-) (Nacalai Tesque). After washing, 500 μL of 0.125% trypsin (Thermo Fisher Scientific) diluted in PBS(-) was added and incubated at 37°C for 20 minutes. A cell suspension was prepared by adding 1 mL of dissection medium containing 35 U / mL DNase Type I (Sigma) and mixing. The cell suspension was passed through a 35 μm cell strainer (Corning) and centrifuged at 590 × g for 3 minutes at 4°C. After centrifugation, the supernatant was removed and the cultured mouse skin epithelium-derived cells were suspended in Advanced DMEM / F-12 medium containing 1% penicillin-streptomycin to recover the cells, and the cell concentration was measured.

[0075] 3) Isolation of mouse Itgb5-positive cells BD FACSAria flow cytometer TM We prepared and analyzed mouse Itgb5-positive cells by cell sorting the Itgb5-positive fraction (black square in the left frame of Figure 1) of cultured mouse skin epithelial cells that were CD34- and CD49f-positive using a Becton Dickinson III (Figure 5C, black box (3)). Note that the isolated mouse Itgb5-positive cells did not express CD86 at a level detectable by flow cytometry. The collected cultured mouse skin epithelial cells (cell number: 1 x 10 5 pcs or 2x10 7After washing once with PBS(-) containing 0.2 mM EDTA (Fujifilm Wako Pure Chemical Industries, Ltd.) and 0.05% BSA (BM Bio Japan), the cells were resuspended in PBS(-) containing 0.2 mM EDTA and 0.05% BSA and eFluor660-labeled CD34 monoclonal antibody (Invitrogen, 1:50 dilution), PE-labeled human / mouse CD49f monoclonal antibody (BioLegend, 1:50 dilution), and Itgb5 antibody (R&D, 1:50 dilution), and incubated at 4°C for 15 minutes. For CD86 expression analysis, FITC-labeled rat anti-mouse CD86 monoclonal antibody (Becton Dickinson, 1:50 dilution) was used. After the reaction, the cells were washed twice and resuspended in PBS(-) containing 0.2 mM EDTA and 0.05% BSA containing BV421-labeled donkey anti-sheep polyclonal antibody (Jackson ImmunoResearch Laboratories, 1:100 dilution), and incubated at 4°C for 15 minutes. After the reaction, the cells were washed twice and resuspended in PBS(-) containing 0.2 mM EDTA and 0.05% BSA at 1x10 7 Resuspended at 500 μL. BD FACSAria TM III, and mouse Itgb5-positive cells were separated from CD34- and CD49f-positive cells. The results are shown in Figure 1. We confirmed that mouse Itgb5-positive cells were isolated from cultured mouse skin epithelium-derived cells (Figure 1).

[0076] 4) Preparation of regenerated hair follicle primordia To examine the differentiation potential of mouse Itgb5-positive cells in hair follicles, we prepared regenerated hair follicle primordia using mouse Itgb5-positive cells according to the organ primordium method (Japanese Patent No. 5932671). First, we prepared skin mesenchymal cells from mouse fetuses using conventional methods. Specifically, skin containing hair primordia was collected from 18.0-18.5-day-old C57BL / 6 mouse fetuses (SLC) and placed in a 35-mm dish (Becton Dickinson). Following a modified method described by Nakao et al. (Nakao K et al., Nat. Methods, 4(3), 227-30, 2007), the cells were incubated with 2 mL of 10 caseinolytic U / mL Dispase (Becton Dickinson) diluted in PBS(-) at 4°C for 1 hour. After incubation, the cells were washed twice with dissection medium, and 2 mL of dissection medium containing 70 U / mL DNase Type I was added. The epidermal and dermal layers were separated using a 25G syringe (Terumo) and placed separately in a 35-mm dish. The epithelial layer was washed twice with PBS(-) and incubated with 1 mL of Acutase (Thermo Fisher Scientific) at room temperature for 45 minutes. After incubation, 2 mL of dissection medium containing 70 U / mL DNase Type I was added and mixed. After mixing, the mixture was passed through a 35 μm cell strainer and centrifuged at 590 x g for 3 minutes at 4°C. After centrifugation, the supernatant was removed, and dissection medium was added and mixed to collect mouse fetal skin epithelial cells. Meanwhile, the dermal layer was transferred to a 50 mL tube (Becton Dickinson) and incubated with 910 U / mL Collagenase I diluted in dissection medium at 37°C for 1 hour with shaking at 55 rpm. After incubation, 10 mL of dissection medium was added, passed through a 10 μm cell strainer (Sysmex), and centrifuged at 590 x g for 3 minutes at 4°C. After centrifugation, the supernatant was removed, and mouse fetal skin mesenchymal cells were collected by adding and mixing with dissection medium containing 70 U / mL DNase Type I. After measuring the cell count of both collected cells, mouse Itgb5-positive cells were mixed with the collected mouse fetal skin epithelial-derived cells at a ratio of 1:9 to prepare mouse mixed epithelial-derived cells.Mouse mixed epithelial-derived cells and mouse fetal skin mesenchymal cells were separately transferred to 1.5 mL microtubes (Eppendorf) coated with silicone grease (Toray Dow Corning) and centrifuged (600 x g, 4°C, 3 minutes) to collect the pellet. The supernatant was then removed using a 0.5-20 μL GELoader Tip (Eppendorf). The centrifugation and supernatant removal procedures were repeated until as much of the supernatant as possible was removed. Next, 30 μL of the reconstitution gel was dropped onto a silicone grease-coated Petri dish (Becton Dickinson) to create a collagen gel drop. Approximately 0.2 μL of the mouse fetal skin mesenchymal cells prepared above were then injected using a 0.1-10 μL pipette tip (Quality Scientific Plastics) to form cell aggregates (cell count: 1 x 10). 4 Next, approximately 0.2 μL of the mouse mixed epithelium-derived cells prepared above was injected into the gel drop using a 0.1-10 μL pipette tip (Quality Scientific Plastics) so that the cells adhered to the aggregate of mouse fetal skin mesenchymal cells, creating a cell aggregate (cell number: 1 × 10 4 Furthermore, a 5 mm nylon thread (Matsuda Medical Industry) was inserted perpendicularly through the contact surface between the mouse fetal skin mesenchymal cell fraction and the mouse mixed epithelial cell fraction from the mouse mixed epithelial cell side of the cell aggregate of mouse fetal skin mesenchymal cells and mouse mixed epithelial cells, without disrupting the structure of the cell aggregate (particularly the contact surface between the mouse mixed epithelial cells and mouse fetal skin mesenchymal cells), after checking under a stereomicroscope (Carl Zeiss). The reconstitution gel was then transferred onto a 0.4 μm pore size Cell Culture Insert (Corning) set in a 6-well plate (Corning) containing 1 mL of DMEM containing 10% FBS and 1% penicillin-streptomycin, and organ culture was performed for 16 to 40 hours in a CO2 incubator set at 37°C and 5% CO2 concentration to produce regenerated hair follicle primordia.

[0077] 5) Intradermal transplantation of regenerated hair follicle primordia into nude mice Regenerated hair follicle primordia were transplanted intracutaneously into mice using conventional methods. Five- to eight-week-old Balb / c nu / nu mice (SLC) were anesthetized according to standard procedures, their backs disinfected with isodine, and then placed in a natural recumbent position. A V-lance microscalpel (Alcon Japan) was used to create a graft wound extending from the epidermis to the lower dermis. The graft wound was approximately 400 μm deep vertically from the body surface and approximately 1 mm horizontally. The regenerated hair follicle primordia, with a nylon thread guide inserted, was inserted using sharp No. 5 forceps (Natsume Seisakusho), with the epithelial-derived cellular components facing the body surface of the graft wound. The graft depth was adjusted so that the upper end of the epithelial-derived cellular components of the regenerated hair follicle primordium were exposed at the upper edge of the graft wound, and the nylon thread guide was positioned so that it was exposed to the body surface. The nylon thread guide was fixed to the skin surface adjacent to the graft wound with Steri-Strips (3M), and the graft wound was then protected with a nurse band (Sun Planet) and surgical tape (3M). The protective tape was removed 5 to 7 days after grafting.

[0078] 6)Histological analysis Three weeks to three months after transplantation, skin tissue containing the grafts was harvested and fixed overnight at 4°C in 4% PFA (Fujifilm Wako Pure Chemical Industries, Ltd.). It was then immersed overnight in 12.5% ​​sucrose (Fujifilm Wako Pure Chemical Industries, Ltd.) in 1x PBS at 4°C. After rinsing with 1x PBS, the tissue was immersed overnight in 25% sucrose in 1x PBS at 4°C and embedded in OCT compound (Sakura Phytec Japan). The embedded tissue was sliced ​​at 50 μm thickness using a Clariostat (Leica). The slices were transferred to a 24-well plate (Becton Dickinson) and washed twice with 500 μL of washing buffer (0.1% Triton X-100 prepared by diluting Triton X-100 (Alfa Aesar) with 1x PBS) at room temperature for 10 minutes. After washing, the cells were resuspended in washing buffer containing Itgb5 antibody (R&D, 1:50 dilution) and shaken overnight at 4°C. The supernatant was removed, and the cells were washed by percolation in 500 μL of washing buffer at room temperature for 1 hour (three washes). After washing, the cells were resuspended in washing buffer containing Alexa Fluor 594-conjugated donkey anti-sheep polyclonal antibody (Thermo Fisher Scientific, 1:100 dilution) and shaken overnight at 4°C. The supernatant was removed, and the cells were washed by percolation in 500 μL of washing buffer at room temperature for 1 hour (three washes). The tissue was transferred to a glass slide and mounted with a coverslip using mounting medium containing antifade. The differentiation potential of mouse Itgb5 cells in hair follicles was analyzed using a confocal laser scanning microscope (LSM-780, Carl Zeiss) based on the localization of EGFP (enhanced green fluorescent protein) fluorescent staining in mouse Itgb5 cells. The results are shown in Figure 2. Mouse Itgb5-positive cells (EGFP-positive cells) were localized in the sebaceous glands (white arrowheads), stem cell niches (white arrowheads), hair follicle variable regions (white arrowheads), and hair bulbs (white arrowheads) that make up hair follicles (Figure 2), suggesting that Itgb5-positive cells have the ability to differentiate into a variety of tissues and regions.

[0079] [Example 2] Functional analysis of Itgb5-positive cells for sustained hair growth 1) Preparation of reagents, 2) experimental animals, and 3) seeding, culture, and collection of mouse skin epithelium-derived cells were carried out by the methods described above.

[0080] 4) Separation of mouse Itgb5-negative / positive cells and mouse Itgb5-negative cells Mouse Itgb5-negative / positive cells were prepared by separating the Itgb5-negative and Itgb5-positive fractions (large black squares in the left frame of Figure 3B) of cultured mouse skin epithelial-derived cells that were positive for CD34 and CD49f using a flow cytometer (Figure 3B, center frame). Mouse Itgb5-negative / positive cells were also prepared by separating (cell sorting) the Itgb5-negative fraction (small black squares in the left frame of Figure 3B) (Figure 3B, right frame). The collected cultured mouse skin epithelial-derived cells (cell number: 5 x 10 7 The cells were incubated with three types of antibodies as described above. The resuspended cells were then analyzed using a BD FACSAria TM III, and Itgb5-negative cells (cell number: 1x10 6 ) and mouse Itgb5 negative / positive cells (cell number: 1x10 6 (pieces) were separated. The results are shown in Figure 3. We confirmed that the cultured mouse skin epithelium-derived cells were positive for CD34 and CD49f (Figure 3A). We also confirmed that Itgb5-negative cells and mouse Itgb5-negative / positive cells were isolated (Figure 3B).

[0081] 5) Evaluation of organogenesis in mouse Itgb5-negative / positive cells To evaluate the organ induction ability of mouse Itgb5-negative / positive cells, we prepared regenerated hair follicle primordia using the organ primordium method. We then performed a functional analysis of Itgb5-positive cells on the sustainability of hair growth using the ability to grow hair after transplantation into animals. Mouse Itgb5-negative cells were used as a control. Regenerated hair follicle primordia were prepared according to the organ primordium method (Japanese Patent No. 5932671). Mouse fetal skin mesenchymal cells were collected using the method described above, and the number of collected cells was measured. Mouse Itgb5-negative / positive cells, mouse Itgb5-negative cells, and mouse fetal skin mesenchymal cells were separately transferred to 1.5 mL microtubes (Eppendorf) coated with silicone grease and collected as pellets by centrifugation (600 x g, 4°C, 3 minutes). The supernatant of the culture medium after centrifugation was removed using a GELoader Tip 0.5-20 μL (Eppendorf). The centrifugation and supernatant removal procedures were repeated until as much of the supernatant as possible was removed. Next, 30 μL of the reconstitution gel was dropped onto a Petri dish coated with silicone grease to create a collagen gel drop, and approximately 0.2 μL of the mouse fetal skin mesenchymal cells prepared above were injected using a 0.1-10 μL pipette tip (Quality Scientific Plastics) to create a cell aggregate (cell number: 1 x 10 4 Next, approximately 0.2 μL of the mouse Itgb5-negative / positive cells or mouse Itgb5-negative cells prepared above was injected into the gel drop using a 0.1-10 μL pipette tip (Quality Scientific Plastics) so that the cells adhered to the aggregate of mouse fetal skin mesenchymal cells, producing a cell aggregate (number of cells: 1 × 10 4Furthermore, a nylon thread with a total length of 5 mm (Matsuda Medical Industries) was inserted from the mouse Itgb5-negative / positive cell or mouse Itgb5-negative cell side of the cell aggregate of mouse fetal skin mesenchymal cells and mouse Itgb5-negative / positive cells or mouse Itgb5-negative cells, after checking under a stereomicroscope that it penetrated the contact surface between the cell fraction of mouse fetal skin mesenchymal cells and mouse Itgb5-negative / positive cells or mouse Itgb5-negative cell fraction without destroying the structure of the cell aggregate (especially the contact surface between the mouse Itgb5-negative / positive cells or mouse Itgb5-negative cells and mouse fetal skin mesenchymal cells), and the thread was placed in a 0.4 μm pore size Cell Culture Microplate (Becton Dickinson) containing 1 mL of DMEM 10% FBS. The collagen gel was transferred onto inserts (Becton Dickinson) and organ culture was carried out for 16 to 40 hours in a CO2 incubator set at 37°C and 5% CO2 concentration to produce regenerated hair follicle primordia. The regenerated hair follicle primordia were transplanted using the method described above. The survival of the transplants was assessed using a SteREO Lumar.V12 (Carl Zeiss) fluorescent stereomicroscope, and then follow-up observations were conducted (once every 3 or 4 days). For follow-up observations, the transplant sites of anesthetized mice were observed and photographed using a SteREO Lumar.V12 (Carl Zeiss) fluorescent stereomicroscope, and the persistence of hair growth was evaluated using the hair growth ability of the regenerated hair follicles as an index. The results are shown in Figure 4. Regenerated hair follicle primordia containing Itgb5-negative / positive cells showed a higher hair growth rate than regenerated hair follicle primordia containing Itgb5-negative cells, and the proportion of hair cycles exceeding three was higher in regenerated hair follicle primordia containing Itgb5-negative / positive cells (Figure 4). This suggests that the inclusion of Itgb5-positive cells in epithelial-derived cells sustains a high level of hair growth potential.

Claims

1. A method for producing a regenerated hair follicle primordium, A manufacturing method comprising the steps of: confirming that epithelial-derived cells express integrin β5 (Itgb5); obtaining an epithelial-derived cell population containing epithelial-derived cells confirmed to express Itgb5 in the above step; and obtaining a regenerated hair follicle primordium by culturing the epithelial-derived cell population in contact with a cell population derived from mesenchymal cells.

2. Epithelial-derived cells further express detectable levels of CD34 and / or CD49f; characterized in that The method for producing the regenerated hair follicle primordium according to claim 1.

3. Epithelial-derived cells The expression of PDGFRα (Platelet derived growth factor receptor-α) and CD86 is not detectable by flow cytometry. The method for producing the regenerated hair follicle primordium according to claim 1.

4. an epithelial-derived cell population containing epithelial-derived cells, In vitro cultures, A method for producing a regenerated hair follicle primordium according to any one of claims 1 to 3.

5. an epithelial-derived cell population containing epithelial-derived cells, At least 2% of the cells express Itgb5; Characterized by A method for producing a regenerated hair follicle primordium according to any one of claims 1 to 3.

6. an epithelial-derived cell population containing epithelial-derived cells, At least 10% of the cells express Itgb5; Characterized by A method for producing a regenerated hair follicle primordium according to any one of claims 1 to 3.

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