Method for producing hair follicle organoid, human hair follicle organoid, and method for enhancing hair shaft-forming potential of hair follicle organoid

Abstract

To provide a novel technique for producing hair follicle organoids, effectively applicable to human cells.SOLUTION: A method for producing hair follicle organoids comprises co-culturing of epithelial cells, mesenchymal cells, and pluripotent stem cells.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a method for producing hair follicle organoids, human hair follicle organoids, and a method for improving the hair shaft formation ability of hair follicle organoids. [Background technology]

[0002] In recent years, hair follicle regeneration technology has been developed as a regenerative medical technology for diseases such as alopecia. For example, hair follicles and hair have been regenerated using epithelial-mesenchymal cell interactions. Patent Document 1 describes a method of transplanting a hair follicle primordium obtained by mixing and culturing mouse epithelial stem cells and hair papilla cells into an animal, and growing hair from the hair follicle primordium. Patent Documents 2 and 3 also describe a method of co-culturing epithelial cells and mesenchymal cells obtained from a mouse to produce a hair follicle primordium.

[0003] Furthermore, Non-Patent Document 1 describes that when epithelial cells collected from mouse fetal skin and mesenchymal cells collected from mouse fetal skin were suspended and cultured in a medium containing a low concentration of Matrigel, the spatial arrangement pattern of the cell aggregates changed and hair germ formation was promoted, and that when the cells were embedded in Matrigel and cultured, the length of the hair shaft reached approximately 3 mm.

[0004] When human cells are used, it has been disclosed that hair follicle-like structures without a hair shaft were successfully formed using fetal epithelial cells and fetal mesenchymal cells, fetal epithelial cells and adult mesenchymal cells, adult epithelial cells and fetal mesenchymal cells, or adult epithelial cells and adult mesenchymal cells (Non-Patent Document 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 7158676 [Patent Document 2] WO2020 / 225934 publication [Patent Document 3] JP 2023-56591 A [Non-patent literature]

[0006] [Non-Patent Document 1] Kageyama et al.,Sci Adv,8,eadd4603,2022 [Non-Patent Document 2] Kageyama et al.,Sci Rep,13(1),4847,2023 Summary of the Invention [Problem to be solved by the invention]

[0007] In view of the above-mentioned prior art, an objective of the present invention is to provide a novel technique for producing hair follicle organoids that can be suitably applied to human cells. [Means for solving the problem]

[0008] As a result of extensive research, the inventors have succeeded in producing hair follicle tissue complete with a hair bulb and hair shaft from human adult-derived cells by co-culturing epithelial cells and mesenchymal cells in the presence of pluripotent stem cells, thereby completing the present invention.

[0009] The present invention and its preferred embodiments for solving the above problems are as follows. [1] A method for producing hair follicle organoids, comprising co-culturing epithelial cells, mesenchymal cells, and pluripotent stem cells. By adding pluripotent stem cells together with epithelial cells and mesenchymal cells and culturing them, it is possible to produce hair follicle organoids, which are hair follicle tissues, in a culture vessel without the need for transplantation into the body.

[0010] [2] The method for producing a hair follicle organoid described in [1], wherein the hair follicle organoid has a hair bulb and a hair shaft. In a preferred embodiment, pluripotent stem cells are added and cultured together with epithelial cells and mesenchymal cells, thereby enabling the production of hair follicle organoids having a hair bulb and a hair shaft in a culture vessel without the need for transplantation in vivo.

[0011] [3] The method for producing a hair follicle organoid described in [2], wherein the hair shaft contains pigment. In a preferred embodiment, pigment-accumulating, highly differentiated hair follicle organoids can be produced.

[0012] [4] The method for producing a hair follicle organoid according to any one of [1] to [3], wherein the hair follicle organoid is a human hair follicle organoid produced from cells derived from a human. According to the present invention, human hair follicle organoids can be efficiently produced.

[0013] [5] A method for producing hair follicle organoids described in any one of [1] to [4], wherein the number of epithelial cells mixed at the start of co-culture is 0.1 to 3 times the number of mesenchymal cells. By making the organoids in such a form, it is possible to promote the formation of hair follicles and hair shafts in the organoids.

[0014] [6] A method for producing hair follicle organoids described in any one of [1] to [5], wherein the number of pluripotent stem cells mixed at the start of co-culture is 0.3 to 3 times the number of epithelial cells. By making the organoids in such a form, it is possible to promote the formation of hair follicles and hair shafts in the organoids.

[0015] [7] A method for producing hair follicle organoids described in any one of [1] to [6], wherein the number of pluripotent stem cells mixed at the start of co-culture is 0.3 to 3 times the number of mesenchymal cells. By making the organoids in such a form, it is possible to promote the formation of hair follicles and hair shafts in the organoids.

[0016] [8] A hair follicle organoid produced by the method for producing a hair follicle organoid described in any one of [1] to [7].

[0017] [9] Human hair follicle organoids contain epithelial and mesenchymal cells and include a hair bulb and hair shaft.

[0018]

[10] The human hair follicle organoid described in [9], wherein the hair shaft contains pigment.

[0019]

[11] A method for producing hair follicle organoids by co-culturing epithelial cells and mesenchymal cells, further comprising co-culturing in the presence of pluripotent stem cells, thereby improving the hair shaft formation ability of the hair follicle organoids. According to the present invention described above, it is possible to promote hair shaft formation from a cell aggregate composed of pluripotent stem cells, epithelial cells, and mesenchymal cells. Effect of the Invention

[0020] According to the present invention, a novel technique for efficiently producing hair follicle organoids can be provided.

[0021] In addition, according to the present invention, hair follicle tissue having a hair shaft structure can be produced in vitro using human cells. That is, according to the present invention, human hair follicle organoids having a hair shaft structure can be efficiently produced.

[0022] Furthermore, according to the present invention, hair follicle organoids having a hair shaft structure can be produced using epithelial cells and mesenchymal cells derived from adults. [Brief description of the drawings]

[0023] [Figure 1] This is a bright-field photograph showing mesenchymal cells proliferating near the hair bulb of a plucked hair. The scale bar is 300 μm. [Diagram 2] Brightfield photograph showing keratinocytes proliferating from the outer root sheath of a plucked hair, scale bar 300 μm. [Diagram 3] Bright-field photographs of hair follicle organoids on day 14 of co-culture (scale bar: 300 μm). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present invention relates to a method for producing hair follicle organoids, comprising co-culturing epithelial cells, mesenchymal cells and pluripotent stem cells. Note that the present invention is not limited to the embodiments described below, and can be appropriately modified within the scope of the invention.

[0025] The origin of the epithelial cells, mesenchymal cells, and pluripotent stem cells used in the present invention is not particularly limited, and for example, cells of mammals such as humans, monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cows, sheep, goats, pigs, etc. can be used in the present invention. In particular, human cells can be used in the present invention.

[0026] The epithelial cells, mesenchymal cells, and pluripotent stem cells used may be those that have been primarily obtained or established, or those that are commercially available.

[0027] The epithelial cells used in the present invention are not particularly limited as long as they are cells derived from epithelial tissue, and include epithelial stem cells. The epithelial cells used can be selected from epithelial hair follicle-derived epithelial cells such as epithelial root sheath (outer root sheath, inner root sheath), epithelial hair follicle stem cells (HFSC) present in the bulge region, hair follicle keratinocytes, and epithelial cells derived from the base of the hair matrix, epithelial cells derived from skin tissue such as keratinocytes, and epithelial cells induced from stem cells or other somatic cells. Furthermore, the epithelial cells used in the present invention may be derived from either a fetus (embryonic) or an adult. From the viewpoint of ease of obtaining the epithelial cells used for production, however, epithelial cells derived from an adult can be preferably selected.

[0028] The mesenchymal cells used in the present invention are not particularly limited as long as they are cells derived from mesenchyme, and include mesenchymal stem cells. The mesenchymal cells used can be selected from hair follicle-derived mesenchymal cells such as hair papilla cells and dermal sheath cells (including hair bulb sheath cells), skin-derived mesenchymal cells, and mesenchymal cells induced from stem cells or other somatic cells. Furthermore, the mesenchymal cells used in the present invention may be derived from either a fetus (embryonic) or an adult. From the viewpoint of ease of obtaining the mesenchymal cells used for production, however, mesenchymal cells derived from an adult can be preferably selected.

[0029] The pluripotent stem cells used in the present invention may be induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells), with the use of induced pluripotent stem cells (iPS cells) being preferred.

[0030] iPS cells are artificial stem cells derived from somatic cells that have almost the same characteristics as ES cells, such as pluripotency and the ability to proliferate through self-renewal, and can be produced by introducing a certain nuclear reprogramming factor into a somatic cell in the form of a nucleic acid or protein encoding the factor, or by increasing the expression of the endogenous mRNA and / or protein of the factor using a drug (K. Takahashi and S. Yamanaka (2006), Cell, 126:663-676; K. Takahashi et al. (2007), Cell, 131:861-872; J. Yu et al. (2007), Science, 318:1917-1920; M. Nakagawa et al. (2008), Nat. Biotechnol., 26:101-106).

[0031] The nuclear reprogramming factor may be a gene specifically expressed in ES cells, or a gene or its gene product that plays an important role in maintaining the undifferentiated state of ES cells, and is not particularly limited, but examples thereof include Oct3 / 4, Klf4, Klf1, Klf2, Klf5, Sox2, Sox1, Sox3, Sox15, Sox17, Sox18, c-Myc, L-Myc, N-Myc, TERT, SV40 large T antigen, HPV16 E6, HPV16 E7, Bmil, Lin28, Lin28b, Nanog, Esrrb, and Esrrg. These nuclear reprogramming factors may be used in combination when establishing iPS cells. For example, the combination includes at least one of the above nuclear reprogramming factors, preferably two or more, and more preferably three or more.

[0032] The culture for reprogramming of pluripotent stem cells is preferably performed by adherent culture. Pluripotent stem cells may be cultured in an adherent manner on a culture surface on which feeder cells such as MEF cells are laid, but it is preferable to perform the adherent culture under feeder-free conditions.

[0033] When pluripotent stem cells are cultured in an adherent manner under feeder-free conditions, it is preferable to coat the culture surface with various culture substrates. Examples of the culture substrates include extracellular matrices such as laminin, collagen, fibronectin, vitronectin, matrigel, fibrin, and thrombin; amino acid polymers such as poly-L-lysine and poly-D-lysine, and fragments thereof, and one or more selected from these may be used. Among the culture substrates, laminin and its fragments are preferred.

[0034] As laminin and its fragments, it is preferable to use laminin 511 (laminin consisting of α5 chain, β1 chain, and γ1 chain) and its fragments. Laminin is a major cell adhesion molecule in the basement membrane, and is a heterotrimer consisting of three subunit chains, α chain, β chain, and γ chain, and is a huge glycoprotein with a molecular weight of about 800,000 Da. It is a heterotrimeric molecule in which the three subunit chains associate at the C-terminus side to form a coiled-coil structure and are stabilized by disulfide bonds. Thus, laminin 511 means laminin whose α chain is α5, whose β chain is β1, and whose γ chain is γ1.

[0035] The laminin may be a mutant, and is not particularly limited as long as it has integrin binding activity. Laminin derived from human is preferable. Laminin and its fragments are preferable to have a binding activity with integrin α6β1 with a dissociation constant of 10 nM or less. It is preferable to use a commercially available product as laminin or laminin fragment.

[0036] Examples of laminin fragments include the E8 fragment (also called laminin 511E8 fragment or laminin 511E8) obtained by digesting laminin 511 with elastase (Ido H, et al., J Biol Chem. 2007, 282, 11144-11154), and recombinant human laminin 511E8 fragment expressed from genetically modified silkworm cocoons. Among laminin and fragments thereof, laminin fragments are preferred, more preferably laminin 511 fragments, even more preferably laminin 511E8 fragments, and even more preferably those derived from humans.

[0037] In carrying out the present invention, an established human iPS cell line may be used as a cell line. For example, a human iPS cell line selected from ChiPSC7, ChiPSC11, ChiPSC12, ChiPSC19, ChiPSC20, ChiPSC21, ChiPSC22, ChiPSC23, 201B7, 201B7-Ff, 253G1, 253G4, 1201C1, 1205D1, 1210B2, and 836B3 may be cultured by the method of the present invention. The above-mentioned human iPS cell lines are available from Cellartis, iPS Academia Japan, Inc., or the Kyoto University iPS Research Institute.

[0038] The iPS cells are preferably derived from cells of adult origin, and fibroblasts and cells present in hair follicles can be preferably used as the cells of adult origin.

[0039] In one embodiment of the production method of the present invention, cells derived from hair follicles can be preferably used, for example, epithelial cells and mesenchymal cells obtained by culturing plucked hairs. Also, iPS cells obtained by initializing cells obtained by culturing plucked hairs can be used. The hair-derived cells used to produce the iPS cells can be epithelial cells obtained by culturing epithelial tissue attached to the plucked hair (epithelial tissue of the hair follicle attached to the area of ​​the hair shaft away from the hair bulb). The subject from which hair is to be obtained is preferably an adult human.

[0040] Next, the method for culturing cells in the production method of the present invention will be described in detail. The production method of the present invention includes a co-culture step of co-culturing epithelial cells, mesenchymal cells, and pluripotent stem cells, and, optionally, a pre-culture step of pre-culturing each of the epithelial cells, mesenchymal cells, and pluripotent stem cells before the co-culture step.

[0041] The cell culture vessel for cells used in the present invention is not particularly limited, and examples thereof include flasks, dishes, petri dishes, bottles, plates, etc. The material of the cell culture vessel is not particularly limited, and is preferably one or more selected from synthetic resins (preferably plastics) such as styrene resins (polystyrene or styrene copolymers, etc.), polycarbonate, polyolefin resins (polyethylene, polypropylene, polyester, ethylene copolymers, etc.), (meth)acrylic resins, silicone resins, amino resins, fluorine resins, and polyimide resins, and glass substrates.

[0042] <Preculture process> In the pre-culture, the epithelial cells, mesenchymal cells, and pluripotent stem cells are each cultured in an appropriate culture medium. The culture conditions for each cell are not particularly limited as long as the characteristics of each cell can be maintained.

[0043] In the pre-culture of epithelial cells, it is preferable to culture the cells in a medium for epithelial cells. The medium for epithelial cells is not particularly limited, and examples thereof include EpiLife TM Examples of suitable medium include keratinocyte growth medium (Gibco), keratinocyte growth medium (Keratinocyte Growth Medium or HuMedia-KG2), minimum essential medium (MEM) containing about 5-20% fetal bovine serum, Dulbecco's modified Eagle medium (DMEM), RPMI1640 medium, 199 medium, F12 medium, etc. Also, suitable components for culturing epithelial cells (calcium, growth factors, antibiotics such as penicillin-streptomycin, etc.) may be added as appropriate.

[0044] In the preculture of epithelial cells, it is preferable to culture the epithelial cells in an adhesion culture medium containing laminin and its fragments. The preferred forms of laminin and its fragments to be used are as described above. This makes it possible to promote the formation of hair follicles during the co-culture process.

[0045] In the pre-culture of epithelial cells, the amount of laminin or a fragment thereof relative to the adhesion area of ​​the cell culture vessel is preferably 0.05 to 1.0 μg / cm 2 , more preferably 0.15 to 0.5 μg / cm 2 , more preferably 0.1 to 0.4 μg / cm 2 , and more preferably 0.2 to 0.3 μg / cm 2 For example, 0.25 μg / cm 2 It is added to the culture medium so that

[0046] In pre-culture of mesenchymal cells, it is preferable to culture the cells in a medium for stem cells. The medium for stem cells is not particularly limited, and may be DMEM, DMEM / F12, Essential 8, or the like. TM Medium (Thermo Fisher Scientific), Essential8 TM Suitable examples of such medium include Flex Medium (Thermo Fisher Scientific), AmnioMAX™ C-100 (Gibco), StemPro MSC SFM (Life Technologies), StemFit For MSC (Ajinomoto), etc. In addition, the culture medium may contain, as desired, additives such as antibiotics such as Normocin, serum, growth factors, hormones, etc.

[0047] In the preculture of mesenchymal cells, it is preferable to culture the mesenchymal cells in an adhesion culture medium containing laminin and its fragments. The preferred forms of laminin and its fragments to be used are as described above. This makes it possible to promote the formation of hair follicles in the co-culture step.

[0048] In the pre-culture of mesenchymal cells, the amount of laminin or a fragment thereof relative to the adhesion area of ​​the cell culture vessel is preferably 0.05 to 1.0 μg / cm 2 , more preferably 0.15 to 0.5 μg / cm 2 , more preferably 0.1 to 0.4 μg / cm 2 , and more preferably 0.2 to 0.3 μg / cm 2 For example, 0.25 μg / cm 2 It is added to the culture medium so that

[0049] The conditions for pre-culturing pluripotent stem cells are not particularly limited as long as they can be cultured while maintaining their differentiation potential. The medium used for pre-culturing pluripotent stem cells is not particularly limited, but may be StemFit. TM AK02N (Ajinomoto), StemSure TM hPSC (Fujifilm Wako Pure Chemical), mTeSR TM 1 (Stemcell Technologies), TeSR TM -E6 (Stemcell Technologies), TeSR TM -E8 (Stemcell Technologies), StemFlex TM (Thermo Fisher Scientific), Essential6 TM Medium (Thermo Fisher Scientific), Essential8 TM Medium (Thermo Fisher Scientific), Essential8 TM It is preferable to use Flex Medium (Thermo Fisher Scientific), which can be supplemented with antibiotics as needed. In addition, homemade media suitable for pluripotent stem cell culture can be used by adding necessary growth factors such as FGF, the antibiotics mentioned above, and various proteins such as HSA and BSA to common cell culture basal media such as αMEM and DMEM.

[0050] In pre-culture of pluripotent stem cells, it is preferable to culture the cells in an adhesion manner in a cell culture vessel coated with laminin or a fragment thereof. The preferred forms of laminin and its fragments to be used are as described above. This makes it possible to promote the formation of hair follicles in the co-culture step.

[0051] In pre-culture of pluripotent stem cells, a Rho-kinase (ROCK) inhibitor may be added. As ROCK inhibitors, (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine dihydrochloride (informal name: H-1152), 1-(5-isoquinolinesulfonyl)piperazine hydrochloride (informal name: HA-100), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (informal name: H-7), 1-(5-isoquinolinesulfonyl)-3-methylpiperazine (informal name: IsoH-7), N-2-(methylamino)ethyl-5-isoquinolinesulfonamide dihydrochloride (informal name: H-8), N-(2-aminoethyl)-5-isoquinolinesulfonamide dihydrochloride (informal name: H-9), N-[2-(p-bromo-cinnaphthalene) Examples of such compounds include, but are not limited to, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide dihydrochloride (informal name: H-89), N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (informal name: H-1004), 1-(5-isoquinolinesulfonyl)homopiperazine dihydrochloride (informal name: H-1077), (S)-(+)-2-methyl-4-glycyl-1-(4-methylisoquinolinyl-5-sulfonyl)homopiperazine dihydrochloride (informal name: Glycyl H-1152), and (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride (informal name: Y-27632). In the present invention, it is preferable to use Y-27632.

[0052] The concentration of the ROCK inhibitor in the medium is preferably 1 to 20 μM, more preferably 3 to 15 μM, and further preferably 5 to 12 μM, and can be, for example, 10 μM.

[0053] <Co-culture process> In the co-culture step, first, epithelial cells, mesenchymal cells, and pluripotent stem cells are seeded in a cell culture vessel to which a culture medium has been added.

[0054] The ratio of the number of epithelial cells to the number of mesenchymal cells (1x) at the start of co-culture is preferably 0.1x or more, more preferably 0.2x or more, more preferably 0.3x or more, more preferably 0.4x or more, even more preferably 0.5x or more, and particularly preferably 0.8x or more.

[0055] The ratio of the number of epithelial cells to the number of mesenchymal cells (1x) at the start of co-culture is preferably 3.0x or less, more preferably 2.5x or less, even more preferably 2.0x or less, even more preferably 1.5x or less, and particularly preferably 1.3x or less.

[0056] For example, the number of epithelial cells relative to the number of mesenchymal cells (1x) at the start of co-culture can be preferably 0.1 to 3x, more preferably 0.3 to 2x, even more preferably 0.5 to 1.5x, and particularly preferably 0.8 to 1.3x. A specific example is that the number of epithelial cells is 1:1 relative to the number of mesenchymal cells (1:1) at the start of co-culture, i.e., the mixing ratio of mesenchymal cells to epithelial cells is 1:1 based on the number of cells.

[0057] The number of pluripotent stem cells relative to the number of epithelial cells (1x) at the start of co-culture is preferably 0.3x or more, more preferably 0.5x or more, more preferably 0.8x or more, more preferably 1.0x or more, more preferably 1.3x or more, even more preferably 1.5x or more, and particularly preferably 1.8x or more.

[0058] The number of pluripotent stem cells relative to the number of epithelial cells (1x) at the start of co-culture is preferably 3.0x or less, more preferably 2.8x or less, more preferably 2.5x or less, even more preferably 2.3x or less, and particularly preferably 2.0x or less.

[0059] For example, the number of pluripotent stem cells relative to the number of epithelial cells (1x) at the start of co-culture can be preferably 0.3 to 3.0 times, more preferably 0.5 to 3.0 times, more preferably 0.8 to 2.8 times, more preferably 1.0 to 2.5 times, more preferably 1.3 to 2.5 times, even more preferably 1.5 to 2.5 times, and particularly preferably 1.8 to 2.3 times. Furthermore, the number of pluripotent stem cells relative to the number of epithelial cells (1x) at the start of co-culture can also be preferably 0.5 to 2 times, more preferably 0.8 to 2 times. As a specific example, the number of pluripotent stem cells at the start of co-culture is twice the number of epithelial cells (1x), i.e., the mixture ratio of epithelial cells to pluripotent stem cells is 1:2 based on the number of cells.

[0060] The number of pluripotent stem cells relative to the number of mesenchymal cells (1x) at the start of co-culture is preferably 0.3x or more, more preferably 0.5x or more, more preferably 0.8x or more, more preferably 1.0x or more, more preferably 1.3x or more, even more preferably 1.5x or more, and particularly preferably 1.8x or more.

[0061] The number of pluripotent stem cells relative to the number of mesenchymal cells (1x) at the start of co-culture is preferably 3.0x or less, more preferably 2.8x or less, more preferably 2.5x or less, even more preferably 2.3x or less, and particularly preferably 2.0x or less.

[0062] For example, the number of pluripotent stem cells relative to the number of mesenchymal cells (1x) at the start of co-culture can be preferably 0.3 to 3.0 times, more preferably 0.5 to 3.0 times, more preferably 0.8 to 2.8 times, more preferably 1.0 to 2.5 times, more preferably 1.3 to 2.5 times, even more preferably 1.5 to 2.5 times, and particularly preferably 1.8 to 2.3 times. In addition, the number of pluripotent stem cells relative to the number of mesenchymal cells (1x) at the start of co-culture can also be preferably 0.5 to 2 times, more preferably 0.8 to 2 times. As a specific example, the number of pluripotent stem cells at the start of co-culture is twice as many as the number of mesenchymal cells (1x), i.e., the mixture ratio of mesenchymal cells to pluripotent stem cells is 1:2 based on the number of cells.

[0063] The order of seeding the cells is not particularly limited, and for example, three types of cells may be seeded in the culture solution in any order. In addition, after the epithelial cells and mesenchymal cells are mixed, pluripotent stem cells may be further added.

[0064] The method of co-culture is not particularly limited as long as it is a method in which epithelial cells and mesenchymal cells are aggregated to form a cell aggregate. Preferably, epithelial cells, mesenchymal cells and pluripotent stem cells are co-cultured on a non-cell-adhesive surface.

[0065] A non-cell-adhesive surface refers to a surface to which cultured cells adhere and do not spread, and in the present invention refers to a surface to which epithelial cells and mesenchymal cells do not adhere and are maintained in a floating state. The non-cell-adhesive surface in the present invention also includes a surface to which cells adhere loosely but do not spread in an adherent state, and from which cells can be easily detached by flowing the culture medium by pipetting or the like, without enzyme treatment or chelating treatment.

[0066] As a cell culture vessel having a cell non-adhesive surface, for example, a commercially available multi-well plate with a cell non-adhesive coating applied to the bottom surface of each well can be used. A preferred example of such a culture vessel is a cell culture plate with micro-concave portions coated with a cell non-adhesive coating. The opening shape of the micro-concave portions of the cell culture plate is not particularly limited, and may be circular, U-shaped, V-shaped, square, hexagonal, linear, etc. In the present invention, the opening shape of the micro-concave portions is preferably circular (U-shaped) or V-shaped. Examples of such cell culture plates include the PrimeSurface 96U plate manufactured by Sumitomo Bakelite Co., Ltd. and the Nunclon 96U plate manufactured by Thermofisher. TM Sphera TM Suitable examples include 96-Well, Nunclon Sphera-Treated, and U-Shaped-Bottom Microplates. Co-culture may also be performed using a cell culture vessel made of an oxygen-permeable material.

[0067] On the non-cell-adhesive surface, a non-adhesive cell aggregate is produced. Here, the non-adhesive state means that the cell aggregate is not attached to the non-cell-adhesive surface and is in a floating state, or that the cell aggregate is loosely attached to the non-cell-adhesive surface but can be easily detached from the non-cell-adhesive surface by an operation of flowing the culture medium, such as pipetting, without performing an enzyme treatment, such as trypsin treatment.

[0068] The culture medium used for co-culture is not particularly limited as long as it can maintain the cells to be cultured, and basal media such as DMEM, DMEM / F12, Advanced DMEM / F12, Minimum Essential Medium (MEM), RPMI-1640, Basal Medium Eagle (BME), Glasgow Minimum Essential Medium (Glasgow MEM), and Neurobasal Medium (Thermo Fisher Scientific) can be used. As the basal medium, it is preferable to use DMEM / F12 or Advanced DMEM / F12, and it is more preferable to use Advanced DMEM / F12. Additives such as antibiotics, serum, growth factors, and hormones may be added to these basal media as desired.

[0069] Suitable components to be added to the basal medium include N2 supplement, B27 supplement, GlutaMAX supplement (Thermo Fisher Scientific), etc. Furthermore, it is preferable to add Normocin as an antibiotic.

[0070] In one embodiment, the culture medium used for co-culture contains an extracellular matrix, preferably, for example, laminin, fibronectin, collagen, laminin / entactin complex, matrigel, or the like.

[0071] In one embodiment, the culture medium used for co-culture preferably contains Matrigel as an extracellular matrix. In the present invention, Matrigel refers to a soluble preparation obtained from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. For the preparation of Matrigel, DMEM is preferably used, for example, DMEM (containing 1 g / L glucose) can be suitably used. The Matrigel used in the present invention may be Growth Factor Reduced (GFR).

[0072] In the present invention, the extracellular matrix contained in Matrigel may be contained alone or in combination with more than one other. For example, one or more selected from collagen I, collagen IV, entactin, and collagen IV may be contained, and more specifically, collagen I, collagen IV, laminin / entactin complex, or laminin / entactin complex and collagen IV may be contained.

[0073] When Matrigel is used, the concentration of Matrigel added to the culture medium may be 0.5 v / v% or more, 1 v / v% or more, 1.5 v / v% or more, or 1.8 v / v% or more, relative to the total volume of the culture medium. The concentration of Matrigel added to the culture medium may be 5 v / v % or less, 3 v / v % or less, or 2.5 v / v % or less, based on the total volume of the culture medium. An example of the concentration of Matrigel added to the culture medium is 2.0 v / v %.

[0074] The culture period of the co-culture can be 1 day or more, can be 5 days or more, can be 10 days or more, can be 12 days or more, or can be 14 days or more. By carrying out the co-culture for the above period, aggregates of epithelial cells and mesenchymal cells can be formed, and further differentiated into organoids with hair follicles.

[0075] When culturing in a culture medium containing an extracellular matrix, low-temperature culture may be performed first. After low-temperature culture, the cells can be cultured at normal culture temperatures (approximately 37°C). Low temperature culture can promote hair follicle and hair shaft formation.

[0076] The temperature for low-temperature culture can be 10° C. or lower, preferably 8° C. or lower, more preferably 5° C. or lower, and even more preferably 4° C. or lower. The temperature for low-temperature culture can be 2° C. or higher, and preferably 3° C. or higher.

[0077] The time for low-temperature culture is preferably 5 minutes or more, more preferably 10 minutes or more, more preferably 15 minutes or more, more preferably 20 minutes or more, even more preferably 25 minutes or more, and particularly preferably 28 minutes or more. The time for low-temperature culture is preferably 120 minutes or less, more preferably 100 minutes or less, more preferably 80 minutes or less, more preferably 60 minutes or less, more preferably 50 minutes or less, even more preferably 40 minutes or less, and particularly preferably 35 minutes or less. The time for low-temperature culture is preferably 5 to 120 minutes, more preferably 10 to 100 minutes, more preferably 20 to 80 minutes, further preferably 25 to 60 minutes, and particularly preferably 28 to 35 minutes.

[0078] The co-culture may also include a period of shaking culture. By performing shaking culture, it is possible to promote the formation of hair follicles and hair shafts.

[0079] Shaking culture may be performed for the entire period of co-culture or for only a part of the period of co-culture. For example, the period for shaking culture may be 1 day or more, 5 days or more, 10 days or more, or 12 days or more from the start of co-culture.

[0080] The conditions for shaking culture are not particularly limited, but the rotation speed is preferably 20 to 90 rpm, more preferably 30 to 80 rpm, further preferably 40 to 70 rpm, and particularly preferably 50 to 60 rpm.

[0081] In the above-mentioned preparation method of the present invention, hair follicle organoids with hair bulb and hair shaft structure can be prepared in vitro, particularly using human cells.In this way, the preparation method of the present invention can provide hair follicle organoids with hair shaft structure, which is effective for clinical application to humans and application as an evaluation tool for human medicines, cosmetics, etc. When hair follicle organoids are used clinically, the cells used may be autologous or allogeneic cells, but autologous cells are preferably used.

[0082] Furthermore, the mesenchymal cells, epithelial cells, and pluripotent stem cells used in the production method of the present invention can be cells obtained from the same donor individual. By using cells derived from the same donor individual, the risk of rejection and the like when transplanting the obtained hair follicle organoids can be reduced. Furthermore, when screening and the like is performed using the obtained hair follicle organoids, it becomes possible to perform screening and the like that reflects the characteristics of the donor individual.

[0083] <Hair follicle organoids> According to the above-mentioned preparation method of the present invention, human-derived hair follicle organoid can be efficiently prepared. That is, the present invention also exists in the hair follicle organoid itself prepared by the above-mentioned preparation method. The preferred embodiment of the hair follicle organoid of the present invention that can be prepared by the above-mentioned preparation method will be described below.

[0084] In the method of the present invention, epithelial cells, mesenchymal cells, and pluripotent stem cells spontaneously aggregate to form a cell aggregate. The cell aggregate is a hair follicle primordium capable of differentiating into a hair follicle. Then, a hair peg is formed from a part of the cell aggregate, and grows into a hair follicle. That is, by performing the co-culture, a hair follicle organoid containing epithelial cells and mesenchymal cells and equipped with a hair follicle can be obtained.

[0085] In the hair follicle organoid, the hair follicle is formed as a structure protruding from the surface of the aggregate of epithelial cells and mesenchymal cells. The hair follicle formed on the surface of the aggregate of cells forms, for example, a hair bulb having a hair papilla-like structure at its free end, i.e., the tip portion.

[0086] In a more preferred embodiment, the hair follicle organoid comprises a hair shaft, which may be formed within the hair follicle protruding from the cell aggregate.

[0087] It is also preferred that the hair shaft contains a pigment, and the pigment that accumulates is preferably melanin.

[0088] The length of the hair shaft is not particularly limited, but may be, for example, 30 μm or more, 50 μm or more, or 100 μm or more.

[0089] <Application> The present inventors discovered that the production method of the present invention promotes hair follicle induction and further stimulates hair shaft formation by further co-culturing pluripotent stem cells during co-culture of epithelial cells and mesenchymal cells in the production of hair follicle organoids. That is, the present invention relates to a method for producing hair follicle organoids by co-culturing epithelial cells and mesenchymal cells, and further to a method for improving the hair shaft formation ability of hair follicle organoids, which includes co-culturing in the presence of induced pluripotent stem cells.

[0090] Furthermore, the hair follicle organoid of the present invention can be used clinically as a transplant composition. That is, the present invention also relates to a transplantable composition comprising the above-mentioned hair follicle organoid.

[0091] Furthermore, as described above, the hair follicle organoid of the present invention can be used as a testing tool. Specifically, by applying a test substance to the hair follicle organoid of the present invention and observing the anatomical and molecular biological reactions, it is possible to screen active ingredients of medicines and cosmetics. Methods for applying the test substance to the hair follicle organoid include coating and injection. In other words, the present invention also relates to a screening method for active ingredients of pharmaceuticals or cosmetics, which is characterized by applying a test substance to the above-mentioned hair follicle organoid. EXAMPLES

[0092] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0093] (1) Cultivation of mesenchymal cells (1-1) Reagents used (a) Antibiotic Mix 247 ml of HBSS was transferred to a sterile container, and 2.5 ml of a penicillin-streptomycin mixed solution (final concentration: 100 U / ml penicillin, 100 μg / ml streptomycin) and 250 μL of Amphotericin B solution (final concentration: 250 ng / ml) were added. (b) Mesenchymal stem cell culture medium (MSC medium) 45 mL of Essential 8 Flex medium (Gibco, supplement added) was transferred to a 50 mL tube, and 0.5 mL of a penicillin-streptomycin mixed solution (final concentration 100 U / mL penicillin, 100 μg / mL streptomycin) and 5 mL of FBS (fetal bovine serum (Gibco)) were added. Then, bFGF (ReproCell) and EGF (epidermal growth factor (EGF), human, Fujifilm Wako Pure Chemical Industries) were added to a final concentration of 10 ng / mL each. (c) iMatrix coating A dilution solution of 5 μL of iMatrix-511 (Nippi) per 1 mL of DPBS (- / -) was added to the bottom of a 24-well cell culture plate at 300 μL / well and left to stand in an incubator at 37°C for 30 minutes or more. The dilution solution was then removed and used.

[0094] (1-2) Cultivation of mesenchymal cells Hair from a subject (adult) was plucked and immediately immersed in Antibiotic Mix for a moment. Next, hair bulbs covered with dermal root sheath tissue were selected, and hairs were cut out to include the hair bulb. The hairs were individually seeded onto a 24-well plate coated with iMatrix containing 150 μL of MSC medium. The day of seeding was considered to be day 0.

[0095] On days 1 and 5 after seeding, 150 μL of MSC medium was replenished, and on day 7 after seeding, 300 μL of MSC medium was replenished. Thereafter, replenishment and replacement of MSC medium was continued depending on the proliferation level of the cells.

[0096] When the cells were grown to 60-70% confluence, they were passaged as follows. That is, 0.2 mL of TrypLESelect was added, and the cells were placed in a 37°C incubator, and the cells were detached by pipetting. After that, the cells were collected in a 1.5 mL tube, centrifuged, the supernatant was removed, and the cells were suspended in 1 mL of MSC medium, and the number of cells was counted. Then, the cells were seeded in MSC medium at 50,000 cells / well on a 6-well plate coated with iMatrix. Next, MSC medium was added to each well so that the medium volume was 2 mL, and 5 μL of iMatrix-511 (Nippi) was added to each well.

[0097] After the medium was added, the cells were cultured in an incubator at 37° C. From the day after seeding, StemFit for Mesenchymal Stem Cells (Ajinomoto, hereafter also referred to as StemFit for MSC) were replenished, and from the 5th day after seeding, the entire amount of StemFit for MSC was replaced every other day to allow the cells to proliferate.

[0098] Figure 1 shows the area around the hair papilla on day 7 after the start of culture, along with the purchased bone marrow-derived MSCs. The cells expanding from the area around the hair papilla had a fibroblast-like appearance, similar to bone marrow-derived MSCs. The obtained mesenchymal cells were cryopreserved until use in co-culture.

[0099] (2) Cultivation of epithelial cells (2-1) Reagents used (a) EpiLife medium EpiLif 500ml capacity TM The entire amount (5 mL) of Human Keratinocyte Growth Supplement (HKGS, Gibco), 5 mL of a penicillin-streptomycin mixed solution (Nacalai Tesque), and 500 μL of amphotericin-B were added to medium with 60 μM calcium (Gibco), and mixed. (b) MEF-Conditioned Medium To 10 mL of EmbryoMax (registered trademark) MEF conditioned medium (Merck), 0.1 mL of penicillin-streptomycin mixed solution (Nacalai Tesque), 10 μL of Amphotericin-B, and 0.1 mL of HKGS (Gibco) were added and mixed. (c)Antibiotic Mix The one prepared in the same manner as in Example 1 was used. (d) Collagen coating Collagen acidic solution I-PC (atelocollagen) 5 mg / mL was diluted 10 times with 1 mM hydrochloric acid. The diluted collagen solution was added to 400 μL / well for 12-well plates for cell culture, and 1000 μL / well for 6-well plates, and left to stand at 37°C for 1 hour or more, then removed with an aspirator. Then, it was washed three times with PBS before use. (e) iMatrix coating A dilution solution of 5 μL of iMatrix-511 (Nippi) per 1 mL of DPBS (- / -) was added to a 6-well plate for cell culture at 1000 μL / well and left to stand in an incubator at 37° C. for 1 hour or overnight at 4° C. The dilution solution was then removed and used.

[0100] (2-2) Cultivation of epithelial cells After removing the hair from the subject (adult), it was immediately immersed in Antibiotic Mix for a moment. Next, 1-2 mm of the hair containing the outer root sheath was cut out in the Antibiotic Mix, and one hair sample was seeded per well in 250 μL of mixed medium (EpiLife+ MEF-Conditioned medium + Antibiotic Mix) in a 12-well plate coated with collagen solution (acidic collagen solution I-PC, final concentration 0.5 mg / mL (atelocollagen)). The day of seeding was considered as seeding day 0.

[0101] On the first or second day after seeding, 250 μL of the above mixed medium was replenished, and on the third day after seeding, 500 μL of the above mixed medium was replenished. Then, it was confirmed that the total liquid volume of the medium was about 0.75 mL on the third day after seeding, and static culture was performed until the sixth day after seeding.

[0102] Six days after seeding, if the cells had not grown to a radius of 1-2 mm or more around the hair shaft, 250 μL of the mixed medium was removed and 250 μL of MEF-Conditioned medium was added. This replacement of MEF-Conditioned medium was continued until the cells had grown to a radius of 1-2 mm or more.

[0103] Next, after confirming that the proliferated cells had spread to a radius of 1-2 mm or more from the hair shaft, the medium was replaced. For the medium replacement, the above mixed medium was removed and 500 μL of EpiLife medium was added. Note that if the cells had spread to a radius of 1-2 mm or more from the hair shaft 6 days after seeding, the medium was replaced with EpiLife medium without replacing with the above MEF-Conditioned medium.

[0104] Two days after the medium replacement, the medium was again replenished with 500 μL of EpiLife medium, and two days after the medium replacement, the medium was replaced with 500 μL of EpiLife medium. Then, medium replacement and replenishment were continued in the same manner every two days.

[0105] In the expansion area of ​​cells proliferating from the outer root sheath, before the cells became overcrowded, specifically when they reached 60-70% confluence, the EpiLife medium was removed and washed with DPBS. Next, 0.5 mL of TrypLESelect (Gibco) was added and the plate was placed in a 37°C incubator, after which the cells were detached from the plate by pipetting and collected in a 15 mL tube. The collected cells were centrifuged, the supernatant was removed, and the cells were suspended in 1 mL of MEF-Conditioned medium. Next, the cells were seeded on a 6-well plate coated with iMatrix-511 (Nippi) at 50,000 cells / well and cultured in an incubator at 37°C. The culture was continued until the cells became 50-60% confluent while replacing the medium with 500 µL of EpiLife medium.

[0106] Figure 2 shows the state of epithelial cells extending from the outer root sheath on the 14th day after seeding. As shown in Figure 2, it was confirmed that a large number of epithelial cells had proliferated around the outer root sheath. The obtained epithelial cells were cryopreserved until use in co-culture.

[0107] (3) Creation of hair follicle organoids (3-1) Preculture [Mesenchymal cells] A medium (Stem-iM) was prepared by adding 12 μL of iMatrix to 30 mL of StemFit for MSC (containing Normocin (final concentration 100 μg / mL)). Thawed mesenchymal cells were added to a T75 flask containing Stem-iM at a concentration of 2.0 × 10 5 cells / flask, or 3.0 x 10 5 The cells were suspended at 100 cells / flask, and then cultured at 37°C with StemFit for MSC (containing Normocin) until the cells reached 70% confluence.

[0108] [Epithelial cells] The thawed hair-derived epithelial cells were suspended in 30 mL of EpiLife medium (containing 1x HKGS, 100 U / mL penicillin, and 100 μg / mL streptomycin), and then dispensed into 15 mL portions at approximately 250,000 cells / T75 flask. 50 μL of iMatrix-511 was then added to the T75 flask, and the cells were cultured at 37°C. The culture was continued until the cells reached 70% confluence, with the medium being replaced every other day.

[0109] [iPS cells] Thawed iPS cells (Cellartis (registered trademark) human iPS cell line P11025 (Takara Bio)) were cultured in a 30-mL flask using Essential 8 TM After suspending in Flex medium (Gibco) (hereafter referred to as E8), the cells were centrifuged, the supernatant was discarded, and E8 containing 10 μM Y27632 (hereafter referred to as E8-Y) was added to resuspend the iPS cells. The obtained cells were seeded on an iMatrix-coated 6-well plate and cultured at 37°C in a 5% CO2 incubator. The next day, the medium was replaced with E8, and the culture was continued until the cells reached 70% confluence.

[0110] (3-2) Co-culture Mesenchymal cells, epithelial cells, and iPS cells were each treated with trypsin, collected in a 15 mL tube, and centrifuged. The supernatant was removed, and Advanced DMEM / F12 (containing Normocin at a final concentration of 100 μg / mL) was added to the tube to suspend the cells, and the number of cells was counted.

[0111] Culture medium 1 or culture medium 2 shown in Table 1 below, which had been cooled on ice, was added to each 6 mL tube. Each cell solution was added to the tube so that the cell numbers were mesenchymal cells: epithelial cells: iPS cells = 120,000: 120,000: 60,000 (2:2:1), 75,000: 75,000: 150,000 (1:1:2), 4000: 4000: 2000 (2:2:1), or 2500: 2500: 5000 (1:1:2). Next, 120 μL of cooled Matrigel GR (Matrigel (registered trademark) Growth Factor Reduced (GFR) Basement Membrane Matrix (Corning)) was added to each tube so that the final concentration was 2% (v / v).

[0112] [Table 1]

[0113] The three types of cell mixtures were placed in a 96-well low-adhesive plate (Nunclon TM Sphera TM 0.2 mL of the solution was added per well to a 96-well Nunclon Sphera-Treated U-Shaped-Bottom Microplate (Thermofisher Scientific). Two types of plates were prepared. After the cells were seeded, the plates were left to stand at 4°C in a refrigerator for 30 minutes. The plates were then moved to a 37°C incubator and cultured. At this time, one plate was cultured stationarily, and the other plate was cultured on a shaker (lab shaker_rotary type BC-740 (Biocraft Co., Ltd.)) at 50 to 60 rpm. Table 2 shows the combinations of culture conditions that were performed.

[0114] [Table 2]

[0115] After the start of culture, the medium was replaced with Medium 1 or Medium 2 supplemented with 2% (v / v) Matrigel GR every 1 or 2 days. Chilled Matrigel GR was added to chilled Medium 1 or 2, which was then returned to room temperature and used for medium replacement.

[0116] (4) Results Figure 3 shows the state of hair follicle organoids on day 14 after the start of co-culture under condition D (MSC:Kc:iPS=1:1:2, medium 2, shaking culture), with the start of co-culture considered to be day 0. The arrowhead indicates a hair peg that sprouted from an aggregate of epithelial cells and mesenchymal cells. A hair bulb was formed at the tip of the hair peg, and a hair shaft with accumulated pigment was confirmed to have formed inside the hair peg. In addition, hair follicle organoids having a hair shaft-like structure were confirmed under conditions A to C and E to H. As described above, it was demonstrated that adding pluripotent stem cells to epithelial cells and mesenchymal cells and culturing them promotes hair follicle induction and enables the production of hair follicle organoids with hair shaft structures.

[0117] (5) Discussion As described above, according to the technology disclosed in Non-Patent Document 1, hair follicle tissue having a hair shaft structure can be produced in vitro using mouse fetal cells. However, due to ethical issues, it is difficult to actively apply human fetal cells. Therefore, although there is a demand for a technology to produce hair follicle organoids using human adult cells, the technology at the time of this application did not succeed in producing hair follicle organoids having a hair shaft structure using human cells (see the discussion in Non-Patent Document 1). In Non-Patent Document 2, when human adult cells were used, only the formation of hair germs was observed, and the formation of hair follicle organoids having a hair shaft structure was not confirmed.

[0118] Under these circumstances, the present inventors have succeeded in producing hair follicle organoids having a hair shaft using cells derived from an adult by co-culturing epithelial cells, mesenchymal cells, and pluripotent stem cells. This is the first case in which hair follicle organoids having a hair shaft have been successfully produced using human-derived cells.

[0119] Furthermore, in the present invention, it is also noteworthy that the hair follicle organoid having a hair shaft was successfully produced using epithelial cells and mesenchymal cells of adult origin, not fetal (embryo) origin cells. Since the hair follicle organoid having a hair shaft can be produced without using fetal origin cells, the technology of the present invention can be said to be a technology that can be widely used in human hair follicle regeneration. [Industrial Applicability]

[0120] The present invention can be applied to the production of organ primordia such as hair follicle primordia and organoids, and to the development of medical materials and the like using these.

Claims

1. A method for producing hair follicle organoids, comprising co-culturing epithelial cells, mesenchymal cells, and pluripotent stem cells.

2. The method for producing a hair follicle organoid according to claim 1, wherein the hair follicle organoid has a hair bulb and a hair shaft.

3. The method for producing a hair follicle organoid according to claim 2, wherein the hair shaft contains a pigment.

4. The method for producing a hair follicle organoid according to claim 1, wherein the hair follicle organoid is a human hair follicle organoid produced from human-derived cells.

5. A method for producing hair follicle organoids according to any one of claims 1 to 4, wherein the number of epithelial cells to the number of mesenchymal cells mixed at the start of co-culture is 0.1 to 3 times.

6. A method for producing hair follicle organoids according to any one of claims 1 to 4, wherein the number of pluripotent stem cells mixed at the start of co-culture is 0.3 to 3 times the number of epithelial cells.

7. A method for producing hair follicle organoids according to any one of claims 1 to 4, wherein the number of pluripotent stem cells to the number of mesenchymal cells mixed at the start of co-culture is 0.3 to 3 times.

8. A hair follicle organoid prepared by the method described in any one of claims 1 to 4.

9. A human hair follicle organoid comprising epithelial cells and mesenchymal cells, and comprising a hair bulb and hair shaft.

10. The hair shaft comprises a pigment, as described in claim 9, for the human hair follicle organoid.

11. A method for producing hair follicle organoids by co-culturing epithelial cells and mesenchymal cells, further comprising co-culturing in the presence of pluripotent stem cells, thereby improving the hair shaft formation ability of the hair follicle organoids.

Images