JP2003238421A - In vivo human hair growth induction method and non- human animal bearing human hair - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of inducing human hair growth in a non-human animal by transplanting mixed cells including human cells, to provide a non-human animal having human hair grown by the method and to provide a screening method using the animal. <P>SOLUTION: In the skin of a non-human animal, hair growth is induced from the hair follicle structure including human-originating cells. Dermal papilla cells, epidermal cells and dermal fibroblasts (at least one of them originates from a human) are mixed and the mixture is transplanted to the skin of a non-human animal whereby the growth of human hair is in vivo induced on the skin. A non-human animal that has human hair grown on its skin. In addition, screening process for hair growth factor and the like including the process that the factors to be tested are brought into contact with the non-human animals and the evaluation method for the activity of the factors. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The invention of the present application relates to a method for inducing hair growth from a non-human animal on the skin of a non-human animal, a non-human animal having human hair grown by this hair growth induction method, and The present invention relates to the invention of utilizing non-human animals.

[0002]

2. Description of the Related Art For hair such as hair, body hair and beard, hair shafts differentiated by hair mother cells in hair follicles grow from the pores of the skin to perform heat retention, sensory organs and cosmetic functions. A schematic view of a hair follicle is shown in FIG. 1, and a development process of the hair follicle is shown in FIG. As shown in Fig. 2, papilla (hair papilla) has a function of inducing hair growth by inducing hair follicles from the epidermis, and determines the properties and hair cycle of hair (Reynolds AJ. Et al, Development, 115: 587. -93, 1
992). In addition, it has been reported that even transplanted papilla cells can induce hair follicle structure (Inamatsu et al. J.
Invest. Dermatol., 111 (5): 767-75, 1998). However, no apparent hair growth (formation of hair shaft and extension of hair shaft from pores) has been confirmed from the hair follicle structure induced by the transplanted papilla cells. That is, as a method for inducing hair follicles by transplanting cultured papilla cells, pellets obtained by aggregating papilla cells by centrifugation, or artificial papilla produced by adding a biodegradable adhesive such as fibrin to the pellets in the skin of experimental animals. The method of direct transplantation to the plant has been reported (Inamatsu et al. J. Invest. Dermatol., 1
11 (5): 767-75, 1998). However, in the transplantation of these human papilla cells, only the construction of the hair follicle structure is observed, and hair shaft formation has not been achieved. Furthermore, the formed hair follicles are often found to have insufficient histological structure.

[0003]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention As mentioned above, clear and stable in vivo hair growth induction by papilla cell skin transplantation has not been successful. For this reason, it has been completely impossible to produce industrially applicable human body hair or non-human animals that grow human hair, such as the development of hair restorers. If an animal that grows human body hair or hair is produced, it can be used industrially such as the development of a hair-growing agent by screening for a drug related to hair growth control.

An object of the invention of this application is to solve the problems of the prior art and to provide a method for inducing human hair growth in non-human animal species by transplanting mixed cells containing human cells.

The invention of the present application also provides a method for screening a non-human animal in which human hair has been grown by inducing hair growth by the above method, and a factor which acts on hair growth such as a hair growth agent using this animal. The challenge is to provide.

[0006]

This application is, as a first invention for solving the above-mentioned problems, a hair follicle structure reconstructed by a cell population containing human-derived cells transplanted in the skin of a non-human animal. A method of inducing hair growth from
In vivo development of human hair, characterized by mixing papilla cells, epidermal cells and skin fibroblasts (at least one of which is a human-derived cell) and transplanting it into the skin of a non-human animal. A hair guidance method is provided.

In the method of the first aspect of the present invention, it is preferable that the papilla cells are of human origin and that the human-derived papilla cells are papilla cells isolated from human scalp.

Furthermore, in the method of the first aspect of the present invention, the non-human animal species is preferably an immunodeficient animal.

This application, as a second invention, relates to a non-human animal in which a mixture of papilla cells, epidermal cells and skin fibroblasts (at least one of which is of human origin) is transplanted into the skin. provide.

This application is the first invention as the third invention.
A non-human animal having hair growth induced by the method of the invention,
Provided is a non-human animal having hair grown from a hair follicle structure containing human-derived cells.

In the non-human animal of the third invention, one of the modes is that the hair is human hair.

Further, the present invention provides a fourth invention, wherein a physiologically active substance which induces androgenetic alopecia is administered to the non-human animal (human hair-grown animal) which is an aspect of the third invention. The present invention provides a model animal for androgenetic alopecia in which human hair is removed.

The present invention also provides, as a fifth invention, a method for screening a factor acting on hair growth, wherein the test factor is a non-human animal of the second invention or the third invention or a model animal of the fourth invention. The present invention provides a screening method, characterized in that a factor affecting the hair growth state of human body hair or human hair, or papilla or hair follicle structure is identified as a hair-growth-acting factor.

In the fifth aspect of the present invention, the hair growth factor is preferably a hair growth inhibitor, hair loss factor or hair growth promoter.

The present invention also provides, as a sixth invention, a method for evaluating the activity of a hair growth factor, comprising the non-human animal of the second invention or the third invention, or the model animal of the fourth invention. A method of evaluation is provided, which comprises contacting a hair growth factor with a hair growth factor and measuring the extent of the effect of the factor on the hair growth state of human hair or human hair, or the papilla or hair follicle structure.

In a preferred embodiment of the evaluation method of the sixth invention, the hair growth-acting factor is a hair growth inhibitor, hair loss factor or hair growth promoter known or identified by the method of the fifth invention. There is.

Embodiments of each invention of this application will be described in detail below.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION In the method of inducing hair growth of the first invention, papilla cells, epidermal cells and dermal fibroblasts are mixed to form a non-human animal (hereinafter sometimes referred to as "recipient animal"). Transplant to the skin. Then, at least one of papilla cells, epidermal cells, and skin fibroblasts is a human-derived cell, and mixed cells containing the human cells reassociate in the skin of the recipient animal to construct a hair follicle structure. , Capillaries are formed from these hair follicles, and human hair grows. In the present invention, “human hair” means hair that has grown from a hair follicle structure containing papilla cells, epidermal cells, and / or dermal fibroblasts, all of which are of human origin. In particular, hair that grows from the hair follicle structure induced to differentiate by human-derived papilla cells is the same as human hair in properties that are determined by papilla, such as developmental processes and hair cycle control, and papilla derived from human scalp. Hair grown from a cell-containing hair follicle structure is substantially the same as human hair.

In the method of the first invention, human-derived cells are used as at least one of papilla cells, epidermal cells and dermal fibroblasts, and it is particularly preferred that papilla cells are human-derived. Furthermore, it is preferable that the human-derived papilla cell is a papilla cell isolated from a human scalp.
In that case, the epidermal cells and / or dermal fibroblasts may be cells derived from a non-human animal (hereinafter sometimes referred to as “donor animal”). Further, the recipient animal and the donor animal may be the same species. Furthermore, the donor animal can be appropriately selected from a neonate or an adult. As the recipient animal and the type of each cell, for example, as shown in Examples, the recipient animal is a mouse (nude mouse), human scalp-derived papilla cells,
A combination of neonatal rat-derived epidermal cells and living rat-derived skin fibroblasts can be exemplified, but it is not limited to this combination. Also, cells grown by primary culture or subculture may be used, but a preferred embodiment is cells that have not undergone malignant transformation such as tumorigenicity.

The papilla cells are primary cells isolated from papilla isolated under sterile conditions from the skin (preferably scalp) of a donor animal (preferably human) under a stereomicroscope, or described in JP-A-7-274950. Cultured papilla cells that have been subcultured by the method described above can be used.

As the epidermal cells and fibroblasts, primary cells isolated by known methods from epidermis and subcutaneous tissues of donor animals including humans, or subcultured cells can be used.

The ratio of epidermal cells, papilla cells, and fibroblasts to be transplanted into the recipient animal is 10: 1, respectively.
It can be set to ~ 10: 9 ~ 0. Further, in order to transplant these mixed cells into the skin, a method in which a recipient animal is equipped with a cell transplant container (graft chamber) and the mixed cells are transplanted into the skin via this container can be adopted. For injection of cells into the transplant container, the mixed cell liquid may be injected using a syringe such as a syringe or a micropipette. The liquid carrier (serum, medium, etc.) may be removed from the mixed cells as much as possible, and about 5 to 100 μl of the mixed cells may be injected.

Epidermal cells transplanted by the above method,
A non-human animal (second invention) having a hair follicle structure composed of a mixture of papilla cells and fibroblasts on the skin is produced. It is also possible to construct a hair follicle structure containing human cells on the skin of a recipient animal and induce hair growth from this hair follicle structure. Then, a non-human animal (third invention) having hairs grown can be produced from the hair follicle structure. In addition, as one aspect of the third invention, a non-human animal having human hair is produced from a hair follicle structure containing papilla cells of human scalp. Furthermore, by administering a physiologically active substance that induces androgenetic alopecia such as dihydrotestosterone or testosterone to the non-human animal that has caused this human hair to grow, androgenetic alopecia model animal in which human hair is alopecia ( Fourth invention) can be created.

The fifth invention of this application is a method for screening a hair growth factor using the non-human animal of the second or third invention and / or the model animal of the fourth invention. The hair-growth-acting factors are, specifically, hair-growth suppressors, hair loss factors, and hair-growth promoting factors. The test factor is, for example, a compound or a natural substance which can be an active ingredient of a hair growth inhibitor, a hair remover, or a hair growth / hair growth agent. These test factors, mixed with a suitable carrier, are systemically administered to a non-human animal or a model animal, or contacted with a skin area where hair growth is induced. A novel factor that acts on hair growth by morphologically or molecularly analyzing the hair growth state and its process, or the hair follicle structure constructed from transplanted cells or the state of papilla in the hair follicle structure. Can be identified. In addition, the test factor has an effect similar to that of hormones or cytokines known to act on hair growth in vivo, or an effect equivalent to that of ultraviolet rays, ionizing radiation, acid rain, etc. It may be an environmental factor exerted. It may also be stress due to the living environment, food, etc.

The sixth invention of this application is a method for evaluating the activity of a hair growth factor using the non-human animal of the second or third invention and / or the model animal of the fourth invention. is there. The test factor may be a known compound or the like used as an active ingredient of a depilatory or a hair regrowth agent, or a known hormone, cytokine, or various known to act on hair growth. Environmental factors, etc. Furthermore, the novel factor identified in the fifth invention can also be targeted. These test factors are brought into contact with the above-mentioned non-human animal or model animal in a state suitable for each, and the state of hair growth or its process, or the state of the hair follicle structure constructed from transplanted cells or the state of papilla in the hair follicle structure The degree of activity of each factor on hair growth can be evaluated by morphologically or molecularly analyzing the above to determine the degree of action.

By the screening method and the activity evaluation method as described above, it is possible to develop new agents such as a depilatory agent and a hair regrowth agent, or a new treatment method for hair regrowth and a depilatory treatment method.

The invention of this application, which shows examples, will be described in more detail and specifically below, but the invention of this application is not limited to the following examples.

[0028]

[Example] 1. Method (A) Isolated culture of papilla from scalp tissue Scalp tissue resected from the subject's head was Isodine solution, 70%
It was disinfected by rucor. 10% test if stored
Dulbecco's modified eag containing human serum or fetal calf serum
Medium (DMEM10) was used. Papilla is carefully dermal hair
Aseptically separate from root sheath and hairy mother, 60 mm culture dish
(Pecton Dickinson)
Medium exchange once every 5 days under back epidermal cell culture supernatant (CM5) conditions
The primary culture was performed for 4 weeks. (B) Preparation of rat epidermal cells From fetuses after 20 days of birth or newborns of 0 to 3 days old
Phosphate extracted skin tissue or adult rat skin tissue
Wash twice with buffered saline (PBS), then 1000 Units / ml
Immerse in DMEM10 containing Dispase (Sankyo Pure Chemical Industries)
Treated overnight at 4 ° C. Treat the skin after dispase treatment with PBS
After washing twice more, it was separated into an epidermis layer and a dermis layer. table
After slicing the cortex with a sharp knife, 0.25% trypsin 1 mM ethi
Enzymatic treatment with diamine tetraacetic acid (EDTA) at 37 ℃ for 10 minutes
I went. After digestion with trypsin, add enough enzyme treatment solution to DMEM.
Nylon filter diluted with 10 and further 100 μm and 40 μm
-Filtered and not digested by trypsin
The layers were removed. (C) Preparation of fibroblasts from adult rat sole dermis Adult rat (Fisher breed, 6-week-old female)
Collect the skin of the sole completely sterilized with 70% ethanol.
Subcutaneous tissue was removed. 1000 Units / ml for epidermis and dermis
Immerse in DMEM10 containing dispase and treat at 4 ° C overnight
separated. This dermis layer is cut into 0.5 mm square pieces, and the 60 mm culture
To the primary culture of fibroblasts
went. After primary culture, this adult rat sole dermis-derived fiber
The blast cells (RSSF) are subcultured and the 4th to 8th passages
The ones up to were used. (D) Human papilla cells, rat neonatal epidermal cells, and R
Mixed port of SSF 4-6 week old male nude mouse (Charles River Japan)
Was anesthetized by intraperitoneal administration of Nemptal
After sterilization with J. Pharmaceuticals, the skin of the flank is circular with a diameter of 7 mm.
All layers were removed. At this site, graft chamber
(J. Invest. Dermatol. Vol. 100, 229-236, 1993)
Attached with 5-0 nylon suture. Fluorescence in advance
Human scalp papilla cells labeled with dye (DiI) 8 × 10⁶ 
1 x 10 cells ⁷cells Rat neonatal epidermal cells, 2 x 10⁶ce
Nude after mixing with lls RSSF and removing enough water
Insert the micropipette into the graft chamber attached to the mouse.
Injection. All the above operations are performed in a sterile environment.
It was. One week after transplantation, use surgical tape (Nichiban)
Protected the transplant site. 1 week after cell transplantation
Remove the chamber and disinfect the transplant site with Isodine.
The animals were bred for 2 weeks, paying attention to infectious diseases. (E) Hair growth observation by cell transplantation Three weeks after the operation, the transplant site was examined by a stereomicroscope (Leica).
Manufactured) and photographed the appearance of hair growth. Same again
Remove the part, fix it with Mild Holm 20N for a day and night, and
It was embedded in a ruffin. This sample was sliced into 5 μm slices and stained with HE.
Fluorescent color labeled on papilla by fluorescent staining of color and nucleus
I confirmed the element DiI. 2. Results Contains primary cultured papilla cells prepared from scalp tissue of all 2 cases.
The mixed cells were transplanted into 5 nude mice. That conclusion
As a result, macroscopic hair growth was observed in 3 cases, but 2 cases were at the transplant site.
The position was dropped due to an infectious disease. Of hair growth cases of these 3 individuals
When the tissue was observed as a tissue section, hair
A hair follicle structure having a trunk was observed. Also, the fluorescence of this site
As a result of the observation, red florescence due to the fluorescent dye DiI was found on the papillae.
It was recognized that it was a phenomenon of hair growth induction by human cultured papilla cells.
It was confirmed that

[0029]

Industrial Applicability As described above in detail, the hair reconstructing method containing human papilla cells according to the invention of this application can induce hair with human papilla cells at high frequency, easily and inexpensively. In addition, the reactivity of hair induced by human papilla cells to a drug can be used for the development of a hair-growth drug, a drug for androgenetic alopecia and the like. Furthermore, by introducing or deleting a specific gene into transplanted cells, it can be used for elucidation of the mechanism that controls hair growth and hair cycle. When applied to humans, treatment of alopecia is also possible.

[Brief description of drawings]

FIG. 1 is a schematic view of a hair follicle.

FIG. 2 is a schematic diagram showing a hair follicle development process (stages A to D). Each stage is A: Hair germ, B: Hair peg, C: Bulbous hair peg, D: Hair follicle.

FIG. 3 is a microscopic image showing the appearance of hair-growth sites induced by cultured human papilla cells and the result of tissue observation of hair follicle structure. A: Appearance of transplant site 3 weeks after transplanting cultured human papilla cells. Skin where the central circular area is reconstituted by the transplanted cells. B: Hair growth. White hair is observed in the area indicated by the arrow. C: HE-stained image of tissue section of hair growth site. D: Nuclear staining image of the same site in serial sections. E: DiI fluorescence observation of the same site in serial sections. Fluorescence was observed at the papilla site indicated by the arrow. The strong fluorescence at the top of the photo is the autofluorescence of the hair shaft. Cells with DiI fluorescence are also found around the hair follicles.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Koei Toyoshima             Hiroshima Prefecture Higashi-Hiroshima City Saijo Chuo 6-26-26-             403 (72) Inventor Matsunaga Mikaru             4776-1-206 Takasu-cho, Onomichi City, Hiroshima Prefecture (72) Inventor Yoshiri Victory             7-22-13 Hachimotomatsunami, Higashihiroshima City, Hiroshima Prefecture F-term (reference) 4C087 AA01 AA02 BB48 MA63 NA14                       ZA92

Claims (12)

[Claims] 1. A method for inducing hair growth from a hair follicle structure containing human-derived cells in the skin of a non-human animal, which comprises papilla cells, epidermal cells and skin fibroblasts (at least one of which is Human hair in v) characterized by being mixed with human-derived cells) and transplanted to the skin of a non-human animal
ivo Hair growth induction method. 2. The method for inducing hair growth according to claim 1, wherein the papilla cells are of human origin. 3. The method for inducing hair growth according to claim 2, wherein the papilla cells are papilla cells isolated from human scalp. 4. The method for inducing hair growth according to claim 1, wherein the non-human animal is an immunodeficient animal. 5. A non-human animal in which a mixture of papilla cells, epidermal cells, and skin fibroblasts (at least one of which is of human origin) is transplanted to the skin. 6. A non-human animal in which hair growth is induced by the method according to any one of claims 1 to 4, wherein the hair is grown from a hair follicle structure containing human-derived cells. 7. The non-human animal according to claim 6, wherein the hair is human hair. 8. A male pattern baldness model animal in which a human hair loss or papilla is denatured by administering a physiologically active substance that induces male pattern baldness to the non-human animal of claim 7. 9. A method for screening a factor that acts on hair growth, which comprises contacting the non-human animal according to any one of claims 5 to 7 or the model animal according to claim 8 with a test factor, and then producing body hair or human hair. A method for screening, characterized in that a factor affecting the hair growth state or papilla or hair follicle structure is identified as a hair growth factor. 10. The screening method according to claim 9, wherein the hair growth factor is a hair growth suppressor, a hair loss factor, or a hair growth promoter. 11. A method for evaluating the activity of a hair growth factor, which comprises contacting the non-human animal according to any one of claims 5 to 7 or the model animal according to claim 8 with the hair growth factor.
An evaluation method characterized by measuring the degree of influence of the factor on the hair growth state of human body hair or human hair, or papilla or hair follicle structure. 12. A hair growth factor is known or claimed in claim 9.
The evaluation method according to claim 11, which is a hair growth inhibitor, hair loss factor, or hair growth promoter identified by the method of.