JP2019199434A - Method for controlling color of regenerated hair, method for regenerating hair, and method for producing hair follicle primordium - Google Patents
Method for controlling color of regenerated hair, method for regenerating hair, and method for producing hair follicle primordium Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/14—Drugs for dermatological disorders for baldness or alopecia
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Abstract
Description
本発明は、再生毛の色制御方法、毛の再生方法及び毛包原基の製造方法に関する。 The present invention relates to a method for controlling the color of regenerated hair, a method for regenerating hair, and a method for producing a hair follicle primordium.
特許文献１には、成体マウスの毛包の毛乳頭から培養毛乳頭細胞を得たこと、当該毛包のバルジ領域からバルジ領域上皮細胞を得たこと、当該毛包のサブバルジ領域からサブバルジ領域細胞を得たこと、及び当該毛包の毛母基底部から毛母基底部細胞を得たことが記載されている。また、特許文献１には、バルジ領域上皮細胞と、サブバルジ領域細胞又は毛母基底部細胞とを混合して遠心分離することにより、当該バルジ領域上皮細胞と、当該サブバルジ領域細胞又は当該毛母基底部細胞とを含む細胞凝集塊を作製するとともに、培養毛乳頭細胞を遠心分離することにより、当該培養毛乳頭細胞の細胞凝集塊を作製したこと、次いで、コラーゲンゲルドロップ内で、当該バルジ領域上皮細胞と、当該サブバルジ領域細胞又は当該毛母基底部細胞とを含む細胞凝集塊の上に、当該培養毛乳頭細胞の細胞凝集塊を密着させたこと、さらに、当該バルジ領域上皮細胞の細胞凝集塊にガイドとしてのナイロン糸を挿入したこと、その後、当該ゲルドロップを固化させて、再生毛包原基を作製したことが記載されている。さらに、特許文献１には、再生毛包原基を、ナイロン糸製ガイドが体表面に露出するようにヌードマウスの皮内に移植した結果、黒色毛の再生毛が得られたことが記載されている。 Patent Document 1 discloses that cultured hair papilla cells were obtained from hair follicles of adult mouse hair follicles, bulge region epithelial cells were obtained from bulge regions of the hair follicles, and subbulge region cells from subbulge regions of the hair follicles. And that the base of the hair matrix was obtained from the base of the hair matrix of the hair follicle. Patent Document 1 discloses that bulge region epithelial cells and sub-bulge region cells or hair matrix basal cells are mixed and centrifuged to obtain the bulge region epithelial cells, sub-bulge region cells or hair matrix base. Cell aggregates containing the somatic cells and centrifuging the cultured hair papilla cells to produce cell aggregates of the cultured hair papilla cells, and then in the collagen gel drop, the bulge region epithelium A cell aggregate of the cultured hair papilla cell is adhered to a cell aggregate including the cell and the subbulge region cell or the hair matrix base cell, and further, the cell aggregate of the bulge region epithelial cell It is described that a nylon thread as a guide was inserted into the gel, and then the gel drop was solidified to produce a regenerated hair follicle primordium. Furthermore, Patent Document 1 describes that as a result of transplanting the regenerated hair follicle primordium into the skin of a nude mouse so that the nylon thread guide is exposed on the body surface, regenerated hair of black hair was obtained. ing.
特許文献２には、規則的な配置の微小凹部からなるマイクロ凹版に、マウス胎児から採取された上皮系細胞及び間葉系細胞の細胞混合懸濁液を加えて、酸素を供給しながら混合培養することにより、再生毛包原基を作製したことが記載されている。また、特許文献２には、再生毛包原基をヌードマウスの皮下に注入した結果、発毛が観察されたことが記載されている。 In Patent Document 2, a mixed suspension of epithelial cells and mesenchymal cells collected from a mouse fetus is added to a micro intaglio plate composed of regularly arranged micro concave portions, and mixed culture is performed while supplying oxygen. It is described that a regenerated hair follicle primordium was produced by doing so. Further, Patent Document 2 describes that hair growth was observed as a result of injecting a regenerated hair follicle primordium into the skin of nude mice.
しかしながら、従来、成体の毛包を細胞源として使用して、再生毛包原基から生える毛の色を制御することは容易ではなかった。 However, conventionally, it has not been easy to control the color of hair grown from a regenerated hair follicle primordium using an adult hair follicle as a cell source.
本発明は、上記課題に鑑みて為されたものであり、毛の色を簡便且つ効果的に制御できる、再生毛の色制御方法、毛の再生方法及び毛包原基の製造方法を提供することをその目的の一つとする。 The present invention has been made in view of the above problems, and provides a method for controlling the color of regenerated hair, a method for regenerating hair, and a method for producing a hair follicle primordium that can easily and effectively control the color of the hair. This is one of its purposes.
上記課題を解決するための本発明の一実施形態に係る再生毛の色制御方法は、恒常部のバルジ領域を含む成体毛包組織から得られた、上皮幹細胞を含む原料細胞を使用すること、分散された毛乳頭細胞と、分散された前記原料細胞とを混合して培養することにより、毛包原基を製造すること、及び前記毛包原基を動物に移植して前記毛包原基から毛を生やすこと、を含み、前記成体毛包組織に含まれる非恒常部の重量割合を変えることにより、前記毛包原基から生える毛の色を変える。本発明によれば、毛の色を簡便且つ効果的に制御できる再生毛の色制御方法が提供される。 The method for controlling the color of regenerated hair according to an embodiment of the present invention for solving the above-described problem uses a raw material cell containing epithelial stem cells obtained from an adult hair follicle tissue containing a bulge region of a constant part, A hair follicle primordium is produced by mixing and culturing dispersed hair papilla cells and the dispersed raw material cells, and transplanting the hair follicle primordia to an animal to produce the hair follicle primordium. And changing the weight ratio of the non-constant part contained in the adult hair follicle tissue to change the color of the hair growing from the hair follicle primordium. ADVANTAGE OF THE INVENTION According to this invention, the color control method of the reproduction | regeneration hair which can control the color of hair easily and effectively is provided.
前記方法において、前記動物は、非ヒト動物であることとしてもよい。また、前記方法においては、前記成体毛包組織に含まれる前記非恒常部の重量割合を増加させることにより、前記毛包原基から生える毛の色を濃くすることとしてもよい。また、前記方法において、前記非恒常部は、毛乳頭及び毛母以外の部分を含むこととしてもよい。 In the method, the animal may be a non-human animal. Moreover, in the said method, it is good also as darkening the color of the hair which grows from the said hair follicle primordium by increasing the weight ratio of the said non-constant part contained in the said adult hair follicle structure | tissue. Moreover, in the said method, the said non-constant part is good also as including parts other than a hair nipple and a hair mother.
上記課題を解決するための本発明の一実施形態に係る毛の再生方法は、恒常部のバルジ領域を含む成体毛包組織から得られた、上皮幹細胞を含む原料細胞を使用すること、分散された毛乳頭細胞と、分散された前記原料細胞とを混合して培養することにより、毛包原基を製造すること、及び前記毛包原基を動物に移植して前記毛包原基から毛を生やすこと、を含み、非恒常部をさらに含む前記成体毛包組織を使用することにより、前記毛包原基から、前記非恒常部を含まない成体毛包組織を使用する場合に比べて色が濃い毛を生やす。本発明によれば、毛の色を簡便且つ効果的に制御できる毛の再生方法が提供される。 The method for regenerating hair according to an embodiment of the present invention for solving the above-mentioned problem is achieved by using raw material cells containing epithelial stem cells obtained from an adult hair follicle tissue containing a bulge region of a constant part. The follicular primordial cells and the dispersed raw material cells are mixed and cultured to produce a hair follicle primordial, and the hair follicle primordial is transplanted into an animal to produce hair from the hair follicle primordial By using the adult hair follicle tissue further comprising a non-constant part, so that the color of the hair follicle primordium compared to the case of using the adult hair follicle tissue not containing the non-constant part is used. Grows dark hair. ADVANTAGE OF THE INVENTION According to this invention, the hair regeneration method which can control the color of hair easily and effectively is provided.
前記方法において、前記動物は、非ヒト動物であることとしてもよい。また、前記方法は、前記非恒常部を第一の重量割合で含む第一の成体毛包組織を使用して製造された第一の毛包原基を前記動物に移植して、前記第一の毛包原基から第一の色の毛を生やすこと、及び前記非恒常部を前記第一の重量割合より大きい第二の重量割合で含む第二の成体毛包組織を使用して製造された第二の細胞凝集塊を前記動物に移植して、前記第二の毛包原基から前記第一の色より濃い第二の色の毛を生やすこと、を含むこととしてもよい。また、前記方法において、前記非恒常部は、毛乳頭及び毛母以外の部分を含むこととしてもよい。 In the method, the animal may be a non-human animal. In the method, the first hair follicle primordium produced using the first adult hair follicle tissue containing the non-constant part at a first weight ratio is transplanted into the animal, Produced from a hair follicle primordium and using a second adult hair follicle tissue comprising the non-constant part in a second weight percentage greater than the first weight percentage. Transplanting the second cell aggregate to the animal to produce a second color hair darker than the first color from the second hair follicle primordium. Moreover, in the said method, the said non-constant part is good also as including parts other than a hair nipple and a hair mother.
上記課題を解決するための本発明の一実施形態に係る毛包原基の製造方法は、恒常部のバルジ領域を含む成体毛包組織から得られた、上皮幹細胞を含む原料細胞を使用すること、及び分散された毛乳頭細胞と、分散された前記原料細胞とを混合して培養することにより、動物に移植されることで毛を生やす毛包原基を製造すること、を含み、非恒常部をさらに含む前記成体毛包組織を使用することにより、前記非恒常部を含まない成体毛包組織を使用する場合に比べて色が濃い毛を生やす前記毛包原基を製造する。本発明によれば、毛の色を簡便且つ効果的に制御できる毛包原基の製造方法が提供される。 The method for producing a hair follicle primordium according to an embodiment of the present invention for solving the above-described problem uses a raw material cell containing epithelial stem cells obtained from an adult hair follicle tissue containing a bulge region of a constant part. And producing a hair follicle primordium that grows hair by being transplanted into an animal by mixing and culturing the dispersed hair papilla cells and the dispersed raw material cells, and is non-constant By using the adult hair follicle tissue further comprising a part, the hair follicle primordium that produces darker hair than the case of using the adult hair follicle tissue not containing the non-constant part is produced. ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of the hair follicle primordium which can control the color of hair easily and effectively is provided.
前記方法は、前記非恒常部を第一の重量割合で含む前記成体毛包組織を使用することにより、前記動物に移植されることで第一の色の毛を生やす第一の毛包原基を製造すること、及び前記非恒常部を前記第一の重量割合より大きい第二の重量割合で含む前記成体毛包組織を使用することにより、前記動物に移植されることで前記第一の色より濃い第二の色の毛を生やす第二の毛包原基を製造すること、を含むこととしてもよい。また、前記方法において、前記非恒常部は、毛乳頭及び毛母以外の部分を含むこととしてもよい。 The method uses a first hair follicle primordium that produces hair of a first color by being transplanted into the animal by using the adult hair follicle tissue comprising the non-constant part in a first weight ratio. And the first color by being implanted into the animal by using the adult hair follicle tissue comprising the non-constant part in a second weight percentage greater than the first weight percentage. Producing a second hair follicle primordium that produces darker second colored hair. Moreover, in the said method, the said non-constant part is good also as including parts other than a hair nipple and a hair mother.
本発明によれば、毛の色を簡便且つ効果的に制御できる、再生毛の色制御方法、毛の再生方法及び毛包原基の製造方法が提供される。 ADVANTAGE OF THE INVENTION According to this invention, the color control method of the reproduction | regeneration hair which can control the color of hair easily and effectively, the reproduction | regeneration method of hair, and the manufacturing method of the hair follicle primordium are provided.
以下に、本発明の一実施形態に係る方法（以下、「本方法」という。）について説明する。なお、本発明は本実施形態に限られるものではない。 Hereinafter, a method according to an embodiment of the present invention (hereinafter referred to as “the present method”) will be described. Note that the present invention is not limited to the present embodiment.
本方法の一側面は、恒常部のバルジ領域を含む成体毛包組織から得られた、上皮幹細胞を含む原料細胞を使用すること、分散された毛乳頭細胞と、分散された当該原料細胞とを混合して培養することにより、毛包原基を製造すること、及び当該毛包原基を動物に移植して当該毛包原基から毛を生やすこと、を含み、当該成体毛包組織に含まれる非恒常部の重量割合を変えることにより、当該毛包原基から生える毛の色を変える、再生毛の色制御方法を含む。 One aspect of this method is to use a raw material cell containing epithelial stem cells obtained from an adult hair follicle tissue containing a bulge region of a homeostatic part, a dispersed hair papilla cell, and the dispersed raw material cell. Including in the adult hair follicle tissue, including producing a hair follicle primordium by mixing and culturing, and transplanting the hair follicle primordia to an animal and growing hair from the follicle primordium A method for controlling the color of regenerated hair, which changes the color of the hair grown from the hair follicle primordium by changing the weight ratio of the non-constant part.
また、本方法の他の側面は、恒常部のバルジ領域を含む成体毛包組織から得られた、上皮幹細胞を含む原料細胞を使用すること、分散された毛乳頭細胞と、分散された当該原料細胞とを混合して培養することにより、毛包原基を製造すること、及び当該毛包原基を動物に移植して当該毛包原基から毛を生やすこと、を含み、非恒常部をさらに含む当該成体毛包組織を使用することにより、当該毛包原基から、当該非恒常部を含まない成体毛包組織を使用する場合に比べて色が濃い毛を生やす、毛の再生方法を含む。 Another aspect of the present method is to use raw material cells containing epithelial stem cells obtained from an adult hair follicle tissue containing a bulge region of a constant part, dispersed hair papilla cells, and the dispersed raw material. Producing a hair follicle primordium by mixing and culturing cells, and transplanting the hair follicle primordia to an animal and growing hair from the hair follicle primordium, Furthermore, by using the adult hair follicle tissue, a hair regeneration method that produces dark hair from the hair follicle primordia as compared to the case of using the adult hair follicle tissue that does not include the non-constant part. Including.
また、本方法のさらに他の側面は、恒常部のバルジ領域を含む成体毛包組織から得られた、上皮幹細胞を含む原料細胞を使用すること、及び分散された毛乳頭細胞と、分散された当該原料細胞とを混合して培養することにより、動物に移植されることで毛を生やす毛包原基を製造すること、を含み、非恒常部をさらに含む当該成体毛包組織を使用することにより、当該非恒常部を含まない成体毛包組織を使用する場合に比べて色が濃い毛を生やす当該毛包原基を製造する、毛包原基の製造方法を含む。 Still another aspect of the method is the use of raw material cells containing epithelial stem cells obtained from adult hair follicle tissue containing a bulge region of the homeostatic region, and dispersed hair papilla cells and dispersed Using the adult hair follicle tissue further comprising a non-constant part, including producing a hair follicle primordium that grows hair by being transplanted to an animal by mixing and culturing the raw material cells The manufacturing method of the hair follicle primordium which manufactures the said hair follicle primordium which grows a hair darker than the case where the adult hair follicle structure | tissue which does not contain the said non-constant part is used is included.
すなわち、本発明の発明者らは、成体毛包組織から得られた細胞を使用して生体外で再構築した毛包原基から生える毛の色を制御する技術的手段について鋭意検討を重ねた結果、意外にも、細胞源として非恒常部を含む成体毛包組織を使用することにより、当該毛包原基から生える毛の色を簡便且つ効果的に制御できることを独自に見出し、本発明を完成するに至った。 That is, the inventors of the present invention have made extensive studies on technical means for controlling the color of hair grown from a hair follicle primordium reconstructed in vitro using cells obtained from adult hair follicle tissue. As a result, surprisingly, by using an adult hair follicle tissue containing a non-constant part as a cell source, it has been found uniquely that the color of hair growing from the hair follicle primordium can be easily and effectively controlled. It came to be completed.
ここで、成体毛包組織の構造について説明する。図１Ａには、成体毛包の構造を模式的に示す。また、図１Ｂには、成体マウスから採取された毛包の構造を示す。図１Ａ及び図１Ｂに示すように、毛幹を包む成体毛包組織は、恒常部と、非恒常部とを含む。 Here, the structure of the adult hair follicle tissue will be described. FIG. 1A schematically shows the structure of an adult hair follicle. FIG. 1B shows the structure of a hair follicle collected from an adult mouse. As shown in FIGS. 1A and 1B, the adult hair follicle tissue that wraps the hair shaft includes a constant part and a non-constant part.
恒常部は、定常部又は不変部とも呼ばれる。恒常部は、成体毛包組織の上側（すなわち、毛幹の先端側）の略半分を占める。恒常部には皮脂腺がある。恒常部は、その皮脂腺より下側（すなわち、毛幹の先端と反対側）に、立毛筋が接着しているバルジ領域を含む。なお、図１Ｂに示すように、マウスにおいては、立毛筋に相当する位置に、リングブルスト(Ｒｉｎｇｗｕｒｓｔ）が存在する。バルジ領域には上皮幹細胞が含まれる。 The constant part is also called a stationary part or an invariant part. The constant part occupies approximately half of the upper side of the adult hair follicle tissue (that is, the tip side of the hair shaft). There is a sebaceous gland in the constant part. The homeostatic part includes a bulge region to which the napped muscles are bonded below the sebaceous glands (that is, opposite to the tip of the hair shaft). As shown in FIG. 1B, in the mouse, there is a ring burst at a position corresponding to the napped muscle. The bulge region contains epithelial stem cells.
このため、恒常部のバルジ領域を含む成体毛包組織から得られた原料細胞は、当該バルジ領域から得られた上皮幹細胞を含む。上皮幹細胞は、例えば、成体毛包組織（特に、バルジ領域）から得られた、ＣＤ３４を発現する細胞として特定される。恒常部はさらに、バルジ領域の下側にサブバルジ領域を含む。サブバルジ領域は、恒常部の最下部を構成する。 For this reason, the raw material cell obtained from the adult hair follicle tissue containing the bulge region of the constant part contains the epithelial stem cell obtained from the bulge region. Epithelial stem cells are identified as cells expressing CD34 obtained from, for example, adult hair follicle tissue (particularly the bulge region). The constant part further includes a sub-bulge region below the bulge region. The sub-bulge area constitutes the lowermost part of the constant part.
非恒常部は、非定常部又は可変部とも呼ばれる。非恒常部は、成体毛包組織の下側の略半分を占める。すなわち、非恒常部は、例えば、成体毛包組織のうち、恒常部のサブバルジ領域より下側の部分である。非恒常部は、毛球部を含む。毛球部は、毛乳頭と毛母とを含む。毛乳頭には毛乳頭細胞が含まれる。毛母には毛母細胞及び色素細胞が含まれる。非恒常部は、毛球部以外の部分（すなわち、恒常部と、毛球部との間の部分）も含む。なお、原料細胞の細胞源としては、毛周期の成長期にある成体毛包組織を使用することが好ましい。 The non-constant part is also called an unsteady part or a variable part. The non-constant part occupies the lower half of the adult hair follicle tissue. That is, the non-constant part is, for example, a part of the adult hair follicle tissue that is below the sub-bulge region of the constant part. The non-constant part includes a hair bulb part. The hair bulb portion includes a hair papilla and a hair mother. The dermal papilla contains dermal papilla cells. The hair matrix includes hair matrix cells and pigment cells. The non-constant part includes a part other than the hair bulb part (that is, a part between the constant part and the hair bulb part). In addition, it is preferable to use the adult hair follicle tissue in the growth phase of the hair cycle as the cell source of the source cells.
本方法において特徴的なことの一つは、上皮幹細胞を含む原料細胞の細胞源として、非恒常部を含む成体毛包組織を利用する点である。成体毛包組織に含まれる非恒常部は、当該成体毛包組織の非恒常部の一部又は全部であれば特に限られないが、毛乳頭以外の部分を含むこととしてもよく、毛母以外の部分を含むこととしてもよく、毛乳頭及び毛母以外の部分を含むこととしてもよく、毛球部以外の部分を含むこととしてもよい。 One of the characteristics of this method is that an adult hair follicle tissue containing a non-constant part is used as a cell source of source cells containing epithelial stem cells. The non-constant part contained in the adult hair follicle tissue is not particularly limited as long as it is a part or all of the non-constant part of the adult hair follicle tissue. It is good also as including a part other than a hair ball part, and it is good also as including parts other than a hair nipple and a hair mother.
成体毛包組織に含まれる非恒常部に対する、毛乳頭以外の部分、毛母以外の部分、毛乳頭及び毛母以外の部分、又は毛球部以外の部分の重量割合は、例えば、５０重量％以上であってもよく、７０重量％以上であってもよく、９０重量％以上であってもよい。 The weight ratio of the portion other than the hair papilla, the portion other than the hair matrix, the portion other than the hair papilla and the hair matrix, or the portion other than the hair bulb relative to the non-constant portion contained in the adult hair follicle tissue is, for example, 50% by weight. The above may be sufficient, 70 weight% or more may be sufficient, and 90 weight% or more may be sufficient.
成体毛包組織に含まれる非恒常部は、毛乳頭を含まないこととしてもよく、毛母を含まないこととしてもよく、毛乳頭及び毛母を含まないこととしてもよく、毛球部を含まないこととしてもよい。 The non-constant part contained in the adult hair follicle tissue may not include the hair papilla, may not include the hair matrix, may not include the hair papilla and the hair matrix, and includes the hair bulb part. It's okay to not.
成体毛包組織に含まれる非恒常部は、恒常部のサブバルジ領域と、当該非恒常部の毛球部との間の部分の一部又は全部を含むこととしてもよい。 The non-constant part contained in the adult hair follicle tissue may include a part or all of a part between the sub-bulge region of the constant part and the hair bulb part of the non-constant part.
成体毛包組織に含まれる非恒常部に対する、恒常部のサブバルジ領域と、当該非恒常部の毛球部との間の部分の重量割合は、例えば、５０重量％以上であってもよく、７０重量％以上であってもよく、９０重量％以上であってもよい。 The weight ratio of the part between the subbulge region of the permanent part and the hair bulb part of the non-constant part with respect to the non-constant part contained in the adult hair follicle tissue may be, for example, 50% by weight or more, 70 It may be 90% by weight or more.
上述のように非恒常部を含む成体毛包組織は、上皮幹細胞を含む原料細胞の細胞源として使用するため、恒常部のバルジ領域を含む。原料細胞の細胞源としての成体毛包組織は、サブバルジ領域をさらに含むこととしてもよい。 As described above, an adult hair follicle tissue containing a non-constant part includes a bulge region of the constant part in order to be used as a cell source of source cells including epithelial stem cells. The adult hair follicle tissue as the cell source of the source cells may further include a subbulge region.
原料細胞の細胞源として使用する成体毛包組織は、成体動物から採取された毛包組織である。成体動物は、皮膚付属器としての毛包を有する成体であれば特に限られず、ヒトであってもよいし、非ヒト動物（ヒト以外の動物）であってもよい。非ヒト動物は、特に限られないが、非ヒト脊椎動物（ヒト以外の脊椎動物）であることが好ましい。非ヒト脊椎動物は、特に限られないが、非ヒト哺乳類であることが好ましい。非ヒト哺乳類は、特に限られないが、例えば、霊長類（例えば、サル）、げっ歯類（例えば、マウス、ラット、ハムスター、モルモット、ウサギ）、食肉類（例えば、イヌ、ネコ）、又は有蹄類（例えば、ブタ、ウシ、ウマ、ヤギ、ヒツジ）であってもよい。 The adult hair follicle tissue used as the cell source of the source cells is a hair follicle tissue collected from an adult animal. The adult animal is not particularly limited as long as it is an adult having a hair follicle as a skin appendage, and may be a human or a non-human animal (an animal other than a human). The non-human animal is not particularly limited, but is preferably a non-human vertebrate (a non-human vertebrate). The non-human vertebrate is not particularly limited, but is preferably a non-human mammal. Non-human mammals are not particularly limited, for example, primates (eg, monkeys), rodents (eg, mice, rats, hamsters, guinea pigs, rabbits), meat (eg, dogs, cats), or It may be a hoof (eg, pig, cow, horse, goat, sheep).
本方法で使用する原料細胞は、後述の毛包原基の製造において、毛乳頭細胞と混合して培養する細胞である。原料細胞は、バルジ領域及び非恒常部を含む成体毛包組織に酵素処理を施すことにより得られる。 The raw material cells used in this method are cells that are mixed and cultured with hair papilla cells in the production of the hair follicle primordium described below. A raw material cell is obtained by performing an enzyme treatment to the adult hair follicle tissue containing a bulge region and a non-constant part.
酵素処理に使用される酵素は、成体毛包組織から原料細胞を遊離させるために有効な酵素（例えば、当該成体毛包組織において当該原料細胞の周囲のマトリクスを分解し、及び／又は当該原料細胞間の結合を切断する分解酵素）であれば特に限られないが、例えば、コラゲナーゼ、ディスパーゼ、及びトリプシンからなる群より選択される１以上であってもよい。 The enzyme used for the enzyme treatment is an enzyme effective for releasing the raw material cells from the adult hair follicle tissue (for example, decomposing a matrix around the raw material cells in the adult hair follicle tissue and / or the raw material cells). For example, it may be one or more selected from the group consisting of collagenase, dispase, and trypsin.
なお、原料細胞は、毛乳頭細胞から独立して、別途調製される。すなわち、原料細胞の細胞源として使用される成体毛包組織は、毛乳頭細胞の細胞源（例えば、生体毛包組織から分離された毛乳頭）とは別に準備される。そして、毛乳頭細胞の調製とは別に、バルジ領域及び非恒常部を含む成体毛包組織から原料細胞を調製する。 The source cells are separately prepared independently from the hair papilla cells. That is, an adult hair follicle tissue used as a cell source of raw material cells is prepared separately from a cell source of hair papilla cells (for example, a hair papilla separated from a living hair follicle tissue). Separately from the preparation of dermal papilla cells, raw material cells are prepared from an adult hair follicle tissue containing a bulge region and a non-constant part.
原料細胞は、生体毛包組織のバルジ領域から得られた上皮幹細胞に加え、当該成体毛包組織の非恒常部から得られた細胞を含む。すなわち、原料細胞は、毛乳頭以外の非恒常部から得られた細胞を含むこととしてもよく、毛母以外の非恒常部から得られた細胞を含むこととしてもよく、毛乳頭及び毛母以外の非恒常部から得られた細胞を含むこととしてもよく、毛球部以外の非恒常部から得られた細胞を含むこととしてもよい。 The source cells include cells obtained from non-constant parts of the adult hair follicle tissue in addition to epithelial stem cells obtained from the bulge region of the living hair follicle tissue. That is, the raw material cells may contain cells obtained from non-constant parts other than the hair papilla, may contain cells obtained from non-constant parts other than the hair matrix, and other than the hair papilla and hair matrix. It is good also as including the cell obtained from non-constant part of this, and good also as including the cell obtained from non-constant part other than a hair bulb part.
また、原料細胞は、非恒常部から分離された毛乳頭から得られた細胞を含まないこととしてもよく、非恒常部から分離された毛母から得られた細胞を含まないこととしてもよく、非恒常部から分離された毛乳頭及び毛母から得られた細胞を含まないこととしてもよく、非恒常部から分離された毛球部から得られた細胞を含まないこととしてもよい。 In addition, the raw material cells may not include cells obtained from the hair papilla separated from the non-constant part, or may not include cells obtained from the hair matrix separated from the non-constant part, The cells obtained from the hair papilla and hair matrix separated from the non-constant part may not be included, and the cells obtained from the hair bulb part separated from the non-constant part may not be included.
また、原料細胞は、毛乳頭から得られた細胞を含まないこととしてもよく、毛母から得られた細胞を含まないこととしてもよく、毛乳頭及び毛母から得られた細胞を含まないこととしてもよく、毛球部から得られた細胞を含まないこととしてもよい。 The source cells may not contain cells obtained from the hair papilla, may not contain cells obtained from the hair matrix, and do not contain cells obtained from the hair papilla and hair matrix. It is good also as not including the cell obtained from the hair bulb part.
毛乳頭細胞は、毛包原基の一部を形成できるものであれば特に限られないが、例えば、成体毛包組織の毛乳頭に由来する細胞であってもよく、皮膚組織（胎児、幼体、成体のいずれの皮膚組織であってもよい。）に由来する細胞であってもよく、予め培養された細胞であってもよく、生体外で幹細胞（例えば、人工多能性（ｉＰＳ）幹細胞、胚性幹（ＥＳ）細胞、又は胚性生殖（ＥＧ）細胞）から誘導された細胞であってもよい。毛乳頭細胞は、例えば、Ｖｅｒｓｉｃａｎを発現する細胞として特定される。 The hair papilla cell is not particularly limited as long as it can form a part of the hair follicle primordium. For example, it may be a cell derived from the hair papilla of an adult hair follicle tissue, and skin tissue (fetus, juvenile body) Cells derived from any adult skin tissue), cells cultured in advance, or stem cells (eg, induced pluripotent (iPS) stem cells) in vitro. , Embryonic stem (ES) cells, or embryonic germ (EG) cells). Papilla cells are identified as, for example, cells that express Versican.
本方法においては、分散された毛乳頭細胞と、分散された原料細胞とを混合して培養することにより、毛包原基を製造する。すなわち、分散された毛乳頭細胞と、分散された原料細胞とを含む混合細胞懸濁液を培養容器に播種することにより、当該毛乳頭細胞及び原料細胞の混合培養を行う。混合細胞懸濁液は、分散された毛乳頭細胞を含む毛乳頭細胞懸濁液と、分散された原料細胞を含む原料細胞懸濁液とを混合することにより調製される。 In this method, a hair follicle primordium is produced by mixing and culturing dispersed hair papilla cells and dispersed raw material cells. That is, mixed culturing of the hair papilla cells and the raw material cells is performed by seeding a mixed cell suspension containing the dispersed hair papilla cells and the dispersed raw material cells in a culture vessel. The mixed cell suspension is prepared by mixing the dermal papilla cell suspension containing dispersed dermal papilla cells and the raw material cell suspension containing dispersed raw material cells.
なお、分散された細胞とは、他の細胞と結合しておらず、又は数個の細胞と結合しているのみであり、溶液中に分散されて浮遊している細胞である。例えば、細胞懸濁液に含まれる細胞と溶媒とを分離するために、分散された細胞を含む当該細胞懸濁液を遠心する場合、遠心後に形成される当該細胞の沈殿物は、当該細胞の凝集塊であり、当該沈殿物を構成する細胞は、分散された細胞ではない。 Note that the dispersed cells are cells that are not bound to other cells or are only bound to several cells, and are dispersed and suspended in the solution. For example, when the cell suspension containing dispersed cells is centrifuged to separate the cells and the solvent contained in the cell suspension, the cell precipitate formed after the centrifugation is The cells that are aggregates and constitute the precipitate are not dispersed cells.
毛乳頭細胞懸濁液は、毛乳頭細胞の培養に適した培養液に、当該毛乳頭細胞を分散させることにより調製される。毛乳頭細胞の培養に適した培養液は、特に限られないが、例えば、ＦＧＦ２（ｂＦＧＦ）を含む培養液を使用することが好ましい。 The dermal papilla cell suspension is prepared by dispersing the dermal papilla cells in a culture medium suitable for culturing the dermal papilla cells. A culture solution suitable for culturing hair papilla cells is not particularly limited, but for example, a culture solution containing FGF2 (bFGF) is preferably used.
毛乳頭の培養に使用される培養液に含まれるＦＧＦ２の濃度は、特に限られないが、例えば、１０ｎｇ／ｍＬ以上であることとしてもよく、３０ｎｇ／ｍＬ以上であることが好ましく、５０ｎｇ／ｍＬ以上であることがより好ましく、７５ｎｇ／ｍＬ以上であることがより一層好ましく、１００ｎｇ／ｍＬ以上であることが特に好ましい。 The concentration of FGF2 contained in the culture medium used for culturing hair papilla is not particularly limited, but may be, for example, 10 ng / mL or more, preferably 30 ng / mL or more, and preferably 50 ng / mL. More preferably, it is more preferably 75 ng / mL or more, and particularly preferably 100 ng / mL or more.
原料細胞懸濁液は、原料細胞の培養に適した培養液に、当該原料細胞を分散させることにより調製される。原料細胞の培養に適した培養液は、上皮幹細胞の培養に適した培養液であることが好ましい。上皮幹細胞の培養に適した培養液は、特に限られず、上皮幹細胞の培養に使用されている公知の培養液を使用してもよい。 The raw material cell suspension is prepared by dispersing the raw material cells in a culture solution suitable for culturing the raw material cells. The culture solution suitable for culturing the source cells is preferably a culture solution suitable for culturing epithelial stem cells. A culture solution suitable for culturing epithelial stem cells is not particularly limited, and a known culture solution used for culturing epithelial stem cells may be used.
混合細胞懸濁液に含まれる毛乳頭細胞と原料細胞との数比は、最終的に毛包原基が製造される範囲内であれば特に限られないが、当該毛乳頭細胞の数に対する当該原料細胞の数の比は、例えば、０．１０以上、１０．００以下であることとしてもよく、０．２０以上、５．００以下であることが好ましく、０．５０以上、２．００以下であることがより好ましく、０．７５以上、１．５０以下であることが特に好ましい。 The number ratio between the dermal papilla cells and the raw material cells contained in the mixed cell suspension is not particularly limited as long as the hair follicle primordium is finally produced. The ratio of the number of source cells may be, for example, 0.10 or more and 10.00 or less, preferably 0.20 or more and 5.00 or less, and 0.50 or more and 2.00 or less. More preferably, it is 0.75 or more and 1.50 or less.
分散された毛乳頭細胞と分散された原料細胞とを混合培養する培養液は、特に限られないが、例えば、ＦＧＦ２を含む培養液を使用することが好ましい。混合培養に使用される培養液に含まれるＦＧＦ２の濃度は、特に限られないが、例えば、１０ｎｇ／ｍＬ以上であることとしてもよく、３０ｎｇ／ｍＬ以上であることが好ましく、５０ｎｇ／ｍＬ以上であることがより好ましく、７５ｎｇ／ｍＬ以上であることがより一層好ましく、１００ｎｇ／ｍＬ以上であることが特に好ましい。 The culture medium for mixing and culturing the dispersed dermal papilla cells and the dispersed raw material cells is not particularly limited. For example, it is preferable to use a culture medium containing FGF2. The concentration of FGF2 contained in the culture medium used for the mixed culture is not particularly limited, but may be, for example, 10 ng / mL or more, preferably 30 ng / mL or more, preferably 50 ng / mL or more. More preferably, it is more preferably 75 ng / mL or more, and particularly preferably 100 ng / mL or more.
毛乳頭細胞と原料細胞との混合培養における細胞密度（混合細胞懸濁液の単位体積あたりに含まれる、毛乳頭細胞と原料細胞との合計数）は、毛包原基が形成される範囲内であれば特に限られないが、例えば、５．０×１０５ｃｅｌｌｓ／ｍＬ以上、１．０×１０８ｃｅｌｌｓ／ｍＬ以下であることとしてもよく、２．５×１０６ｃｅｌｌｓ／ｍＬ以上、５．０×１０７ｃｅｌｌｓ／ｍＬ以下であることが好ましく、２．５×１０６ｃｅｌｌｓ／ｍＬ以上、２．５×１０７ｃｅｌｌｓ／ｍＬ以下であることが特に好ましい。 Cell density (total number of dermal papilla cells and raw material cells contained per unit volume of the mixed cell suspension) in the mixed culture of dermal papilla cells and raw material cells is within the range in which the hair follicle primordium is formed If it is not particularly limited, for example, it may be 5.0 × 10 5 cells / mL or more, 1.0 × 10 8 cells / mL or less, 2.5 × 10 6 cells / mL or more, It is preferably 5.0 × 10 7 cells / mL or less, particularly preferably 2.5 × 10 6 cells / mL or more and 2.5 × 10 7 cells / mL or less.
毛乳頭細胞と原料細胞との混合培養に使用する培養容器は、特に限られないが、例えば、細胞非接着性の培養容器が好ましく使用される。細胞非接着性の培養容器は、細胞非接着性の表面を有する培養容器である。すなわち、例えば、培養容器が、基材の表面に形成された凹部（有底穴）である場合、当該凹部は、細胞非接着性の底面を有し、細胞非接着性の側面をさらに有することが好ましい。 The culture vessel used for the mixed culture of the hair papilla cells and the raw material cells is not particularly limited. For example, a cell non-adhesive culture vessel is preferably used. A non-cell-adhesive culture vessel is a culture vessel having a non-cell-adhesive surface. That is, for example, when the culture vessel is a recess (bottomed hole) formed on the surface of the substrate, the recess has a cell non-adhesive bottom surface and further has a cell non-adhesive side surface. Is preferred.
細胞非接着性の表面は、培養される細胞が接着して伸展しない表面である。すなわち、細胞非接着性の表面を有する培養容器で培養された細胞は、当該表面に弱く接着することはあるが、当該表面に伸展はしない。このため、細胞非接着性表面に接着した細胞は、例えば、酵素処理やキレート処理を行うことなく、培養液を流動させることにより、当該表面から脱離して浮遊する。 A cell non-adhesive surface is a surface to which cultured cells do not adhere and extend. That is, cells cultured in a culture vessel having a non-cell-adhesive surface may adhere weakly to the surface but do not spread on the surface. For this reason, the cells adhered to the non-cell-adhesive surface are detached from the surface and floated, for example, by flowing the culture solution without performing enzyme treatment or chelation treatment.
毛乳頭細胞と原料細胞との混合培養に使用する培養容器は、例えば、酸素透過性が比較的大きい基材の表面に形成された凹部であることが好ましい。すなわち、培養容器としては、例えば、酸素透過率が１００ｃｍ３／（ｍ２・２４ｈ・ａｔｍ）以上の基材に形成された凹部であることとが好ましく、酸素透過率が５００ｃｍ３／（ｍ２・２４ｈ・ａｔｍ）以上の基材に形成された凹部であることとがより好ましく、酸素透過率が１０００ｃｍ３／（ｍ２・２４ｈ・ａｔｍ）以上の基材に形成された凹部であることとが特に好ましい。 The culture vessel used for the mixed culture of the hair papilla cells and the raw material cells is preferably, for example, a recess formed on the surface of the base material having a relatively large oxygen permeability. That is, the culture vessel is preferably, for example, a recess formed in a substrate having an oxygen permeability of 100 cm 3 / (m 2 · 24 h · atm) or more, and the oxygen permeability is 500 cm 3 / (m 2. -It is more preferable that it is a recessed part formed in the base material more than 24h * atm), and it is a recessed part formed in the base material whose oxygen permeability is 1000cm < 3 > / (m < 2 > * 24h * atm) or more. Is particularly preferred.
酸素透過性が大きい基材を構成する材料としては、例えば、シリコン系材料（例えば、ポリジメチルシロキサン（ｐｏｌｙ（ｄｉｍｅｔｈｙｌｓｉｌｏｘａｎｅ）：ＰＤＭＳ））が好ましく使用される。 For example, a silicon-based material (for example, polydimethylsiloxane (PDMS)) is preferably used as a material constituting the base material having high oxygen permeability.
毛乳頭細胞と原料細胞との混合培養に使用する培養容器として、基材の表面に形成された凹部を使用する場合、当該凹部の容積や、当該凹部の底面の形状及び面積は特に限られない。凹部の容積は、例えば、１ｎＬ以上、１ｍＬ以下であることとしてもよく、１０ｎＬ以上、１００μＬ以下であることが好ましく、１００ｎＬ以上、１０μＬ以下であることが特に好ましい。凹部の底面の形状は、例えば、円形、楕円形、又は多角形であることとしてもよい。凹部の底面の面積は、例えば、１μｍ２以上、１００ｍｍ２以下であることとしてもよく、１０μｍ２以上、１０ｍｍ２以下であることが好ましく、１００μｍ２以上、１ｍｍ２以下であることが特に好ましい。 When a recess formed on the surface of the substrate is used as a culture container used for mixed culture of follicular papilla cells and raw material cells, the volume of the recess and the shape and area of the bottom surface of the recess are not particularly limited. . The volume of the recess may be, for example, 1 nL or more and 1 mL or less, preferably 10 nL or more and 100 μL or less, and particularly preferably 100 nL or more and 10 μL or less. The shape of the bottom surface of the recess may be, for example, a circle, an ellipse, or a polygon. The area of the bottom surface of the recess may be, for example, 1 μm 2 or more and 100 mm 2 or less, preferably 10 μm 2 or more and 10 mm 2 or less, and particularly preferably 100 μm 2 or more and 1 mm 2 or less.
毛乳頭細胞と原料細胞との混合培養に使用する培養容器としては、基材の表面に規則的に形成された複数の凹部（例えば、一定の間隔で直線的又は網目状に配置された複数の凹部）を使用することとしてもよい。 The culture vessel used for the mixed culture of follicular dermal papilla cells and raw material cells includes a plurality of recesses regularly formed on the surface of the substrate (for example, a plurality of linearly or meshedly arranged at regular intervals). It is good also as using a recessed part.
毛乳頭細胞と原料細胞との混合培養時間は、毛包原基を形成できる範囲内であれば特に限られないが、例えば、１２時間以上、１０日以下であってもよく、１日以上、７日以下であることが好ましい。培養温度は、毛包原基を形成できる範囲内であれば特に限られないが、例えば、２５℃以上、４０℃以下であることとしてもよく、３５℃以上、３９℃以下であることが好ましい。 The mixed culture time of the hair papilla cells and the raw material cells is not particularly limited as long as the follicle primordium can be formed. For example, it may be 12 hours or more, 10 days or less, or 1 day or more, It is preferable that it is 7 days or less. The culture temperature is not particularly limited as long as it is within the range in which the hair follicle primordium can be formed. For example, the culture temperature may be 25 ° C. or more and 40 ° C. or less, and preferably 35 ° C. or more and 39 ° C. or less. .
混合細胞懸濁液中で、混合された毛乳頭細胞及び原料細胞を培養することにより、当該毛乳頭細胞の凝集部と、当該原料細胞の凝集部とを含む複合細胞凝集体である、毛包原基が形成される。 A hair follicle that is a composite cell aggregate comprising the aggregated portion of the hair papilla cell and the aggregated portion of the raw material cell by culturing the mixed hair papilla cell and the raw material cell in the mixed cell suspension A primordial is formed.
すなわち、この毛包原基は、毛乳頭細胞が互いに結合し凝集することにより形成された毛乳頭細胞凝集部と、原料細胞が互いに結合し凝集することにより形成された原料細胞凝集部とを含む。原料細胞凝集部は、上皮幹細胞が互いに結合し凝集して形成された上皮幹細胞凝集部を含んでもよい。 That is, the hair follicle primordium includes a hair papilla cell aggregation part formed by binding and aggregation of hair follicle cells to each other, and a raw material cell aggregation part formed by binding and aggregation of raw material cells to each other. . The source cell aggregated part may include an epithelial stem cell aggregated part formed by combining and aggregating epithelial stem cells.
毛包原基において、毛乳頭細胞凝集部の一部と原料細胞凝集部の一部とは結合している。すなわち、毛乳頭細胞凝集部に含まれる一部の毛乳頭細胞と、原料細胞凝集部に含まれる一部の原料細胞とが結合している。この場合、毛乳頭細胞凝集部に含まれる一部の毛乳頭細胞と、原料細胞凝集部に含まれる一部の上皮幹細胞とが結合していてもよい。 In the hair follicle primordium, a part of the hair papilla cell aggregation part and a part of the raw material cell aggregation part are bonded. That is, a part of hair papilla cells contained in the hair papilla cell aggregation part and a part of raw material cells contained in the raw material cell aggregation part are bonded. In this case, a part of the hair papilla cells contained in the hair papilla cell aggregation part and a part of the epithelial stem cells contained in the raw material cell aggregation part may be combined.
本方法における毛包原基の形成は、毛乳頭細胞及び原料細胞が分散され、且つ混合された状態で培養を開始した後、当該毛乳頭細胞同士が結合して自発的に凝集し、且つ当該原料細胞同士（例えば、上皮幹細胞同士）が結合して自発的に凝集するとともに、一部の毛乳頭細胞と一部の原料細胞（例えば、一部の上皮幹細胞）とが結合することにより、達成される。 The formation of the hair follicle primordium in this method is carried out after the dermal papilla cells and the raw material cells are dispersed and mixed, and then the dermal papilla cells bind to each other and spontaneously aggregate, Achieved by combining source cells (for example, epithelial stem cells) and spontaneously agglutinating, and combining some of the dermal papilla cells and some of the source cells (for example, some epithelial stem cells). Is done.
本方法において形成される毛包原基の形状は、特に限られないが、例えば、球状、又は長球状であることとしてもよい。本方法において形成される毛包原基の体積は、特に限られないが、例えば、１×１０−５ｍｍ３以上、１０ｍｍ３以下であることとしてもよく、１×１０−４ｍｍ３以上、１ｍｍ３以下であることが好ましく、１×１０−３ｍｍ３以上、０．１ｍｍ３以下であることが特に好ましい。 The shape of the hair follicle primordium formed in this method is not particularly limited, and may be, for example, spherical or oblong. The volume of the hair follicle primordium formed in this method is not particularly limited, and may be, for example, 1 × 10 −5 mm 3 or more and 10 mm 3 or less, and 1 × 10 −4 mm 3 or more, is preferably 1 mm 3 or less, 1 × 10 -3 mm 3 or more, and particularly preferably 0.1 mm 3 or less.
毛包原基に含まれる細胞の密度（毛包原基の単位体積あたりに含まれる細胞の合計数）は、特に限られないが、例えば、１．０×１０６ｃｅｌｌｓ／ｃｍ３以上、７．０×１０１０ｃｅｌｌｓ／ｃｍ３以下であることとしてもよく、５．０×１０６ｃｅｌｌｓ／ｃｍ３以上、６．５×１０１０ｃｅｌｌｓ／ｃｍ３以下であることが好ましく、１．０×１０７ｃｅｌｌｓ／ｃｍ３以上、５．０×１０１０ｃｅｌｌｓ／ｃｍ３以下であることが特に好ましい。 The density of cells contained in the hair follicle primordium (total number of cells contained per unit volume of the hair follicle primordium) is not particularly limited, but is, for example, 1.0 × 10 6 cells / cm 3 or more, 7 0.0 × 10 10 cells / cm 3 or less, preferably 5.0 × 10 6 cells / cm 3 or more, and preferably 6.5 × 10 10 cells / cm 3 or less, and 1.0 × It is particularly preferably 10 7 cells / cm 3 or more and 5.0 × 10 10 cells / cm 3 or less.
本方法においては、分散された毛乳頭細胞と、分散された原料細胞とを混合して培養することにより、動物に移植されることで毛を生やす毛包原基を製造する。すなわち、本方法における混合培養で形成された毛包原基は、後述するように、動物に移植することができ、且つ当該動物に移植された後に、当該毛包原基から毛が生える。 In this method, the hair follicle primordium which grows hair by being transplanted to an animal is produced by mixing and cultivating dispersed hair papilla cells and dispersed raw material cells. That is, the hair follicle primordium formed by the mixed culture in this method can be transplanted to an animal as described later, and hair is grown from the hair follicle primordium after being transplanted to the animal.
本方法において、製造された毛包原基を動物に移植して当該毛包原基から毛を生やす場合、当該動物は特に限られず、ヒトであってもよいし、非ヒト動物であってもよい。非ヒト動物は特に限られないが、非ヒト哺乳類であることが好ましい。非ヒト哺乳類は、特に限られないが、例えば、霊長類（例えば、サル）、げっ歯類（例えば、マウス、ラット、ハムスター、モルモット、ウサギ）、食肉類（例えば、イヌ、ネコ）、又は有蹄類（例えば、ブタ、ウシ、ウマ、ヤギ、ヒツジ）であってもよい。 In the present method, when the produced hair follicle primordium is transplanted into an animal to produce hair from the hair follicle primordium, the animal is not particularly limited and may be a human or a non-human animal. Good. The non-human animal is not particularly limited, but is preferably a non-human mammal. Non-human mammals are not particularly limited, for example, primates (eg, monkeys), rodents (eg, mice, rats, hamsters, guinea pigs, rabbits), meat (eg, dogs, cats), or It may be a hoof (eg, pig, cow, horse, goat, sheep).
毛包原基の動物への移植は、当該動物の皮膚への移植であることが好ましい。皮膚への移植は、例えば、皮下移植であってもよいし、皮内移植であってもよい。 The transplantation of the hair follicle primordium to the animal is preferably a transplantation to the skin of the animal. The skin transplantation may be, for example, a subcutaneous transplantation or an intradermal transplantation.
本方法における毛包原基の製造及びその動物への移植は、医学的用途であってもよいし、研究用途であってもよい。すなわち、本方法においては、例えば、脱毛を伴う疾患の治療又は予防のために、当該疾患を患っている又は患う可能性のあるヒト患者に移植する目的で毛包原基を製造し、又は当該毛包原基を当該ヒト患者に移植することとしてもよい。 The production of the hair follicle primordium and its implantation into animals in this method may be for medical use or research use. That is, in this method, for example, for the treatment or prevention of diseases associated with hair loss, a hair follicle primordium is produced for the purpose of transplanting to a human patient suffering from or likely to suffer from the disease, or The hair follicle primordium may be transplanted into the human patient.
脱毛を伴う疾患は、特に限られないが、例えば、男性型脱毛症（Ａｎｄｒｏｇｅｎｅｔｉｃ Ａｌｏｐｅｃｉａ：ＡＧＡ）、女子男性型脱毛症（Ｆｅｍａｌｅ Ａｎｄｒｏｇｅｎｅｔｉｃ Ａｌｏｐｅｃｉａ：ＦＡＧＡ）、分娩後脱毛症、びまん性脱毛症、脂漏性脱毛症、粃糠性脱毛症、牽引性脱毛症、代謝異常性脱毛症、圧迫性脱毛症、円形脱毛症、神経性脱毛症、抜毛症、全身性脱毛症、及び症候性脱毛症からなる群より選択される１以上であることとしてもよい。 Diseases associated with hair loss are not particularly limited. For example, androgenetic alopecia (AGA), female androgenetic alopecia (FAGA), postpartum alopecia, diffuse alopecia, seborrhea Alopecia, alopecia areata, traction alopecia, metabolic alopecia, pressure alopecia, alopecia areata, neurogenic alopecia, hair loss, systemic alopecia, and symptomatic alopecia It may be one or more selected from the group.
また、本方法においては、例えば、脱毛を伴う疾患の治療又は予防に使用され得る物質の探索、及び／又は当該疾患の機構に関与する物質の探索のために、毛包原基を製造し、又は当該毛包原基を非ヒト動物に移植することとしてもよい。 In this method, for example, a hair follicle primordium is produced in order to search for a substance that can be used for treatment or prevention of a disease associated with hair loss and / or to search for a substance involved in the mechanism of the disease. Alternatively, the hair follicle primordium may be transplanted into a non-human animal.
そして、本実施形態に係る再生毛の色制御方法においては、成体毛包組織に含まれる非恒常部の重量割合（すなわち、原料細胞の細胞源として使用される成体毛包組織の重量に対する、当該成体毛包組織に含まれる非恒常部の重量の割合）を変えることにより、毛包原基から生える毛の色を変える。 And in the color control method of regenerated hair according to the present embodiment, the weight ratio of the non-constant part contained in the adult hair follicle tissue (that is, the weight relative to the weight of the adult hair follicle tissue used as the cell source of the source cells) By changing the weight ratio of the non-constant part contained in the adult hair follicle tissue, the color of the hair growing from the hair follicle primordium is changed.
具体的に、例えば、成体毛包組織に含まれる非恒常部の重量割合を増加させることにより、移植された毛包原基から生える毛の色を濃くする。ここで、毛の色の濃さは、目視でも評価できるが、例えば、当該毛の単位重量当たりに含まれるメラニン色素の量として、定量的に評価してもよい。毛に含まれるメラニン色素の定量は、例えば、測定対象となる当該毛を水酸化ナトリウム溶液中で６０℃３０分間インキュベートしてメラニンを抽出し、次いで、吸光度計を用いて、４０５ｎｍの吸光度を測定することで、抽出された当該メラニンを定量することができる。 Specifically, for example, by increasing the weight ratio of the non-constant part contained in the adult hair follicle tissue, the color of hair growing from the transplanted hair follicle primordium is increased. Here, the darkness of the hair color can be evaluated visually, but for example, it may be quantitatively evaluated as the amount of the melanin pigment contained per unit weight of the hair. The melanin pigment contained in the hair is quantified by, for example, incubating the hair to be measured in a sodium hydroxide solution at 60 ° C. for 30 minutes to extract melanin, and then measuring the absorbance at 405 nm using an absorptiometer. By doing so, the extracted melanin can be quantified.
また、本実施形態に係る毛の再生方法においては、非恒常部を含む成体毛包組織を使用することにより、移植された毛包原基から、当該非恒常部を含まない成体毛包組織を使用する場合に比べて色が濃い毛を生やす。 Further, in the hair regeneration method according to the present embodiment, by using an adult hair follicle tissue containing a non-constant part, an adult hair follicle tissue not containing the non-constant part is obtained from the transplanted hair follicle primordia. Produces darker hair than when used.
すなわち、例えば、毛乳頭以外の部分を含む非恒常部、毛母以外の部分を含む非恒常部、毛乳頭及び毛母以外の部分を含む非恒常部、又は毛球部以外の部分を含む非恒常部を含む成体毛包組織から得られた原料細胞を使用して製造された第一の毛包原基を動物に移植することにより、当該非恒常部を含まない成体毛包組織から得られた原料細胞を使用して製造された第二の毛包原基を動物に移植する場合に比べて、色が濃い毛を当該移植された第一の毛包原基から生やす。 That is, for example, a non-constant part including a part other than the hair papilla, a non-constant part including a part other than the hair matrix, a non-constant part including a part other than the hair papilla and the hair matrix, or a non-constant part including a part other than the hair bulb part It is obtained from an adult hair follicle tissue that does not contain the non-constant part by transplanting the first hair follicle primordium produced using the raw material cells obtained from the adult hair follicle tissue containing the constant part to an animal. Compared with the case where the second hair follicle primordium produced using the raw material cells is transplanted into the animal, dark hair is grown from the transplanted first hair follicle primordium.
また、非恒常部を第一の重量割合で含む第一の成体毛包組織を使用して製造された第一の毛包原基を動物に移植して、当該第一の毛包原基から第一の色の毛を生やすとともに、当該非恒常部を当該第一の重量割合より大きい第二の重量割合で含む第二の成体毛包組織を使用して製造された第二の細胞凝集塊を動物に移植して、当該第二の毛包原基から当該第一の色より濃い第二の色の毛を生やすこととしてもよい。 In addition, the first hair follicle primordium produced using the first adult hair follicle tissue containing the non-constant part at a first weight ratio is transplanted into an animal, A second cell aggregate produced using a second adult hair follicle tissue that grows hair of the first color and includes the non-constant part at a second weight percentage greater than the first weight percentage. May be transplanted into an animal to grow second colored hair darker than the first color from the second hair follicle primordium.
この場合、原料細胞の細胞源として使用する生体毛包組織に含まれる非恒常部の重量割合として、複数の重量割合を採用することにより、移植された複数の毛包原基から、色の濃さが異なる複数の毛を生やすことができる。 In this case, by adopting a plurality of weight ratios as the weight ratio of the non-constant part contained in the living hair follicle tissue used as the cell source of the source cells, the color concentration is increased from the plurality of transplanted hair follicle primaries. A plurality of different hairs can be grown.
また、本実施形態に係る毛包原基の製造方法においては、非恒常部を含む成体毛包組織を使用することにより、当該非恒常部を含まない成体毛包組織を使用する場合に比べて色が濃い毛を生やす毛包原基を製造する。 Further, in the method for producing a hair follicle primordium according to the present embodiment, by using an adult hair follicle tissue containing a non-constant part, compared to a case where an adult hair follicle tissue not containing the non-constant part is used. Produces a hair follicle primordium that produces dark hair.
すなわち、例えば、毛乳頭以外の部分を含む非恒常部、毛母以外の部分を含む非恒常部、毛乳頭及び毛母以外の部分を含む非恒常部、又は毛球部以外の部分を含む非恒常部を含む成体毛包組織から得られた原料細胞を使用することにより、当該恒常部を含まない成体毛包組織から得られた原料細胞を使用する場合に比べて、動物に移植されることで色が濃い毛を生やす毛包原基を製造する。 That is, for example, a non-constant part including a part other than the hair papilla, a non-constant part including a part other than the hair matrix, a non-constant part including a part other than the hair papilla and the hair matrix, or a non-constant part including a part other than the hair bulb part By using a raw material cell obtained from an adult hair follicle tissue containing a constant part, it is transplanted to an animal as compared with a case where a raw material cell obtained from an adult hair follicle tissue not containing the constant part is used. To produce a hair follicle primordium that produces dark hair.
また、非恒常部を第一の重量割合で含む第一の成体毛包組織を使用することにより、動物に移植されることで第一の色の毛を生やす第一の毛包原基を製造するとともに、当該非恒常部を当該第一の重量割合より大きい第二の重量割合で含む第二の成体毛包組織を使用することにより、当該動物に移植されることで当該第一の色より濃い第二の色の毛を生やす第二の毛包原基を製造する。 In addition, by using a first adult hair follicle tissue containing a non-constant part in a first weight ratio, a first hair follicle primordium that produces hair of the first color by being transplanted to an animal is produced. And using the second adult hair follicle tissue containing the non-constant part at a second weight ratio that is greater than the first weight ratio, so that the non-constant part can be transplanted into the animal by the first color. A second follicle primordium that produces dark second colored hair is produced.
この場合、原料細胞の細胞源として使用する生体毛包組織に含まれる非恒常部の重量割合として、複数の重量割合を採用することにより、移植された場合に色の濃さが異なる毛を生やす複数の毛包原基を製造することができる。 In this case, by adopting a plurality of weight ratios as the weight ratio of the non-constant part contained in the living hair follicle tissue used as the cell source of the raw material cells, hair having different color densities can be produced when transplanted. Multiple hair follicle primordia can be produced.
生体毛包組織に含まれる非恒常部の重量割合は、０％以上、１００％以下の範囲内であれば特に限られない。すなわち、第一の重量割合が０％である場合（すなわち、非恒常部を含まない成体毛包組織を使用する場合）、第二の重量割合は、例えば、５％以上であることとしてもよく、１０％以上であることとしてもよく、２０％以上であることとしてもよく、３０％以上であることとしてもよい。通常、生体毛包組織から非恒常部を除去しない場合（すなわち、生体毛包組織が恒常部及び非恒常部の全部を含む場合）、当該成体毛包組織の重量に対する、当該非恒常部の重量の割合は、４０％程度となる。 The weight ratio of the non-constant part contained in the living hair follicle tissue is not particularly limited as long as it is in the range of 0% to 100%. That is, when the first weight ratio is 0% (that is, when an adult hair follicle tissue not including a non-constant part is used), the second weight ratio may be, for example, 5% or more. It may be 10% or more, may be 20% or more, and may be 30% or more. Usually, when the non-constant part is not removed from the living hair follicle tissue (that is, when the living hair follicle tissue includes both the constant part and the non-constant part), the weight of the non-constant part relative to the weight of the adult hair follicle tissue The ratio is about 40%.
なお、本方法において、生体毛包組織に含まれる非恒常部の重量割合が０％である場合、すなわち、原料細胞の細胞源として非恒常部を含まない成体毛包組織を使用する場合、上述のようにして、分散された毛乳頭細胞と、分散された当該原料細胞とを混合して培養することにより、動物に移植された場合に白色の毛が生える毛包原基を製造することができる。 In this method, when the weight ratio of the non-constant part contained in the living hair follicle tissue is 0%, that is, when an adult hair follicle tissue that does not contain a non-constant part is used as the cell source of the source cells, Thus, by mixing and cultivating the dispersed hair papilla cells and the dispersed raw material cells, a hair follicle primordium that produces white hair when transplanted to an animal can be produced. it can.
すなわち、本方法によれば、バルジ領域に加え、サブバルジ領域を含む成体毛包組織から得られた原料細胞を使用することにより、実質的に着色されていない毛が生える毛包原基が製造される。 That is, according to this method, by using raw material cells obtained from an adult hair follicle tissue containing a sub-bulge region in addition to a bulge region, a hair follicle primordium in which hairs that are substantially uncolored grow is produced. The
したがって、例えば、このように白色の毛が生える条件と、着色された毛が生える条件とを採用する場合、本方法は、毛の色の制御に関する知見を得るための研究ツールとして利用することができる。また、本方法は、白色の毛を人工的に製造するために利用することもできる。 Therefore, for example, when adopting the conditions under which white hair grows and the conditions under which colored hair grows, the present method can be used as a research tool for obtaining knowledge relating to hair color control. it can. The method can also be used to artificially produce white hair.
また、第一の重量割合が０％超である場合、当該第一の重量割合に対する第二の重量割合の比は、１超であれば特に限られないが、例えば、１．５以上であることとしてもよく、２．０以上であることとしてもよく、５．０以上であることとしてもよく、１０．０以上であることとしてもよい。 Further, when the first weight ratio is greater than 0%, the ratio of the second weight ratio to the first weight ratio is not particularly limited as long as it is greater than 1, but is, for example, 1.5 or more. It is good also as being 2.0 or more, it is good also as being 5.0 or more, and good also as being 10.0 or more.
次に、本実施形態に係る具体的な実施例について説明する。 Next, specific examples according to the present embodiment will be described.
［毛乳頭細胞の採取］
成体毛包組織の毛乳頭から毛乳頭細胞を採取した。すなわち、まず７週齢の成体Ｃ５７ＢＬ／６マウスより髭を採取し、当該髭の周囲の脂肪組織を取り除いた。次いで、髭毛包より毛乳頭を切り分けた。この毛乳頭を１００Ｕ／ｍＬのコラゲナーゼと４．８Ｕ／ｍＬのディスパーゼとを含む酵素溶液に浸漬して、ＣＯ２インキュベータ内、３７℃で４０分間、酵素処理を行った。酵素処理によって毛乳頭から単離された細胞を培養ディッシュに播種して、１０ｎｇ／ｍＬ又は１００ｎｇ／ｍＬのＦＧＦ２を含む培地中で培養した。培養後の細胞をトリプシン−ＥＤＴＡ溶液を用いて回収し、実験に使用した。
[Collecting dermal papilla cells]
Papilla cells were collected from the dermal papilla of adult hair follicle tissue. That is, first, pupae were collected from 7-week-old adult C57BL / 6 mice, and adipose tissue around the pupae was removed. Next, the hair papilla was cut out from the eyelash follicle. The hair papilla was immersed in an enzyme solution containing 100 U / mL collagenase and 4.8 U / mL dispase and subjected to enzyme treatment in a CO 2 incubator at 37 ° C. for 40 minutes. Cells isolated from the hair papilla by enzyme treatment were seeded in a culture dish and cultured in a medium containing 10 ng / mL or 100 ng / mL FGF2. The cultured cells were collected using a trypsin-EDTA solution and used for experiments.
［原料細胞の採取］
バルジ領域を含む恒常部と、毛乳頭及び毛母が除去された非恒常部とを含む成体毛包組織から、上皮幹細胞を含む原料細胞を採取した。すなわち、まず７週齢の成体Ｃ５７ＢＬ／６マウスより髭を採取し、当該髭の周囲の脂肪組織を取り除いた。次いで、髭毛包より毛乳頭及び毛母を除去した。毛乳頭及び毛母が除去された毛包組織を、１００Ｕ／ｍＬのコラゲナーゼと４．８Ｕ／ｍＬのディスパーゼとを含む酵素溶液に浸漬して、ＣＯ２インキュベータ内、３７℃で１０分間、酵素処理を行った。その後、毛包組織を、ＤＭＥＭ培地にＨＥＰＥＳを混合して調製されたＤＭＥＭ／ＨＥＰＥＳ培地中に移し、当該毛包組織からコラーゲン鞘を取り除いた。さらに、毛包組織にトリプシン溶液に浸漬して、ＣＯ２インキュベータ内、３７℃で６０分間、酵素処理を行った。その後、酵素処理によって毛包組織から単離された細胞をピペッティングで分散させ、さらに７０μｍセルストレーナーによる単一化処理を施し、得られた細胞を実験に使用した。
[Collection of source cells]
Raw material cells containing epithelial stem cells were collected from an adult hair follicle tissue containing a constant part including a bulge region and a non-constant part from which the hair papilla and hair matrix were removed. That is, first, pupae were collected from 7-week-old adult C57BL / 6 mice, and adipose tissue around the pupae was removed. Next, the hair papilla and hair matrix were removed from the eyelash follicle. The hair follicle tissue from which the hair papilla and hair matrix have been removed is immersed in an enzyme solution containing 100 U / mL collagenase and 4.8 U / mL dispase, and the enzyme treatment is performed at 37 ° C. for 10 minutes in a CO 2 incubator. Went. Thereafter, the hair follicle tissue was transferred to a DMEM / HEPES medium prepared by mixing HEPES in a DMEM medium, and the collagen sheath was removed from the hair follicle tissue. Furthermore, it was immersed in a trypsin solution in a hair follicle tissue, and an enzyme treatment was performed at 37 ° C. for 60 minutes in a CO 2 incubator. Thereafter, the cells isolated from the hair follicle tissue by the enzyme treatment were dispersed by pipetting, and further subjected to a unification treatment with a 70 μm cell strainer, and the obtained cells were used for experiments.
［培養毛乳頭細胞の顕微鏡観察］
図２Ａ及び図２Ｂには、それぞれ１０ｎｇ／ｍＬ及び１００ｎｇ／ｍＬのＦＧＦ２を含む培地中で２日間培養した毛乳頭細胞の位相差顕微鏡写真を示す。また、図２Ｃ及び図２Ｄには、それぞれ１０ｎｇ／ｍＬ及び１００ｎｇ／ｍＬのＦＧＦ２を含む培地中で９日間培養した毛乳頭細胞の位相差顕微鏡写真を示す。図２Ａ〜図２Ｄのいずれにおいても、毛乳頭から同心円状に広がるように細胞が増殖する様子が観察された。
[Microscopic observation of cultured dermal papilla cells]
2A and 2B show phase contrast micrographs of hair papilla cells cultured for 2 days in a medium containing 10 ng / mL and 100 ng / mL FGF2, respectively. 2C and 2D show phase contrast micrographs of hair papilla cells cultured for 9 days in a medium containing 10 ng / mL and 100 ng / mL FGF2, respectively. In any of FIGS. 2A to 2D, it was observed that the cells proliferated so as to spread concentrically from the dermal papilla.
［毛乳頭細胞及び上皮幹細胞の特定］
図３Ａには、上述のようにして成体毛包組織の毛乳頭から採取され培養された細胞に、毛乳頭細胞のマーカータンパク質であるＶｅｒｓｉｃａｎの免疫組織化学染色を施して、蛍光顕微鏡で観察した結果を示す。図３Ａに示すように、Ｖｅｒｓｉｃａｎ／ＤＡＰＩの蛍光二重染色によって、Ｖｅｒｓｉｃａｎを発現する細胞の存在が確認された（図中の矢印は、Ｖｅｒｓｉｃａｎを発現する細胞の一つを指している。）。すなわち、成体毛包組織の毛乳頭から毛乳頭細胞が得られたことが確認された。
[Identification of dermal papilla cells and epithelial stem cells]
FIG. 3A shows a result obtained by applying immunohistochemical staining of Versican, a marker protein of hair papilla cells, to cells collected from the hair papilla of adult hair follicle tissue and cultured as described above, and observed with a fluorescence microscope. Indicates. As shown in FIG. 3A, the presence of cells expressing Versican was confirmed by fluorescent double staining of Versican / DAPI (the arrows in the figure indicate one of the cells expressing Versican). That is, it was confirmed that dermal papilla cells were obtained from the dermal papilla of the adult hair follicle tissue.
図３Ｂには、上述のようにして毛乳頭及び毛母が除去された成体毛包組織から採取された細胞に、毛包上皮幹細胞のマーカータンパク質であるＣＤ３４の免疫組織化学染色を施して、蛍光顕微鏡で観察した結果を示す。図３Ｂに示すように、ＣＤ３４／ＤＡＰＩの蛍光二重染色によって、ＣＤ３４を発現する細胞の存在が確認された（図中の矢印は、ＣＤ３４を発現する細胞の一つを指している。）。すなわち、バルジ領域を含む恒常部と、毛乳頭及び毛母が除去された非恒常部とを含む成体毛包組織から得られた原料細胞には、上皮幹細胞が含まれていることが確認された。 FIG. 3B shows that cells collected from the adult hair follicle tissue from which the hair papilla and hair matrix were removed as described above were subjected to immunohistochemical staining of CD34, which is a marker protein of hair follicle epithelial stem cells. The result observed with the microscope is shown. As shown in FIG. 3B, the presence of cells expressing CD34 was confirmed by fluorescent double staining of CD34 / DAPI (the arrow in the figure indicates one of the cells expressing CD34). That is, it was confirmed that epithelial stem cells were contained in the raw material cells obtained from the adult hair follicle tissue including the constitutive part including the bulge region and the non-constant part from which the hair papilla and hair matrix were removed. .
［マルチウェル培養容器の作製］
毛乳頭細胞と原料細胞とを共培養するためのマルチウェル培養容器を、上述した特許文献２と同様にして作製した。すなわち、まずＣＡＤソフト（Ｖ Ｃａｒｖｅ Ｐｒｏ ６．５）を用いて、作製するマルチウェルのパターンをコンピューターで設計した。次いで、切削機を用いて、設計したパターン通りにオレフィン系樹脂基板を切削することで、当該パターンを有する凹鋳型を作製した。この鋳型にエポキシ樹脂（クリスタルリジン、日新レジン株式会社製）を流し込み、１日硬化させ、その後、離型することで、上述の設計されたパターンを有する凸鋳型を形成した。形成した凸鋳型を２４ウェルプレートの底面に固定し、ポリジメチルシロキサン（ＰＤＭＳ）を流し込んで固化し、その後、離型することで、ＰＤＭＳ基板に規則的なパターンで形成されたマルチウェル（各ウェルの直径は１ｍｍ、深さは１ｍｍ）を有するマルチウェル培養容器（以下、「ＰＤＭＳスフェロイドチップ」という。）を作製した。
[Production of multiwell culture vessel]
A multi-well culture container for co-culturing hair papilla cells and raw material cells was prepared in the same manner as in Patent Document 2 described above. That is, first, CAD software (V Carve Pro 6.5) was used to design a multiwell pattern to be produced by a computer. Next, a concave mold having the pattern was manufactured by cutting the olefin resin substrate according to the designed pattern using a cutting machine. An epoxy resin (Crystal lysine, manufactured by Nissin Resin Co., Ltd.) was poured into this mold, cured for 1 day, and then released to form a convex mold having the above designed pattern. The formed convex mold is fixed to the bottom surface of a 24-well plate, and polydimethylsiloxane (PDMS) is poured and solidified, and then released to form a multiwell (each well formed in a regular pattern on the PDMS substrate). Was prepared in a multiwell culture container (hereinafter referred to as “PDMS spheroid chip”) having a diameter of 1 mm and a depth of 1 mm.
このＰＤＭＳスフェロイドチップは、酸素透過性に優れたＰＤＭＳ製の基板にウェルが形成されているため、当該ウェル内で培養される細胞、及び細胞凝集塊には、培養期間を通じて、適切な量の酸素が供給された。 In this PDMS spheroid chip, since a well is formed on a PDMS substrate having excellent oxygen permeability, an appropriate amount of oxygen is contained in cells and cell aggregates cultured in the well throughout the culture period. Was supplied.
［毛乳頭細胞の採取］
上述の実施例１と同様に、成体毛包組織の毛乳頭から毛乳頭細胞を採取し培養した。
[Collecting dermal papilla cells]
As in Example 1 above, dermal papilla cells were collected from the dermal papilla of adult hair follicle tissue and cultured.
［原料細胞の採取］
上述の実施例１と同様に、バルジ領域を含む恒常部と、毛乳頭及び毛母が除去された非恒常部とを含む成体毛包組織から、上皮幹細胞を含む原料細胞（本実施例において「原料細胞（Ｆｕｌｌ）」という。）を採取した。
[Collection of source cells]
Similar to Example 1 above, from an adult hair follicle tissue containing a constitutive part containing a bulge region and a non-constant part from which the hair papilla and hair matrix were removed, a source cell containing epithelial stem cells (in this example, “ "Source cells (Full)") were collected.
また、非恒常部が除去された成体毛包組織（すなわち、バルジ領域及びサブバルジ領域を含み、非恒常部を含まない成体毛包組織）から、上皮幹細胞を含む原料細胞（本実施例において「原料細胞（１／２）」という。）を採取した。すなわち、まず７週齢の成体Ｃ５７ＢＬ／６マウスより髭を採取し、当該髭の周囲の脂肪組織を取り除いた。次いで、髭毛包の恒常部より下側の部分（すなわち、サブバルジ領域より下の部分）を切断することにより、当該髭毛包から毛乳頭及び毛母を含む非恒常部を除去した。 In addition, from an adult hair follicle tissue from which a non-constant part has been removed (that is, an adult hair follicle tissue including a bulge region and a sub-bulge region but not including a non-constant part), a source cell containing epithelial stem cells (in this example, “source material” Cells (1/2) ") were collected. That is, first, pupae were collected from 7-week-old adult C57BL / 6 mice, and adipose tissue around the pupae was removed. Subsequently, the non-constant part including a hair papilla and a hair mother was removed from the eyelash follicle by cutting a part below the constant part of the eyelash follicle (that is, a part below the subbulge region).
次いで、非恒常部が除去された毛包組織を、１００Ｕ／ｍＬのコラゲナーゼと４．８Ｕ／ｍＬのディスパーゼとを含む酵素溶液に浸漬して、ＣＯ２インキュベータ内、３７℃で１０分間、酵素処理を行った。その後、毛包組織を、ＤＭＥＭ培地にＨＥＰＥＳを混合して調製されたＤＭＥＭ／ＨＥＰＥＳ培地中に移し、当該毛包組織からコラーゲン鞘を取り除いた。さらに、毛包組織にトリプシン溶液に浸漬して、ＣＯ２インキュベータ内、３７℃で６０分間、酵素処理を行った。その後、酵素処理によって毛包組織から単離された細胞をピペッティングで分散させ、さらに７０μｍセルストレーナーによる単一化処理を施し、得られた細胞を「原料細胞（１／２）」として実験に使用した。 Next, the hair follicle tissue from which the non-constant parts have been removed is immersed in an enzyme solution containing 100 U / mL collagenase and 4.8 U / mL dispase, and the enzyme treatment is performed at 37 ° C. for 10 minutes in a CO 2 incubator. Went. Thereafter, the hair follicle tissue was transferred to a DMEM / HEPES medium prepared by mixing HEPES in a DMEM medium, and the collagen sheath was removed from the hair follicle tissue. Furthermore, it was immersed in a trypsin solution in a hair follicle tissue, and an enzyme treatment was performed at 37 ° C. for 60 minutes in a CO 2 incubator. Thereafter, the cells isolated from the hair follicle tissue by enzyme treatment are dispersed by pipetting, and further subjected to the unification treatment by a 70 μm cell strainer, and the obtained cells are used as “source cells (1/2)” for experiments used.
［毛包原基の製造］
毛包原基の培養に使用する培地として、間葉細胞培養培地（ＤＭＥＭ＋１０％ＦＢＳ＋１％Ｐ／Ｓ（Ｐｅｎｉｃｉｌｌｉｎ−Ｓｔｒｅｐｔｏｍｙｃｉｎ））とＨｕＭｅｄｉａＫＧ２培地とを体積比１：１で混合し、ＦＧＦ２を１０ｎｇ／ｍＬ添加することにより、混合培地を調製した。
[Manufacture of hair follicle primordium]
As a medium used for culturing hair follicle primordia, a mesenchymal cell culture medium (DMEM + 10% FBS + 1% P / S (Penicillin-Streptomycin)) and HuMediaKG2 medium are mixed at a volume ratio of 1: 1, and FGF2 is mixed with 10 ng / mL. A mixed medium was prepared by addition.
上述のようにして得られた毛乳頭細胞を混合培地に分散して、毛乳頭細胞懸濁液を調製した。また、上述のようにして得られた原料細胞を混合培地に分散して、原料細胞懸濁液を調製した。そして、毛乳頭細胞懸濁液と原料細胞懸濁液とを混合して、分散された毛乳頭細胞及び原料細胞を含む混合細胞懸濁液を調製した。 The dermal papilla cells obtained as described above were dispersed in a mixed medium to prepare a dermal papilla cell suspension. Moreover, the raw material cells obtained as described above were dispersed in a mixed medium to prepare a raw material cell suspension. Then, the dermal papilla cell suspension and the raw material cell suspension were mixed to prepare a mixed cell suspension containing the dispersed dermal papilla cells and the raw material cells.
上述のようにして作製したマルチウェル培養容器の各ウェルに、毛乳頭細胞及び毛包上皮幹細胞それぞれの細胞密度が１×１０４ｃｅｌｌｓ／ｗｅｌｌ（全細胞数２×１０３ｃｅｌｌｓ／ｗｅｌｌ）となるように、混合細胞懸濁液を播種し、ＣＯ２インキュベータ内、３７℃で３日間、培養を行った。 In each well of the multi-well culture container prepared as described above, the cell density of the hair papilla cells and hair follicle epithelial stem cells is 1 × 10 4 cells / well (total number of cells 2 × 10 3 cells / well). As described above, the mixed cell suspension was seeded and cultured in a CO 2 incubator at 37 ° C. for 3 days.
培養３日目にウェル内を位相差顕微鏡で観察したところ、当該ウェル内に播種された細胞が凝集して球状の細胞凝集塊（いわゆるスフェロイドと呼ばれる。）が形成されていることが確認された。なお、予備的な検討において、この細胞凝集塊は、主に毛乳頭細胞を含む毛乳頭細胞凝集部と、当該毛乳頭細胞凝集部と結合し、主に上皮幹細胞を含む上皮系細胞凝集部とを含むことが確認された。 When the inside of the well was observed with a phase-contrast microscope on the third day of culture, it was confirmed that the cells seeded in the well aggregated to form a spherical cell aggregate (so-called spheroid). . In a preliminary study, this cell aggregate is mainly composed of a hair papilla cell aggregate part containing hair papilla cells, and a epithelial cell aggregate part mainly binding to the hair papilla cell aggregate part and mainly containing epithelial stem cells. It was confirmed to contain.
このような細胞凝集塊の形成は、上述のように、分散された毛乳頭細胞と、分散された原料細胞とを混合して、非接着性の培養容器内で培養することにより、例えば、同種の細胞同士が自発的に集合して各細胞凝集部を形成することにより達成されるものと推測される。 As described above, such cell aggregates can be formed by mixing dispersed hair papilla cells and dispersed raw material cells and culturing them in a non-adhesive culture vessel. This is presumed to be achieved by spontaneously gathering cells together to form each cell aggregate.
［毛包原基の移植］
３日間の培養により形成された毛包原基を回収し、注射器を用いて、５週齢のＳＣＩＤ−ｎｕマウスの皮下に直接注入することにより、当該毛包原基を移植した。
[Transplantation of hair follicle primordia]
The hair follicle primordium formed by culturing for 3 days was collected, and the hair follicle primordium was transplanted by direct injection into a 5-week-old SCID-nu mouse subcutaneously using a syringe.
［再生毛の観察］
移植後３週間目に、マウスの皮下に移植された毛包原基をデジタルマイクロスコープにより観察した。
[Observation of regenerated hair]
Three weeks after transplantation, the hair follicle primordium implanted subcutaneously in the mouse was observed with a digital microscope.
図４Ａ及び図４Ｂには、それぞれ原料細胞（１／２）を使用して製造された毛包原基、及び原料細胞（Ｆｕｌｌ）を使用して製造された毛包原基を観察した結果を示す。各図において、矢印は、移植された毛包原基から生えた毛を指している。 FIG. 4A and FIG. 4B show the results of observing the hair follicle primordium produced using the raw material cells (1/2) and the hair follicle primordium produced using the raw material cells (Full), respectively. Show. In each figure, the arrow points to the hair grown from the transplanted hair follicle primordium.
図４Ａに示すように、非恒常部を含まない成体毛包組織から得られた原料細胞（１／２）を使用して製造された毛包原基からは、白色の毛が生えていることが確認された。一方、図４Ｂに示すように、毛乳頭及び毛母が除去された非恒常部を含む成体毛包組織から得られた原料細胞（Ｆｕｌｌ）を使用して製造された毛包原基からは、着色された黒っぽい毛が生えていることが確認された。 As shown in FIG. 4A, white hair grows from the hair follicle primordium produced using the raw material cells (1/2) obtained from the adult hair follicle tissue not containing the non-constant part. Was confirmed. On the other hand, as shown in FIG. 4B, from a hair follicle primordium produced using raw material cells (Full) obtained from an adult hair follicle tissue containing a non-constant part from which the hair papilla and hair matrix are removed, It was confirmed that colored dark hair grew.
すなわち、毛乳頭及び毛母以外の非恒常部を含む成体毛包組織から得られた原料細胞（Ｆｕｌｌ）を使用することにより、非恒常部を含まない成体毛包組織から得られた原料細胞（１／２）を使用する場合よりも色が濃い毛が生える毛包原基を製造できることが確認された。 That is, by using a raw material cell (Full) obtained from an adult hair follicle tissue containing non-constant parts other than the hair papilla and hair matrix, a raw material cell obtained from an adult hair follicle tissue containing no non-constant part ( It was confirmed that a hair follicle primordium having darker hair than that in the case of using 1/2) can be produced.
このように、原料細胞の細胞源である成体毛包組織における非恒常部（特に、毛乳頭及び毛母以外の非恒常部）の含有量が、当該原料細胞を使用して形成される毛包原基から生える毛の色を変化させるメカニズムは明らかではないが、例えば、当該非恒常部の含有量によって、当該毛包原基に含まれる、色素細胞等の毛色を制御する細胞の含有量及び／又は分布が変化することによるものと推測される。 Thus, the hair follicle formed by using the raw material cells for the content of non-constant parts (especially non-constant parts other than the hair papilla and hair matrix) in the adult hair follicle tissue that is the cell source of the raw material cells. Although the mechanism for changing the color of hair growing from the primordia is not clear, for example, depending on the content of the non-constant part, the content of cells that control hair color such as pigment cells contained in the hair follicle primordium and It is presumed that the distribution is changed.
［毛乳頭細胞の採取］
上述の実施例１と同様に、成体毛包組織の毛乳頭から毛乳頭細胞を採取し培養した。
[Collecting dermal papilla cells]
As in Example 1 above, dermal papilla cells were collected from the dermal papilla of adult hair follicle tissue and cultured.
［原料細胞の採取］
上述の実施例１と同様に、バルジ領域を含む恒常部と、毛乳頭及び毛母が除去された非恒常部とを含む成体毛包組織から、上皮幹細胞を含む原料細胞を採取した。
[Collection of source cells]
In the same manner as in Example 1 above, raw material cells containing epithelial stem cells were collected from an adult hair follicle tissue containing a constant part including the bulge region and a non-constant part from which the hair papilla and hair matrix were removed.
［毛包原基の製造］
毛包原基の培養に使用する培地として、上述の実施例２で使用した混合培地にＦＧＦ２を１０ｎｇ／ｍＬ、５０ｎｇ／ｍＬ、又は１００ｎｇ／ｍＬ添加したＦＧＦ２含有混合培地を使用したこと以外は上述の実施例２と同様にして、マルチウェル培養容器を使用して、毛包原基を製造した。
[Manufacture of hair follicle primordium]
As described above, except that FGF2 containing mixed medium in which 10 ng / mL, 50 ng / mL, or 100 ng / mL of FGF2 was added to the mixed medium used in Example 2 was used as the medium used for culturing hair follicle primordium. In the same manner as in Example 2, a hair follicle primordium was produced using a multiwell culture vessel.
［毛包原基の顕微鏡観察］
図５Ａ、図５Ｂ及び図５Ｃには、それぞれ１０ｎｇ／ｍＬ、５０ｎｇ／ｍＬ、及び１００ｎｇ／ｍＬのＦＧＦ２を含む混合培地中の培養で形成された毛包原基を位相差顕微鏡で観察した結果を示す。図５Ａ〜図５Ｃにおけるスケールバーは５００μｍを示す。
[Microscopic observation of hair follicle primordium]
FIG. 5A, FIG. 5B, and FIG. 5C show the results of observation of the hair follicle primordium formed by culturing in a mixed medium containing 10 ng / mL, 50 ng / mL, and 100 ng / mL of FGF2 with a phase contrast microscope, respectively. Show. The scale bar in FIGS. 5A to 5C indicates 500 μm.
図５Ｄ、図５Ｅ及び図５Ｆには、それぞれ１０ｎｇ／ｍＬ、５０ｎｇ／ｍＬ、及び１００ｎｇ／ｍＬのＦＧＦ２を含む混合培地中の培養で形成された毛包原基を位相差顕微鏡で観察した結果を示す。図５Ｄ〜図５Ｆにおけるスケールバーは１ｍｍを示す。 FIG. 5D, FIG. 5E, and FIG. 5F show the results of observation of the hair follicle primordium formed by culturing in a mixed medium containing 10 ng / mL, 50 ng / mL, and 100 ng / mL FGF2 with a phase contrast microscope, respectively. Show. The scale bar in FIGS. 5D to 5F indicates 1 mm.
図５Ａ〜図５Ｆに示すように、３日間の培養で形成された毛包原基の形態は、混合培地のＦＧＦ２含有量によって違いは見られなかった。 As shown in FIGS. 5A to 5F, no difference was observed in the form of the hair follicle primordium formed in the culture for 3 days depending on the FGF2 content of the mixed medium.
［発毛に関する遺伝子発現の解析］
３日間の培養で形成された毛包原基における発毛マーカー遺伝子（Ｖｅｒｓｉｃａｎ及びＷｎｔ−１０ｂ）の発現について、ＲＴ−ＰＣＲ解析を行った。すなわち、まず毛包原基からＲＮＡを抽出した。具体的に、毛包原基を１５ｍＬチューブに回収し、当該毛包原基が沈殿したら、溶液の体積が１ｍＬとなるように上清を除去した。次いで、毛包原基を含む溶液１ｍＬを１．５ｍＬマイクロチューブに移し替えて、４℃、５０００ｒｐｍで、３分間遠心した。
[Analysis of gene expression related to hair growth]
RT-PCR analysis was performed on the expression of hair growth marker genes (Versican and Wnt-10b) in the hair follicle primordium formed by 3 days of culture. That is, RNA was first extracted from the hair follicle primordium. Specifically, the hair follicle primordium was collected in a 15 mL tube, and when the hair follicle primordium was precipitated, the supernatant was removed so that the volume of the solution was 1 mL. Next, 1 mL of the solution containing the hair follicle primordium was transferred to a 1.5 mL microtube and centrifuged at 4 ° C. and 5000 rpm for 3 minutes.
遠心後の溶液の上清を捨てた後、３５０μＬのＢｕｆｆｅｒ ＲＬＴを加え、よくピペッティングした。さらに、ピペッティング後の溶液をＱＩＡ Ｓｈｒｅｄｄｅｒスピンカラムに回収し、４℃、１００００ｒｐｍで、２分間遠心した。ＱＩＡ Ｓｈｒｅｄｄｅｒスピンカラムの上部を捨て、コレクションチューブ内の溶液に７０％エタノールを３５０μＬ加えた。溶液をＲＮｅａｓｙスピンカラムに移し、４℃、１００００ｒｐｍで、１５秒間遠心した。 After discarding the supernatant of the solution after centrifugation, 350 μL of Buffer RLT was added and well pipetted. Further, the solution after pipetting was collected on a QIA Shredder spin column and centrifuged at 4 ° C. and 10,000 rpm for 2 minutes. The top of the QIA Shredder spin column was discarded and 350 μL of 70% ethanol was added to the solution in the collection tube. The solution was transferred to an RNeasy spin column and centrifuged at 10000 rpm at 4 ° C. for 15 seconds.
コレクションチューブ内の濾液を捨て、７００μＬのＢｕｆｆｅｒ ＲＷ１を加え、４℃、１００００ｒｐｍで、１５秒間遠心した。コレクションチューブ内の濾液を捨て、５００μＬのＢｕｆｆｅｒ ＲＰＥを加え、４℃、１００００ｒｐｍで、１５秒間遠心した。コレクションチューブ内の濾液を捨て、５００μＬのＢｕｆｆｅｒ ＲＰＥを加え、４℃、１００００ｒｐｍで、２分間遠心した。遠心後のカラムを新しい２ｍＬコレクションチューブに移し、４℃、１００００ｒｐｍで、１分間遠心した。これは、残存するＢｕｆｆｅｒ ＲＰＥを除去するために行った。 The filtrate in the collection tube was discarded, 700 μL of Buffer RW1 was added, and the mixture was centrifuged at 4 ° C. and 10,000 rpm for 15 seconds. The filtrate in the collection tube was discarded, 500 μL of Buffer RPE was added, and the mixture was centrifuged at 4 ° C. and 10,000 rpm for 15 seconds. The filtrate in the collection tube was discarded, 500 μL of Buffer RPE was added, and the mixture was centrifuged at 4 ° C. and 10,000 rpm for 2 minutes. The column after centrifugation was transferred to a new 2 mL collection tube and centrifuged at 4 ° C. and 10,000 rpm for 1 minute. This was done to remove any remaining Buffer RPE.
遠心後のカラムを１ｍＬマイクロチューブに移し、ＲＮａｓｅ ｆｒｅｅ ｗａｔｅｒを３０μＬ加え、４℃、１００００ｒｐｍで、１分間遠心した。遠心後のカラムが設置された１ｍＬマイクロチューブに、再度ＲＮａｓｅ ｆｒｅｅ ｗａｔｅｒを３０μＬ加え、４℃、１００００ｒｐｍで、１分間遠心し、ＲＮＡ溶解液を得た。 The column after centrifugation was transferred to a 1 mL microtube, 30 μL of RNase free water was added, and the mixture was centrifuged at 10000 rpm at 4 ° C. for 1 minute. 30 μL of RNase free water was again added to the 1 mL microtube on which the column after centrifugation was installed, and centrifuged at 10000 rpm at 4 ° C. for 1 minute to obtain an RNA solution.
次いで、分光光度計によりＲＮＡ濃度を測定した。すなわち、分光光度計（Ｎａｎｏ Ｖｕｅ）の電源を付け、希釈率を６０．０に設定した。ここで、希釈率とは、ＲＮＡ抽出における最終容量である。測定板を７０％エタノールで拭いた後、２μＬのＲＮａｓｅ ｆｒｅｅ ｗａｔｅｒを当該測定板の中心にアプライし、［ＯＡ／１００％Ｔ］のボタンを押した。この操作でベースラインを得た。 The RNA concentration was then measured with a spectrophotometer. That is, the spectrophotometer (Nano Vue) was turned on and the dilution rate was set to 60.0. Here, the dilution rate is the final volume in RNA extraction. After the measurement plate was wiped with 70% ethanol, 2 μL of RNase free water was applied to the center of the measurement plate, and the [OA / 100% T] button was pressed. A baseline was obtained by this operation.
上述のようにして毛包原基から得られたＲＮＡ溶解液２μＬを測定板の中心にアプライし、測定ボタンを押した。Ａ２６０／Ａ２８０はサンプルの純度を表し、２．０に近い方が好ましい。 2 μL of RNA lysate obtained from the hair follicle primordium as described above was applied to the center of the measurement plate, and the measurement button was pressed. A260 / A280 represents the purity of the sample and is preferably close to 2.0.
その後、ＲＴ−ＰＣＲを行った。すなわち、上述のようにしてＲＮＡ濃度を測定したＲＮＡ溶解液を、そのＲＮＡ濃度が１５０μｇ／ｍＬとなるように希釈した。希釈後のＲＮＡ溶解液を、６５℃で、５分間インキュベートし、氷上で冷却した。 Thereafter, RT-PCR was performed. That is, the RNA lysate whose RNA concentration was measured as described above was diluted so that the RNA concentration became 150 μg / mL. The diluted RNA lysate was incubated at 65 ° C. for 5 minutes and cooled on ice.
その後、２μＬのＲＮＡ溶解液と、１２μＬのＮｕｃｌｅａｒ ｆｒｅｅ ｗａｔｅｒと、４μＬの５×ＲＴ Ｂｕｆｆｅｒと、１μＬのＰｒｉｍｉｅｒ ｍｉｘと、１μＬのＥｎｚｙｍｅ ｍｉｘとをマイクロチューブに入れて、透明フィルムで覆った。 Thereafter, 2 μL of RNA lysate, 12 μL of Nuclear free water, 4 μL of 5 × RT Buffer, 1 μL of Primer mix, and 1 μL of Enzyme mix were placed in a microtube and covered with a transparent film.
マイクロチューブをサーマルサイクラーにセットし、しっかり閉まっていることを確認した。その後、３７℃で１５分間、９８℃で５分間の逆転写反応を行い、毛包原基由来のＲＮＡの逆転写産物であるｃＤＮＡを得た。 The microtube was set on a thermal cycler and confirmed to be tightly closed. Thereafter, reverse transcription was performed at 37 ° C. for 15 minutes and at 98 ° C. for 5 minutes to obtain cDNA, which is a reverse transcription product of RNA derived from hair follicle primordia.
その後、１μＬのｃＤＮＡ（毛包原基由来のＲＮＡの逆転写産物）と、１０μＬのＳＹＢＲ Ｇｒｅｅｎ ｍａｓｔｅｒ ｍｉｘと、０．４μＬのＦｏｒｗａｒｄ Ｐｒｉｍｅｒと、０．４μＬのＲｅｖｅｒｓｅ Ｐｒｉｍｅｒと、０．４μＬのＤｙｅと、７．８μＬのＮｕｃｌｅａｒ ｆｒｅｅ ｗａｔｅｒとをマイクロチューブに入れて、透明フィルムで覆った。 Thereafter, 1 μL of cDNA (reverse transcript of RNA derived from hair follicle primordium), 10 μL of SYBR Green master mix, 0.4 μL of Forward Primer, 0.4 μL of Reverse Primer, 0.4 μL of Dye, , 7.8 μL of Nuclear free water was placed in a microtube and covered with a transparent film.
なお、ＰＣＲで用いたプライマーの塩基配列は、Ｖｅｒｓｉｃａｎについて、Ｆｏｒｗａｒｄ Ｐｒｉｍｅｒは「５‘−ＧＡＣＧＡＣＴＧＴＣＴＴＧＧＴＧＧ−３’」、Ｒｅｖｅｒｓｅ Ｐｒｉｍｅｒは「５‘−ＡＴＡＴＣＣＡＡＡＣＡＡＧＣＣＴＧ−３’」、Ｗｎｔ−１０ｂについて、Ｆｏｒｗａｒｄ Ｐｒｉｍｅｒは「５‘−ＣＣＡＡＧＡＧＣＣＧＧＧＣＣＣＧＡＧＴＧＡ−３’」、Ｒｅｖｅｒｓｅ Ｐｒｉｍｅｒは「５‘−ＡＡＧＧＧＣＧＧＡＧＧＣＣＧＡＧＡＣＣＧ−３’」、コントロールとして用いたＧＡＰＤＨについて、Ｆｏｒｗａｒｄ Ｐｒｉｍｅｒは「５‘−ＡＧＡＡＣＡＴＣＡＴＣＣＣＴＧＣＡＴＣＣ−３’」、Ｒｅｖｅｒｓｅ Ｐｒｉｍｅｒは「５‘−ＴＣＣＡＣＣＡＣＣＣＴＧＴＴＧＣＴＧＴＡ−３’」であった。 The base sequences of the primers used in the PCR are Versican, Forward Primer is “5′-GACGACTGTTCTGTGGG-3 ′”, Reverse Primer is “5′-ATATCCAAACAAGCCTG-3 ′”, and Wnt-10b is Forward Prime. 5'-CCAAGAGCCGGGCCCGAGTGA-3 '", Reverse Primer is" 5'-AAGGGCGGAGGGCCGAGACCG-3' ", Forward Primer is" 5'-AGAACATCATCCCTCATCSECC-3CT " -3 ′ ”.
マイクロチューブをサーマルサイクラーにセットし、しっかり閉まっていることを確認した。その後、９５℃で４分間、（９５℃で５秒間、６０℃で６０秒間）×４５サイクル、７２℃で１０分間、のプロトコールにてＰＣＲを行った。コントロールとして、ＧＡＰＤＨの発現量を測定し、ＧＡＰＤＨの発現量に対するＶｅｒｓｉｃａｎ及びＷｎｔ−１０ｂの相対的な発現量を算出した。 The microtube was set on a thermal cycler and confirmed to be tightly closed. Thereafter, PCR was performed with a protocol of 95 ° C. for 4 minutes, (95 ° C. for 5 seconds, 60 ° C. for 60 seconds) × 45 cycles, 72 ° C. for 10 minutes. As a control, the expression level of GAPDH was measured, and the relative expression levels of Versican and Wnt-10b with respect to the expression level of GAPDH were calculated.
図６Ａ及び図６Ｂには、それぞれ毛包原基におけるＶｅｒｓｉｃａｎ及びＷｎｔ−１０ｂの相対的な遺伝子発現量を示す。図６Ａ及び図６Ｂより、毛包原基におけるＶｅｒｓｉｃａｎ及びＷｎｔ−１０ｂの遺伝子発現量が、培地のＦＧＦ２濃度に依存して増加する傾向が確認された。 6A and 6B show the relative gene expression levels of Versican and Wnt-10b in the hair follicle primordia, respectively. 6A and 6B confirmed that the gene expression levels of Versican and Wnt-10b in the hair follicle primordia increased depending on the FGF2 concentration of the medium.
［毛包原基の移植］
３日間の培養により形成された毛包原基を回収し、パッチ法で、５週齢のＳＣＩＤ−ｎｕマウスの皮膚に移植した。すなわち、マウスの皮膚の真皮又は筋膜の付近に複数の毛包原基を注入した。
[Transplantation of hair follicle primordia]
The hair follicle primordium formed by the culture for 3 days was collected and transplanted to the skin of a 5-week-old SCID-nu mouse by the patch method. That is, a plurality of hair follicle primordia were injected in the vicinity of the dermis or fascia of mouse skin.
［再生毛の観察］
移植後６週間目に、マウスに移植された毛包原基をデジタルマイクロスコープにより観察した。図７Ａ及び図７Ｂには、それぞれ１０ｎｇ／ｍＬ及び１００ｎｇ／ｍＬのＦＧＦ２を含む培地中で形成された毛包原基を観察した結果を示す。各図において、矢印は、移植された毛包原基を指している。
[Observation of regenerated hair]
Six weeks after the transplantation, the hair follicle primordium transplanted into the mice was observed with a digital microscope. FIG. 7A and FIG. 7B show the results of observing hair follicle primordia formed in a medium containing 10 ng / mL and 100 ng / mL FGF2, respectively. In each figure, the arrow points to the transplanted hair follicle primordium.
図７Ａ及び図７Ｂより、１０ｎｇ／ｍＬのＦＧＦを含む培地中で形成された毛包原基に比べて、１００ｎｇ／ｍＬのＦＧＦを含む培地中で形成された毛包原基の方が、より高い毛髪再生能を示すことが確認された。 From FIG. 7A and FIG. 7B, the hair follicle primordium formed in the medium containing 100 ng / mL FGF is more preferable than the hair follicle primordium formed in the medium containing 10 ng / mL FGF. It was confirmed to show high hair regeneration ability.
Claims (11)
分散された毛乳頭細胞と、分散された前記原料細胞とを混合して培養することにより、毛包原基を製造すること、及び
前記毛包原基を動物に移植して前記毛包原基から毛を生やすこと、
を含み、
前記成体毛包組織に含まれる非恒常部の重量割合を変えることにより、前記毛包原基から生える毛の色を変える、再生毛の色制御方法。 Using raw material cells containing epithelial stem cells obtained from adult hair follicle tissue containing the bulge region of the homeostasis,
A hair follicle primordium is produced by mixing and culturing the dispersed hair papilla cells and the dispersed raw material cells, and transplanting the hair follicle primordia to an animal to produce the hair follicle primordium. Grow hair from
Including
A method for controlling the color of regenerated hair, wherein the color of hair grown from the hair follicle primordium is changed by changing the weight ratio of the non-constant part contained in the adult hair follicle tissue.
を含む、請求項１乃至３のいずれかに記載の再生毛の色制御方法。 The non-constant part includes parts other than the hair papilla and the hair mother,
The method for controlling the color of regenerated hair according to claim 1, comprising:
分散された毛乳頭細胞と、分散された前記原料細胞とを混合して培養することにより、毛包原基を製造すること、及び
前記毛包原基を動物に移植して前記毛包原基から毛を生やすこと、
を含み、
非恒常部をさらに含む前記成体毛包組織を使用することにより、前記毛包原基から、前記非恒常部を含まない成体毛包組織を使用する場合に比べて色が濃い毛を生やす、毛の再生方法。 Using raw material cells containing epithelial stem cells obtained from adult hair follicle tissue containing the bulge region of the homeostasis,
A hair follicle primordium is produced by mixing and culturing the dispersed hair papilla cells and the dispersed raw material cells, and transplanting the hair follicle primordia to an animal to produce the hair follicle primordium. Grow hair from
Including
By using the adult hair follicle tissue further comprising a non-constant part, hair having a darker color than the case of using the adult hair follicle tissue not containing the non-constant part from the hair follicle primordium is obtained. How to play.
前記非恒常部を前記第一の重量割合より大きい第二の重量割合で含む第二の成体毛包組織を使用して製造された第二の細胞凝集塊を前記動物に移植して、前記第二の毛包原基から前記第一の色より濃い第二の色の毛を生やすこと、
を含む、請求項５又は６に記載の毛の再生方法。 Transplanting the first hair follicle primordium produced using the first adult hair follicle tissue containing the non-constant part in a first weight ratio into the animal, Growing a first color hair, and a second cell aggregation produced using a second adult hair follicle tissue comprising the non-constant part in a second weight percentage greater than the first weight percentage. Transplanting a lump into the animal to grow a second colored hair darker than the first color from the second follicle primordium,
The method for regenerating hair according to claim 5 or 6, comprising:
を含む、請求項５乃至７のいずれかに記載の毛の再生方法。 The non-constant part includes parts other than the hair papilla and the hair mother,
The method for regenerating hair according to any one of claims 5 to 7, comprising:
分散された毛乳頭細胞と、分散された前記原料細胞とを混合して培養することにより、動物に移植されることで毛を生やす毛包原基を製造すること、
を含み、
非恒常部をさらに含む前記成体毛包組織を使用することにより、前記非恒常部を含まない成体毛包組織を使用する場合に比べて色が濃い毛を生やす前記毛包原基を製造する、毛包原基の製造方法。 Use of raw material cells including epithelial stem cells obtained from adult hair follicle tissue containing a bulge region in the homeostatic region, and mixing and culturing the dispersed hair papilla cells and the dispersed raw material cells To produce a hair follicle primordium that grows hair by being transplanted into an animal,
Including
By using the adult hair follicle tissue further comprising a non-constant part, the hair follicle primordium that produces darker hair than when using the adult hair follicle tissue not containing the non-constant part is produced. Manufacturing method of hair follicle primordium.
前記非恒常部を前記第一の重量割合より大きい第二の重量割合で含む前記成体毛包組織を使用することにより、前記動物に移植されることで前記第一の色より濃い第二の色の毛を生やす第二の毛包原基を製造すること、
を含む、請求項９に記載の毛包原基の製造方法。 By using the adult hair follicle tissue containing the non-constant part in a first weight ratio, a first hair follicle primordium that produces hair of the first color by being transplanted to the animal is produced. And a second darker than the first color by being transplanted into the animal by using the adult hair follicle tissue comprising the non-constant part in a second weight percentage greater than the first weight percentage. Producing a second hair follicle primordium that produces
The manufacturing method of the hair follicle primordium of Claim 9 containing this.
を含む、請求項９又は１０に記載の毛包原基の製造方法。
The non-constant part includes parts other than the hair papilla and the hair mother,
The manufacturing method of the hair follicle primordium of Claim 9 or 10 containing this.
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| WO2012115079A1 (en) * | 2011-02-24 | 2012-08-30 | 株式会社オーガンテクノロジーズ | Method of preparing regenerated hair follicle germ for transplantation in which hair color is controlled, composition including regenerated hair follicle germ for transplantation, and method of transplanting regenerated hair follicle germ |
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