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WO2019221307A1 - Méthode de contrôle de la couleur de poils régénérés, méthode de régénération des poils, et méthode de production d'ébauches de follicules pileux - Google Patents

Méthode de contrôle de la couleur de poils régénérés, méthode de régénération des poils, et méthode de production d'ébauches de follicules pileux Download PDF

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Publication number
WO2019221307A1
WO2019221307A1 PCT/JP2019/020185 JP2019020185W WO2019221307A1 WO 2019221307 A1 WO2019221307 A1 WO 2019221307A1 JP 2019020185 W JP2019020185 W JP 2019020185W WO 2019221307 A1 WO2019221307 A1 WO 2019221307A1
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Prior art keywords
hair
hair follicle
cells
adult
constant part
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PCT/JP2019/020185
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English (en)
Japanese (ja)
Inventor
福田　淳二
達斗 景山
知紗 吉村
陸満 中嶋
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Yokohama National University NUC
Kanagawa Institute of Industrial Science and Technology
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Yokohama National University NUC
Kanagawa Institute of Industrial Science and Technology
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Publication of WO2019221307A1 publication Critical patent/WO2019221307A1/fr
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Ceased legal-status Critical Current

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • the method for controlling the color of regenerated hair 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.
  • regeneration hair which can control the color of hair easily and effectively.
  • the animal may be a non-human animal.
  • 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
  • the said non-constant part is good also as including parts other than a hair nipple and a hair mother.
  • 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
  • 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.
  • the hair regeneration method which can control the color of hair easily and effectively.
  • the animal may be a non-human animal.
  • 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.
  • the said non-constant part is good also as including parts other than a hair nipple and a hair mother.
  • 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.
  • the manufacturing method of the hair follicle primordium which can control the color of hair easily and effectively is provided.
  • 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.
  • a color control method for regenerated hair a method for regenerating hair, and a method for producing a hair follicle primordium capable of easily and effectively controlling the color of the hair.
  • phase contrast micrograph of a hair follicle primordium formed in the presence of 10 ng / mL FGF2 using cells obtained from an adult hair follicle tissue containing a non-constant part in Example 3 according to the present embodiment. is there.
  • phase contrast micrograph of a hair follicle primordium formed in the presence of 50 ng / mL FGF2 using cells obtained from an adult hair follicle tissue containing a non-constant part in Example 3 according to the present embodiment. is there.
  • phase contrast micrograph of a hair follicle primordium formed in the presence of 100 ng / mL FGF2 using cells obtained from an adult hair follicle tissue containing a non-constant part in Example 3 according to the present embodiment. is there.
  • Other phase contrast micrograph of hair follicle primordium formed in the presence of 10 ng / mL FGF2 using cells obtained from adult hair follicle tissue containing non-constant part in Example 3 according to this embodiment It is an example.
  • Other phase contrast micrographs of hair follicle primordium formed in the presence of 50 ng / mL FGF2 using cells obtained from adult hair follicle tissue containing non-constant parts in Example 3 according to this embodiment It is an example.
  • phase contrast micrographs of hair follicle primordium formed in the presence of 100 ng / mL FGF2 using cells obtained from adult hair follicle tissue containing non-constant parts in Example 3 according to this embodiment It is an example. It is an example of the result of having evaluated the Versican gene expression in the hair follicle primordium formed using the cell obtained from the adult hair follicle structure
  • the present method 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.
  • 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.
  • 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.
  • still another aspect of the present 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 primordial which manufactures the said hair follicle primordium which grows a darker hair compared with the case where the adult hair follicle structure
  • 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.
  • the adult hair follicle tissue that wraps the hair shaft includes a constant part and a non-constant part.
  • 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).
  • 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).
  • FIG. 1B in the mouse, there is a ring wurst (Ringwurst) at a position corresponding to the napped muscle.
  • the bulge region contains epithelial stem cells.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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) carnivores (eg, dogs, cats), or ungulates (Eg, pig, cow, horse, goat, sheep).
  • 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.
  • 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).
  • it may be one or more selected from the group consisting of collagenase, dispase, and trypsin.
  • 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).
  • raw material cells are prepared from an adult hair follicle tissue containing a bulge region and a non-constant part.
  • 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.
  • 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.
  • 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.
  • the hair papilla cell is not particularly limited as long as it can form a part of the hair follicle primordium.
  • 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.
  • a hair follicle primordium is produced by mixing and culturing dispersed hair papilla cells and dispersed raw material cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the culture medium for mixing and culturing the dispersed dermal papilla cells and the dispersed raw material cells is not particularly limited.
  • 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.
  • 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 there is no particular limitation, for example, 5.0 ⁇ 10 5 cells / mL or more, 1.0 ⁇ 10 8 cells / mL or less, 2.5 ⁇ 10 6 cells / mL or more, 5.0 ⁇ 10 7 cells / mL or less, preferably 2.5 ⁇ 10 6 cells / mL or more, 2.5 ⁇ 10 7
  • the cell / mL or less is particularly preferable.
  • the culture vessel used for the mixed culture of the hair papilla cells and the raw material cells is not particularly limited.
  • 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.
  • 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.
  • 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, as a culture container, for example, the oxygen permeability is 100 cm. 3 / (M 2 It is preferably a recess formed in a base material of 24 h ⁇ atm) or more, and the oxygen permeability is 500 cm.
  • the recess is a recess formed in a base material of 24 h ⁇ atm) or more, and the oxygen permeability is 1000 cm. 3 / (M 2 It is particularly preferable that the recess is formed on a base material of 24 h ⁇ atm) or more.
  • a silicon-based material for example, polydimethylsiloxane (PDMS)
  • PDMS polydimethylsiloxane
  • 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 ⁇ L or less, and particularly preferably 100 nL or more and 10 ⁇ L 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 is, for example, 1 ⁇ m 2 100 mm 2 It may be less than 10 ⁇ m 2 10 mm 2 Or less, preferably 100 ⁇ m 2 1mm or more 2 It is particularly preferred that
  • 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.
  • 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.
  • the culture temperature is not particularly limited as long as it is within the range in which the hair follicle primordium can be formed.
  • 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.
  • 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.
  • 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.
  • a part of the hair papilla cell aggregation part and a part of the raw material cell aggregation part are bonded.
  • 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.
  • 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.
  • 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.
  • 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 the present method is not particularly limited.
  • the density of cells contained in the hair follicle primordium is not particularly limited.
  • 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.
  • the animal 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).
  • 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.
  • 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. Diseases associated with hair loss are not particularly limited.
  • AGA androgenetic alopecia
  • FGA female androgenetic alopecia
  • postpartum alopecia diffuse alopecia, seborrhea Alopecia, alopecia areata, traction alopecia, metabolic alopecia, pressure alopecia, alopecia areata, alopecia areata, alopecia, systemic alopecia, and symptomatic alopecia
  • 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.
  • the hair follicle primordium may be transplanted into a non-human animal.
  • 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
  • the ratio of the weight of the non-constant part contained in the adult hair follicle tissue By changing the ratio of the weight 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. 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.
  • 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. 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.
  • 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.
  • 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.
  • 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.
  • the color concentration is increased from the plurality of transplanted hair follicle primaries.
  • a plurality of different hairs can be grown.
  • 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.
  • 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
  • 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.
  • a hair follicle primordium that produces dark hair.
  • a first hair follicle primordium that produces hair of the first color by being transplanted to an animal is produced.
  • 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.
  • 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% or more and 100% or less. 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.
  • the weight of the non-constant part relative to the weight of the adult hair follicle tissue is about 40%.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIGS. 1A 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.
  • 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).
  • 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.
  • CD34 is a marker protein of hair follicle epithelial stem cells. The result observed with the microscope is shown.
  • 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. .
  • 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 CarvePro 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).
  • PDMS spheroid chip a multiwell culture container having a diameter of 1 mm and a depth of 1 mm.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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 a singularization treatment by a 70 ⁇ m cell strainer, and the obtained cells are used as “source cells (1/2)” for the experiment. used.
  • 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.
  • the dermal papilla cells obtained as described above were dispersed in a mixed medium to prepare a dermal papilla cell suspension.
  • the raw material cells obtained as described above were dispersed in a mixed medium to prepare a raw material cell suspension.
  • 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.
  • 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).
  • the mixed cell suspension was seeded and cultured in a CO 2 incubator at 37 ° C. for 3 days.
  • 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.
  • 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.
  • 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. 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.
  • non-constant parts especially non-constant parts other than the hair papilla and hair matrix
  • 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.
  • FIGS. 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 ⁇ m.
  • 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.
  • the scale bar in FIGS. 5D to 5F indicates 1 mm.
  • the form of the hair follicle primordium formed by the culture for 3 days did not differ depending on the FGF2 content of the mixed medium.
  • 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.
  • the filtrate in the collection tube was discarded, 700 ⁇ L 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 ⁇ L 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 ⁇ L 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.
  • 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.
  • the dilution rate is the final volume in RNA extraction.
  • 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.
  • 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 ⁇ g / mL. The diluted RNA lysate was incubated at 65 ° C.
  • RNA lysate 12 ⁇ L of Nuclear free water
  • 4 ⁇ L of 5 ⁇ RT Buffer 4 ⁇ L of 5 ⁇ RT Buffer
  • 1 ⁇ L of Primer mix 1 ⁇ L of Enzyme mix
  • the microtube was set on a thermal cycler and confirmed to be tightly closed.
  • 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.
  • cDNA reverse transcript of RNA derived from hair follicle primordium
  • 10 ⁇ L of SYBR Green master mix 0.4 ⁇ L of Forward Primer
  • 0.4 ⁇ L of Reverse Primer 0.4 ⁇ L of Dye
  • 7.8 ⁇ L of Nuclear free water was placed in a microtube and covered with a transparent film.
  • the base sequences of the primers used in the PCR are Versican, Forward Primer is “5′-GACGACTGTTCTTGGTGGG-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 ′ ”.
  • 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.
  • FIG. 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.
  • 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.
  • the arrow points to the transplanted hair follicle primordium.
  • 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.
  • This application claims the priority on the basis of Japanese application Japanese Patent Application No. 2018-094498 for which it applied on May 16, 2018, and takes in those the indications of all here.

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Abstract

Le problème que cherche à résoudre la présente invention est de fournir : une méthode de contrôle de la couleur de poils régénérés qui permette de contrôler la couleur des poils de manière simple et efficace ; une méthode de régénération des poils ; et une méthode de production d'ébauches de follicules pileux. La solution selon l'invention concerne une méthode de contrôle de la couleur de poils régénérés qui consiste : à utiliser des cellules source comprenant des cellules souches épithéliales obtenues à partir de tissus de follicules pileux adultes contenant une zone renflée qui est une partie constante ; à mélanger et à cultiver les cellules de papille pilaire dispersées et les cellules source dispersées afin de préparer des ébauches de follicules pileux ; et à greffer l'ébauche de follicule pileux à un animal pour faire pousser les poils à partir de l'ébauche de follicule pileux, le rapport en poids de la partie non constante, qui fait partie des tissus de follicules pileux adultes, étant modifié pour altérer la couleur des poils cultivés à partir des ébauches de follicules pileux.
PCT/JP2019/020185 2018-05-16 2019-05-15 Méthode de contrôle de la couleur de poils régénérés, méthode de régénération des poils, et méthode de production d'ébauches de follicules pileux Ceased WO2019221307A1 (fr)

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JP2018094498A JP7158676B2 (ja) 2018-05-16 2018-05-16 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
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WO2012115079A1 (fr) * 2011-02-24 2012-08-30 株式会社オーガンテクノロジーズ Procédé de préparation d'un germe de follicule pileux régénéré transplantable avec maîtrise de la couleur, composition contenant un germe de follicule pileux régénéré transplantable et procédé de transplantation d'un germe de follicule pileux régénéré
WO2014178438A1 (fr) * 2013-05-02 2014-11-06 Saeki Masanori Préparation de cellules pour la régénération capillaire

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WO2012115079A1 (fr) * 2011-02-24 2012-08-30 株式会社オーガンテクノロジーズ Procédé de préparation d'un germe de follicule pileux régénéré transplantable avec maîtrise de la couleur, composition contenant un germe de follicule pileux régénéré transplantable et procédé de transplantation d'un germe de follicule pileux régénéré
WO2014178438A1 (fr) * 2013-05-02 2014-11-06 Saeki Masanori Préparation de cellules pour la régénération capillaire

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