Composition for reconstructing human skin tissue having a hair follicle, human skin tissue model animal, and method for producing the same

Abstract

To provide evaluation systems for correctly evaluating a hair growth effect of a newly developed hair regrowth technique.SOLUTION: Provided are a composition for reconstructing human skin tissues having hair follicles, which comprise human epidermal cells and human dermal cells, the human dermal cells comprising a group of human dermal papilla cells derived from non-fetus, which has undergone spheroid formation; a human skin tissue model animal to which it is applied; a method for producing the composition that comprises a step of preparing a spheroid comprising human hair papilla cells derived from non-fetus to mix and culture the spheroid, human epidermal cells, and human dermal cells; and a method for producing the human skin tissue model animal comprising a step of applying the composition to a non-human mammal.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a composition for reconstructing a human skin tissue having hair follicles and a method for producing the same. Further, the present invention relates to a human skin tissue model animal having hair follicles to which the composition for reconstructing a human skin tissue having hair follicles is applied and a method for producing the same.

Background Art

[0002] Hair is produced by hair follicles present in the skin. A hair follicle is a tissue layer surrounding the hair and is composed of an outer ectoderm-derived hair matrix (hair matrix cells), an inner root sheath, an outer root sheath, and a mesoderm-derived dermal root sheath, a hair papilla, and the like. The hair matrix cells surrounding the hair papilla are induced by nutrients and proteins supplied from the hair papilla to repeatedly divide and form hair by keratinization.

[0003] When there are problems in hair growth, hair loss or alopecia occurs. Hair loss or alopecia includes male pattern alopecia, alopecia areata, telogen effluvium, etc., and the most frequent one is male pattern alopecia. Male pattern alopecia is mainly caused by the influence of male hormones in men and is also called androgenetic alopecia. Hair regrows while repeating a hair cycle consisting of anagen, catagen, and telogen. Male pattern alopecia is a symptom caused by a shortening of the anagen phase in this hair cycle and an increase in the proportion of thin and short hair.

[0004] Currently, as a treatment for male pattern alopecia, topical minoxidil and oral finasteride are mainly used. Although the effectiveness and safety of these treatment methods have been confirmed, they are not necessarily effective for all cases of male pattern alopecia.

[0005] In addition, as a treatment for male pattern alopecia, autologous hair transplantation is performed. Autologous hair transplantation is a surgical procedure in which hair including hair roots collected from the temporal region or the occipital region is transplanted to the patient's own alopecic area. However, since this surgical procedure is a method of transplanting one's own hair to another place, it does not increase the total number of hairs.

[0006] In recent years, regenerative medicine technologies for various diseases have been developed, and new technologies are also being developed in the field of hair regeneration. In order to develop a new hair regeneration technology, it is important to establish an evaluation system for correctly evaluating the hair growth effect, and various research groups are developing reconstituted skin having hair follicles (for example, Patent Document 1 and Non-Patent Documents 1 and 2, etc.).

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0009] In the field of hair regeneration, therapeutic methods using follicle-derived cells are being developed. However, in order to correctly evaluate the effects of the developed therapeutic methods, it is important to establish an evaluation system using reconstituted human skin containing human hair follicles that can maintain its structure for a long period of time. However, to date, it has still been difficult to provide reconstituted human skin containing human hair follicles with good reproducibility by using stably available materials.

[0010] Therefore, an object of the present invention is to provide a composition and a method for producing the same for reconstituting a human skin tissue having a human hair follicle with good reproducibility by using stably available materials. Another object of the present invention is to provide a human skin tissue model animal having a hair follicle to which a composition for reconstituting a human skin tissue having a hair follicle is applied and a method for producing the same.

Means for Solving the Problems

[0011] As a result of intensive studies by the present inventors, it has been found that by including a cell group of non-fetal-derived human dermal papilla cells that have undergone spheroid formation in a composition, a human skin tissue having a hair follicle can be stably and reproducibly reconstituted, and the present invention has been completed. That is, the present invention includes the following inventions.

[0012] [1] A composition for reconstituting a human skin tissue having a hair follicle, comprising: human epidermal cells; human dermal cells, and wherein the human dermal cells include a cell group of non-fetal-derived human dermal papilla cells that have undergone spheroid formation. [2] The composition according to [1], wherein the non-fetal-derived human dermal papilla cells are non-fetal-derived human dermal papilla cells stimulated with a Wnt signal activator. [3] The composition according to [2], wherein the Wnt signal activator is a Wnt ligand family protein or a Wnt Signal transmission agonist. [4] The composition according to [3], wherein the Wnt ligand family protein is selected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, and combinations thereof. [5] The Wnt Signal transmission agonist is CHIR99021 or BIO, and the composition according to [3]. [6] The composition according to any one of [1] to [5], wherein the human dermal cells include human fibroblasts.

[0013] [7] A human skin tissue model animal having hair follicles, to which the composition according to any one of [1] to [6] is applied to a non-human mammal.

[0014] [8] A method for producing a composition for reconstructing human skin tissue having hair follicles, comprising: (1) a step of preparing a spheroid containing non-fetal-derived human dermal papilla cells; (2) a step of mixing and culturing the spheroid, human epidermal cells, and human dermal cells. The method comprising the above steps. [9] The method according to [8], wherein the step (1) is carried out in the presence of a Wnt signal activator.

[10] The method according to [9], wherein the Wnt signal activator is a Wnt ligand family protein or a Wnt Signal transmission agonist.

[11] The method according to

[10] , wherein the Wnt ligand family protein is selected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, and combinations thereof.

[12] The Wnt Signal transmission agonist is CHIR99021 or BIO, and the method according to

[10] .

[13] The method according to any one of [8] to

[12] , wherein the step (1) is carried out by the hanging drop method.

[14] The method according to any one of [8] to

[13] , wherein the non-fetal-derived human dermal papilla cells are adult-derived human dermal papilla cells.

[15] The method according to any one of [8] to

[14] , wherein the human dermal cells include human fibroblasts.

[16] The method according to any one of [8] to

[15] , wherein the human epidermal cells are the human epidermal cells stimulated by an extracellular matrix protein or a derivative thereof.

[17] The method according to

[16] , wherein the extracellular matrix protein includes laminin or a fragment thereof.

[18] The method according to any one of [8] to

[17] , wherein the step (2) is a step of culturing on a culture substrate provided with a porous membrane.

[0015]

[19] A step of applying the composition obtained by the method according to any one of [8] to

[18] to a non-human mammal, A method for producing a human skin tissue model animal having a hair follicle, comprising the above steps.

[0016]

[20] A composition for reconstructing a human skin tissue having a hair follicle, obtained by the method according to any one of [8] to

[18] .

[0017]

[21] A human skin tissue model animal having a hair follicle, obtained by the method of

[19] .

Advantages of the Invention

[0018] According to the present invention, it is possible to provide a composition and a method for producing the same, which can stably and reproducibly reconstruct a human skin tissue having a hair follicle. In addition, it is possible to provide a human skin tissue model animal having a hair follicle to which the composition is applied and a method for producing the same. The evaluation system provided by the present invention enables the effects of drugs, regenerative medical technologies, or cosmetic technologies under development, particularly the hair growth effect, to be evaluated with good reproducibility.

Brief Description of the Drawings

[0019]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Mode for Carrying Out the Invention

[0020] Hereinafter, embodiments for carrying out the present invention will be described. However, the technical scope of the present invention is not limited only to the following embodiments. In addition, the content described in the following embodiments for carrying out the present invention is mutually applicable in a composition for reconstructing a human skin tissue having a hair follicle and a method for producing the same, and a human skin tissue model animal having a hair follicle and a method for producing the same, unless otherwise particularly limited.

[0021] 1. Composition for Reconstructing Human Skin Tissue Having a Hair Follicle

[0022] In one embodiment, the composition for reconstructing a human skin tissue having a hair follicle of the present invention is human epidermal cells; human dermal cells, and wherein the human dermal cells include a cell group of non-fetal-derived human dermal papilla cells that have undergone spheroid formation.

[0023] As used herein, the "composition for reconstructing a human skin tissue having a hair follicle" refers to a composition that, when applied to the skin, has the ability to reconstruct a human skin-like tissue having a hair follicle structure at that site.

[0024] The "hair follicle (HF)" generally refers to the tissue layer surrounding the hair, including the hair matrix (hair matrix cells) which are epithelial cells, the inner root sheath, the outer root sheath, and the dermal root sheath and dermal papilla which are mesenchymal cells. By applying the composition of the present invention, a human skin-like tissue having such human hair follicles can be reconstituted at the application site. Moreover, hair grows from the reconstituted hair follicles.

[0025] In the present invention, the "epidermal cells" are the cells constituting the epidermis and include cells with different differentiation stages (mainly keratinocytes). In a living body, the epidermis is formed by epidermal cells with different differentiation stages overlapping in layers. The deepest part of the epidermis is called the basal layer, which forms a single layer of columnar cells. As the differentiation stage of epidermal cells progresses, they change to a flat shape and move to the outer layer. The basal layer is located at the interface with the dermis, and the spinous layer exists above it. Epidermal cells that have advanced further in the differentiation stage than the spinous layer form the granular layer having keratohyalin granules and lamellar granules. The granular layer is composed of about 2 to 3 layers in a living tissue. When the differentiation stage further progresses from the granular layer, the cell nucleus disappears and the stratum corneum is formed. In addition to epidermal cells, the epidermis also contains melanocytes, Langerhans cells, Merkel cells, etc. The composition of the present invention may contain other cells contained in the epidermis in addition to epidermal cells.

[0026] In one embodiment, the epidermal cells contained in the composition of the present invention are human-derived epidermal cells. The human epidermal cells may be commercially available human epidermal cells, or may be primary human epidermal cells obtained by finely cutting a biological tissue and treating it with proteolytic enzymes such as collagenase and trypsin, or may be fibroblasts obtained by subculturing and proliferating the obtained primary human epidermal cells. In one embodiment, the epidermal cells contained in the composition of the present invention may be derived from a human fetus, a newborn (e.g., 0 to 2 years old), a minor (e.g., 3 to 17 years old), or an adult (e.g., 18 years old or older, 20 years old or older, 25 years old or older, 30 years old or older, 35 years old or older, 40 years old or older), etc. Further, in one embodiment, the epidermal cells contained in the composition of the present invention may be epidermal cells differentiated from pluripotent stem cells or tissue stem cells. The pluripotent stem cells may be, for example, iPS cells, ES cells, or Muse cells, etc., and epidermal cells obtained by inducing differentiation from these pluripotent stem cells by a known method may also be used.

[0027] In one embodiment, the epidermal cells contained in the composition of the present invention are epidermal cells stimulated by an extracellular matrix protein or a derivative thereof. Examples of the extracellular matrix protein include laminin, nidogen, tenascin, thrombospondin, fibronectin, vitronectin, collagen, elastin, etc. The extracellular matrix protein for stimulating epidermal cells may be a natural-type extracellular matrix protein or a derivative (recombinant) thereof. Further, it may be a fragment of an extracellular matrix protein having a function of stimulating epidermal cells. The inclusion of epidermal cells stimulated by an extracellular matrix protein in the composition of the present invention makes the composition have a higher ability to reconstruct a human skin-like tissue having human hair follicles, which is preferable.

[0028] In one embodiment, the extracellular matrix protein for stimulating epidermal cells contains laminin or a fragment thereof. Laminin is a protein composed of a combination of α chains (LAMA1, LAMA2, LAMA3, LAMA4, LAMA5), β chains (LAMB1, LAMB2, LAMB3, LAMB4), and γ chains (LAMC1, LAMC2, LAMC3). To date, 17 combinations (laminin 1-15, as well as laminin 212 / 222 and laminin 522) are known. For example, a laminin with a combination of α, β, and γ chains of LAMA5, LAMB1, and LAMC1 is called "laminin 511". Laminin 511 is also known as laminin 10. In one embodiment, the extracellular matrix protein for stimulating epidermal cells may be a fragment of laminin. For example, the laminin 511-E8 fragment can be used.

[0029] As used herein, the term "dermal cell" refers to a cell contained in the dermis that exists between the epidermis and the subcutaneous tissue in the skin and mainly includes fibroblasts. The dermis is composed of, in addition to fibroblasts, collagen, elastic fibers (elastin), extracellular matrix, hyaluronic acid, and the like.

[0030] In one embodiment, the dermal cells contained in the composition of the present invention are human-derived dermal cells. The human dermal cells may be commercially available human dermal cells, or may be primary human dermal cells obtained by finely cutting a biological tissue and treating it with proteolytic enzymes such as collagenase and trypsin, or may be dermal cells obtained by subculturing and proliferating the obtained primary human dermal cells. In one embodiment, the dermal cells contained in the composition of the present invention may be derived from a human fetus, a neonate (e.g., 0 to 2 years old), a minor (e.g., 3 to 17 years old), or an adult (e.g., 18 years old or older, 20 years old or older, 25 years old or older, 30 years old or older, 35 years old or older, 40 years old or older), etc. Further, in one embodiment, the dermal cells contained in the composition of the present invention may be epidermal cells differentiated from pluripotent stem cells or tissue stem cells. The pluripotent stem cells may be iPS cells, ES cells, Muse cells, etc., and dermal cells obtained by inducing differentiation from these pluripotent stem cells by a known method may also be used.

[0031] The swollen part deepest inside the skin of the hair is called the hair bulb, and the part composed of mesenchymal cells in the center of the hair bulb is called the dermal papilla (DP). Also, the cells constituting the dermal papilla are called "dermal papilla cells". Capillaries and nerves enter the dermal papilla, taking in nutrients and oxygen from food and controlling hair generation and growth. There are hair matrix cells in contact with the dermal papilla, and hair is produced at this site. Hair matrix cells take in nutrients and oxygen from the capillaries that enter the dermal papilla and form hair by repeating division.

[0032] In one embodiment, the dermal papilla cells contained in the composition of the present invention are dermal papilla cells derived from humans. In one embodiment, human dermal papilla cells can be isolated from human hair follicles (preferably human scalp hair follicles), and the tissue in the dermal papilla region can be further isolated from the isolated human hair follicles and then cultured to proliferate. Specifically, for example, the scalp is cut into strips about 5 mm long, washed with phosphate buffered saline (PBS) or the like, and then, if necessary, treated with a proteolytic enzyme or surgically, removing the epidermal layer and the dermal layer to leave only the subcutaneous fat layer, and then the hair follicles are isolated by physical means such as forceps. The hair bulb can be cut off from the hair follicle, and the dermal papilla can be exposed from the lower part of the hair bulb to isolate the dermal papilla (cells).

[0033] The dermal papilla cells can be cultured (primary culture and subculture) using a commercially available nutrient medium as it is or a modified one used for culturing animal cells. Representative media that can be used for culturing dermal papilla cells include Dulbecco's modified Eagle's medium containing fetal bovine serum, Chang's medium, MesenPRO medium (Thermo fisher), and the like. Cell growth factors, hormones, and other micronutrients can be further added to the medium as needed. Specific examples of these include transferrin, insulin, triiodothyronine, glucagon, hydrocortisone, testosterone, estradiol, progesterone, selenium, and the like.

[0034] The human dermal papilla cells contained in the composition of the present invention are non-fetal-derived human dermal papilla cells, for example, dermal papilla cells derived from minors (e.g., 3 to 17 years old) or adults (e.g., 18 years old or older). According to the present invention, it becomes possible to reconstruct human skin tissue having hair follicles even using non-fetal-derived human dermal papilla cells, which was difficult in the past.

[0035] In other embodiments, the dermal papilla cells contained in the composition of the present invention may be dermal papilla cells differentiated from pluripotent stem cells or tissue stem cells. The pluripotent stem cells may be, for example, iPS cells, ES cells, or Muse cells, etc., and dermal papilla cells obtained by inducing differentiation from these pluripotent stem cells may also be used.

[0036] In one embodiment, the composition of the present invention contains a cell group of non-fetal-derived human dermal papilla cells that have undergone spheroid formation. As used herein, "spheroid" refers to a spherical cell aggregate in which cells aggregate or agglomerate. Examples of methods for forming spheroids include, for example, a method of forming spheroids in a U-bottom well having a low cell adhesion surface with cells suspended, a method of forming spheroids with cells adhered to a surface with a predetermined uneven structure, the hanging drop method, and a method of producing spheroids by culturing a large number of cells while rotating a cell culture chamber containing a medium. The cell group of human dermal papilla cells contained in the composition of the present invention is preferably a spheroid formed by the hanging drop method. The inclusion of a cell group of non-fetal-derived human dermal papilla cells that have undergone spheroid formation in the composition increases the efficiency of reconstructing human skin tissue having hair follicles.

[0037] The number of human dermal papilla cells contained in a cell group of human dermal papilla cells that have formed spheroids per one may be, for example, 1×10 to 1×10 5 and may be, preferably 1×10 2 to 1×10 4 and more preferably 1×10 2 to 5×10 3 . Also, the average diameter of the cell group of human dermal papilla cells that have formed spheroids is 20 μm to 1000 μm, preferably 50 μm to 300 μm, and more preferably 70 μm to 200 μm.

[0038] In one embodiment, the non-fetal-derived human dermal papilla cells contained in the composition of the present invention are non-fetal-derived human dermal papilla cells stimulated with a Wnt signal activator. The Wnt signal refers to a series of actions that promote the nuclear translocation of β-catenin and exert its function as a transcription factor. This signal is caused by cell-cell interactions. For example, a protein called Wnt3A secreted from a certain cell acts on another cell, and a series of processes occur in which intracellular β-catenin undergoes nuclear translocation and acts as a transcription factor. This series of processes causes the first phenomenon of organogenesis, taking epithelial-mesenchymal interaction as an example. The Wnt signal is known to control various cellular functions such as cell proliferation, differentiation, organ formation, and cell movement during early development by activating three pathways: the β-catenin pathway, the PCP pathway, and the Ca 2+ pathway. Due to its function of maintaining the undifferentiated state, the Wnt signal is used in the culture of ES cells for the purpose of suppressing differentiation (for example, Noburo Sato et al., Nature Medicine Vol.10, No.1, Jan. 2004). When the composition of the present invention contains non-fetal-derived human dermal papilla cells stimulated with a Wnt signal activator, the efficiency of reconstructing human skin tissue having hair follicles is increased.

[0039] The Wnt signal activator may be a Wnt ligand family protein or a Wnt Signal transmission agonist. Examples of the Wnt ligand family proteins include Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, etc. These Wnt ligand family proteins may be used in combination.

[0040] Wnt Signal transmissionExamples of agonists include, for example, glycogen synthase kinase-3 (GSK-3) inhibitors, R-spondin 1-4, Norrin, etc. Examples of GSK-3 inhibitors include, for example, CHIR99021, bis-indolo (indirubin) compound (BIO) ((2’Z,3’E)-6-bromoindirubin-3’-oxime), its acetoxime analog BIO-acetoxime ((2’Z,3’E)-6-bromoindirubin-3’-acetoxime), thiazolidine (TDZD) analog (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione), oxothiadiazolidine-3-thione analog (2,4-dibenzyl-5-oxothiadiazolidine-3-thione), thienyl α-chloromethyl ketone compound (2-chloro-1-(4,4-dibromo-thiophen-2-yl)-ethanone), phenyl α-bromomethyl ketone compound (α-4-dibromoacetophenone), thiazole-containing urea compound (N-(4-methoxybenzyl)-N’-(5-nitro-1,3-thiazol-2-yl)urea), and GSK-3β peptide inhibitors such as H-KEAPPAPPQSpP-NH2, etc.

[0041] The addition amount of the Wnt signal activator is not particularly limited and can be appropriately determined by those skilled in the art. For example, when using CHIR99021 as a Wnt signal activator for dermal papilla cells, the amount can stimulate, for example, at about 0.1 μM to 10 μM, but is not limited to such an amount.

[0042] In addition to the above cells, the composition of the present invention may contain, for example, a biocompatible substance. Biocompatible substances may be, for example, water, physiological saline, phosphate buffer solution, cell culture medium, biocompatible hydrogels (such as chitosan gel, collagen gel, gelatin, peptide gel, laminin gel, and fibrin gel, etc.), and are not limited thereto.

[0043] The composition of the present invention is preferably prepared on a culture substrate provided with a porous membrane. As the culture substrate provided with the porous membrane, for example, a cell culture insert can be used. A method for producing the composition of the present invention will be described later.

[0044] By applying the composition of the present invention to humans or non-human mammals, it is possible to reconstruct human skin tissue having hair follicles at the applied site.

[0045] 2. Human Skin Tissue Model Animal Having Hair Follicles

[0046] In one embodiment, the composition of the present invention is applied to a non-human mammal. Thereby, a human skin tissue model animal having hair follicles can be obtained. Examples of non-human mammals include rats, mice, guinea pigs, marmosets, rabbits, dogs, cats, sheep, pigs, goats, monkeys, chimpanzees, or non-human mammalian animals with suppressed immune systems, etc., but preferably non-human mammalian animals with suppressed immune systems. A method for producing a human skin tissue model animal having hair follicles of the invention will be described later.

[0047] A candidate substance expected to have a hair growth or hair promoting effect is applied to the human skin tissue model animal having hair follicles of the present invention, and for example, by observing the hair growth state from the hair follicles, the state of the hair follicle tissue, markers related to hair growth, etc., the hair growth or hair promoting effect of the candidate substance can be evaluated. Examples of candidate factors include low molecular weight compounds, peptides, nucleic acids, proteins, cells, tissue extracts or cell culture supernatants of mammals (e.g., mice, rats, pigs, cows, sheep, monkeys, humans, etc.), plant-derived compounds or extracts (e.g., crude drug extracts, compounds derived from crude drugs), and compounds, extracts or culture products derived from microorganisms. Also, for example, it may be for evaluating the influence on the hair follicles formed and / or the hair growth from the hair follicles due to differences such as the donor of human dermal papilla cells contained in the composition of the present invention and the culture conditions.

[0048] 3. Method for producing a composition for reconstructing human skin tissue having hair follicles, and composition for reconstructing human skin tissue having hair follicles obtained thereby

[0049] In one embodiment, the method for producing a composition for reconstructing human skin tissue having hair follicles of the present invention comprises: (1) A step of preparing a spheroid containing non-fetal-derived human dermal papilla cells; (2) A step of mixing and culturing the spheroid, human epidermal cells, and human dermal cells. It includes.

[0050] In the method for producing the composition of the present invention, the step of preparing a spheroid containing non-fetal-derived human dermal papilla cells includes, for example, a method of forming a spheroid by suspending cells in a U-bottom well having a low cell adhesion surface, a method of forming a spheroid by adhering cells on a surface with a predetermined concavo-convex structure, the hanging drop method, a method of producing a spheroid by culturing a large number of cells while rotating a cell culture chamber containing a medium, etc. are applicable, but preferably, the hanging drop method.

[0051] The hanging drop method is a method of spotting a droplet of a culture solution containing cells on the ceiling side of the lid of a culture dish, or producing a droplet with a special culture instrument (for example, Elplasia MPc (Kuraray Co., Ltd., Japan)), and culturing the cells in the culture solution in a droplet state by surface tension. By culturing in this way, the influence received by the cells due to contact with the culture substrate surface can be minimized.

[0052] The number of each cell (or the number of spheroids) of the spheroid containing non-fetal-derived human dermal papilla cells, human epidermal cells, and human dermal cells can be adjusted according to the purpose. For example, spheroid: human epidermal cells: human dermal cells is 10 to 10000: 0.1×10 6 ~100×10 6 : 0.1×10 6 ~100×10 6, preferably 20 to 3000: 1×10 6 ~10×10 6 : 1×10 6 ~10×10 6 , more preferably 50 to 1000: 3×10 6 ~6×10 6 : 3×10 6 ~6×10 6 They may be mixed and cultured.

[0053] In one embodiment, the above step (1) is carried out in the presence of a Wnt signal activator. Human dermal papilla cells activated by a Wnt signal activator are more promoted in hair follicle reconstruction. The time for treating human dermal papilla cells with a Wnt signal activator is 6 hours to 120 hours, preferably 12 hours to 108 hours, more preferably 24 hours to 96 hours, and still more preferably 36 hours to 84 hours.

[0054] In one embodiment, the human epidermal cells used in the method of the present invention are human epidermal cells stimulated with an extracellular matrix protein or a derivative thereof. Examples of the method for stimulating human epidermal cells include culturing in a medium supplemented with an extracellular matrix protein or a derivative thereof, and culturing on a culture substrate coated with an extracellular matrix protein or a derivative thereof. Preferably, it is a method of culturing on a culture substrate coated with an extracellular matrix protein or a derivative thereof. The coating method may follow a known method and is not particularly limited. By using human epidermal cells stimulated with an extracellular matrix protein or a derivative thereof, hair follicle reconstruction is more promoted.

[0055] In one embodiment, step (2) included in the method of the present invention is a step of culturing on a culture substrate provided with a porous membrane. As the culture substrate provided with a porous membrane, for example, a commercially available cell culture insert can be used. The average pore size of the porous membrane can be appropriately selected, for example, about 0.01 μm to about 100 μm, preferably 0.05 μm to 50 μm, more preferably 0.1 μm to 25 μm, and most preferably 1 μm to 10 μm.

[0056] In one embodiment, in step (2), a suspension obtained by mixing the spheroid, human epidermal cells, and human dermal cells is applied onto a culture substrate provided with a porous membrane and cultured for a predetermined period of time (for example, the time during which liquid components such as a culture medium are discharged, for example, 0.5 to 3 hours). Thereby, a sheet-like composition is formed on the culture substrate.

[0057] In one embodiment, by implementing the above method, a composition for reconstructing human skin tissue having hair follicles can be obtained.

[0058] 4. Method for producing a human skin tissue model animal having hair follicles The present invention provides a method for producing a human skin tissue model animal having hair follicles. In one embodiment, the human skin tissue model animal having hair follicles is produced by a method including the step of applying the above composition to a non-human mammalian animal.

[0059] In one embodiment, the step of applying the composition of the present invention to a non-human mammalian animal can be implemented, for example, by incising the skin at an arbitrary site (for example, the back) of the non-human mammalian animal to an arbitrary size and transplanting the composition to the incised site. Since the size of the incised site can be appropriately set according to the size and form (suspension form, sheet form, etc.) of the composition to be applied, it is not particularly limited. Further, the depth of the incised site is not limited because it depends on the thickness of the skin of the non-human mammalian animal to which it is applied. The composition of the present invention is applied to the site where the skin has been removed by the incision, and the composition can be transplanted by suturing it together with the skin around the incised site. After applying the composition, the applied site is protected with an arbitrary dressing material or the like to physically prevent the composition from falling off.

Examples

[0060] Hereinafter, the present invention will be described in more detail based on examples, but these do not limit the present invention in any way.

[0061] <Example 1: Preparation of Reconstituted Skin> (1) Preparation of Cells Dermal papilla cells were isolated from human hair follicles (aged 20 - 65, male) using scissors and forceps to isolate the dermal papilla region, then placed in a culture dish and cultured in MesenPRO medium (Thermo fisher) to obtain dermal papilla cells. Human fibroblasts and epidermal cells were purchased and prepared from commercially available cells from Iwai Chemical Co., Ltd.

[0062] (2) Cell Preparation After adding 3 μM of CHIR99021 (Axon Medchem) to the isolated human dermal papilla cells and culturing, spheroids were formed by the hanging drop method using Elplasia MPc (Kuraray Co., Ltd., Japan).

[0063] Human epidermal cells were cultured on a culture flask coated with iMatrix - 511 (Nippi) in Epilife medium (Thermo fisher).

[0064] Human fibroblasts were cultured in a medium containing a 3:1 mixture of DMEM medium and F12 medium, 5% FBS, 40 ng / ml bFGF (PeproTech), 20 ng / ml EGF (PeproTech), and B27 (registered trademark) supplement (Thermo Fisher Scientific K.K.).

[0065] (3) Formation of Reconstituted Skin A mixture containing 3 - 6 million human fibroblasts, 3 - 6 million human epidermal cells, and 100 - 1000 human dermal papilla spheroids was placed in a commercially available cell culture insert (Corning), incubated in a 37°C incubator for a short time until the solution was drained and only the cells remained, and the graft was prepared.

[0066] (4) Transplantation of Graft The back skin of SCID individuals (Charles River) under anesthesia was incised to a size of approximately 1 - 2 cm × approximately 1 - 2 cm, the prepared graft was applied to the incision site, and it was sutured to the surrounding skin. Thereafter, the graft site was protected with a dressing material and a bandage. Approximately 10 days later, the dressing material was removed, and approximately 2 - 3 months later, the presence or absence of reconstituted skin was confirmed, and photography was performed with a stereomicroscope equipped with a camera. As a result, hair growth was observed at the graft site of the graft (Figure 1).

[0067] <Example 2: Histological Analysis of Reconstituted Skin> The reconstituted skin was excised, fixed overnight with a 4% paraformaldehyde solution, and then a paraffin-embedded tissue block was prepared. After preparing tissue sections with a thickness of 4 μm using a microtome, various histological stains were performed. The hydrophilized tissue sections were stained with H.&E. by a general method and photographed with an AxioScan (Zeiss) (Figure 2).

[0068] In addition, in order to confirm whether the tissue containing the reconstituted hair follicles is a tissue composed of human cells, in situ hybridization (hereinafter ISH) using a human genome-specific repetitive sequence (Alu sequence) as a probe was performed by a general method, and photography was similarly performed (Figure 3).

[0069] Furthermore, double staining of immunostaining with an antibody AE13 (Abcam) that specifically recognizes the hair shaft and Alu-ISH was performed (Figure 4). The basic procedures for immunostaining and ISH were performed with reference to "Immunostaining / In Situ Hybridization in the Post-Genomic Research Era" (Yodosha).

[0070] As a result, it was confirmed that the reconstituted skin tissue containing hair follicles was derived from the transplanted human cells. It was also confirmed that hair shafts were formed.

[0071] <Example 3: Tracking Experiment of Fluorescently Labeled Dermal Papilla Spheroids> Dermal papilla cells cultured in the presence of 3 μM CHIR99021 were labeled with the red fluorescent dye PKH-26 (Sigma-Aldrich) according to the attached protocol (Figure 5). As a result of preparing reconstituted skin containing the labeled dermal papilla spheroids, red fluorescence was observed at the dermal papilla site that serves as the basis for hair follicle formation. From this, it was concluded that the hair follicles observed in the reconstituted skin were induced and formed by the dermal papilla spheroids (Figure 5).

[0072] <Example 4: Confirmation of the ability to reconstruct human skin tissue with hair follicles depending on the presence or absence of spheroidized dermal papilla cells and the presence or absence of CHIR99021 stimulation> Except for the conditions of the presence or absence of spheroidized dermal papilla cells and the presence or absence of CHIR99021 stimulation, an attempt was made to reconstruct human skin tissue with hair follicles in SCID mice according to the method described in Example 1. The results are shown below.

Table 1

Claims

1. A method for producing a composition for reconstructing a human skin tissue having a hair follicle, comprising: (1) a step of preparing a spheroid containing non-transfected human dermal papilla cells derived from an adult, which are primary cells or subcultured cells thereof; (2) a step of mixing and culturing the spheroid, human epidermal cells, and human dermal fibroblasts, wherein this step is a step of culturing on a culture substrate provided with a porous membrane; comprising: The method, wherein the step (1) is carried out in a medium supplemented with a Wnt signal activator.

2. The method according to claim 1, wherein the Wnt signal activator is a Wnt ligand family protein or a Wnt signal transduction agonist.

3. The method according to claim 2, wherein the Wnt ligand family protein is selected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, and combinations thereof.

4. The method according to claim 2, wherein the Wnt signal transduction agonist is CHIR99021 or BIO.

5. The method according to any one of claims 1 to 4, wherein the step (1) is carried out by the hanging drop method.

6. The method according to any one of claims 1 to 5, wherein the human epidermal cells are the human epidermal cells stimulated with an extracellular matrix protein or a derivative thereof.

7. The method according to claim 6, wherein the extracellular matrix protein contains laminin or a fragment thereof.

8. A step of applying the composition obtained by the method according to any one of claims 1 to 7 to a non-human mammal. A method for producing a human skin tissue model animal having a hair follicle, comprising:

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