WO2012042618A1 - Cd36発現性結合組織鞘細胞を含有する毛包を再生するための組成物 - Google Patents

Cd36発現性結合組織鞘細胞を含有する毛包を再生するための組成物 Download PDF

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WO2012042618A1
WO2012042618A1 PCT/JP2010/066999 JP2010066999W WO2012042618A1 WO 2012042618 A1 WO2012042618 A1 WO 2012042618A1 JP 2010066999 W JP2010066999 W JP 2010066999W WO 2012042618 A1 WO2012042618 A1 WO 2012042618A1
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cells
animal
dsc
hair
hair follicle
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French (fr)
Japanese (ja)
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雄三 吉田
勤 相馬
重良 藤原
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Shiseido Co Ltd
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Shiseido Co Ltd
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Priority to CN201080069392.7A priority Critical patent/CN103237554B/zh
Priority to KR1020137007049A priority patent/KR101730216B1/ko
Priority to IN607MUN2013 priority patent/IN2013MN00607A/en
Priority to SG2013023619A priority patent/SG189167A1/en
Priority to PCT/JP2010/066999 priority patent/WO2012042618A1/ja
Priority to US13/876,608 priority patent/US20130212724A1/en
Application filed by Shiseido Co Ltd filed Critical Shiseido Co Ltd
Priority to EP10857831.1A priority patent/EP2623106B1/en
Priority to HK13110994.0A priority patent/HK1183790B/xx
Publication of WO2012042618A1 publication Critical patent/WO2012042618A1/ja
Anticipated expiration legal-status Critical
Priority to US15/076,836 priority patent/US10172332B2/en
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    • 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
    • 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
    • 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
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
    • A61K8/985—Skin or skin outgrowth, e.g. hair, nails
    • 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
    • A61L27/3804—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 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3813—Epithelial cells, e.g. keratinocytes, urothelial 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q7/00—Preparations for affecting hair growth
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0627—Hair cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0627—Hair cells
    • C12N5/0628—Hair stem cells; Hair progenitors
    • 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
    • A01K2207/00—Modified animals
    • A01K2207/12—Animals modified by administration of exogenous cells
    • 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
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • 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
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/70—Biological properties of the composition as a whole
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/18—Materials or treatment for tissue regeneration for hair reconstruction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0666—Mesenchymal stem cells from hair follicles

Definitions

  • the present invention relates to connective tissue sheath (“DS”: dermal sheath) cells (hereinafter, also referred to as “CD-expressing DSc”) expressing CD36 antigen (thrombospondin receptor) for regenerating hair follicles and any A composition containing dermal papilla (“DP”) cells (hereinafter also referred to as “DPc”), a method for regenerating a hair follicle using the composition, and a hair regenerated by such a method An animal or three-dimensional skin model carrying a sac is provided.
  • DS connective tissue sheath
  • CD36 antigen thrombospondin receptor
  • Hair is very important for aesthetic appearance. Therefore, hair loss due to congenital or acquired factors is a serious concern for many people. Especially in the modern society, which is called an aging society and a stress society, the hair of the head is exposed to a risk of hair loss due to various acquired causes. Corresponding to this, various attempts have been made to provide a better hair-restoring agent that exhibits a hair-restoring effect including promotion of hair growth and hair thickening.
  • the hair follicle is an exceptional organ that repeats self-regeneration for almost a lifetime in a mature organism. Elucidating the mechanism of self-regeneration will lead to clinical applications with high needs such as hair removal treatment by tissue and cell transplantation, construction of skin sheets that are functionally superior in nature containing hair follicles and sebaceous glands. Be expected.
  • hair follicle epithelial stem cells epithelial cells
  • the properties of hair follicle papilla cells which are hair follicle-specific mesenchymal cells, have gradually become known. .
  • Papilla cells play the role of a control tower that sends activation signals to hair follicle epithelial stem cells for self-renewal of hair follicles, and are essential cells with hair follicle epithelial stem cells in the evaluation system for hair follicle reconstitution (Kishimoto et al., Proc. Natl. AcaDSci. USA (1999), Vol.96, pp. 7336-7341; Non-Patent Document 1).
  • Both DP and DS surrounding the hair follicle are composed of a group of cells derived from the mesenchymal system, unlike the epithelial cells that make up most of the hair follicle. Numerous findings have been reported in recent years that suggest the importance of DS for hair follicle formation.
  • DP is regenerated from DS (1), and hair follicles are transplanted with DS of hair follicles cut at the lower half in mice. It has also been reported that regeneration is induced (2).
  • Jahoda et al. Jahoda et al. (Development.
  • Non-Patent Document 2 1992 Apr: 114 (4): 887-97; Non-Patent Document 2 also reported that hair follicle remodeling can be induced by transplanting DS into humans (Horne). KA and Jahoda CA.Development.1992 Nov: 116 (3): 563-71; Non-Patent Document 3).
  • Tobin Paus et al., In the mouse hair cycle, cell migration between DS and DP occurs, and connective tissue sheath cells (DSc) start to proliferate before DPc, which begins to grow in the hair growth phase. (Tobin DJ et al., J.Invest.Dermatol., 120: 895-904, 2003; Non-Patent Document 4).
  • DS is likely to play an important role in hair follicle formation, but the mechanism of action is largely unknown, and even the nature of DSC is unknown. Therefore, we investigated the gene expression profile that characterizes DSC and analyzed it to clarify the mechanism of action on hair follicle formation.
  • An object of the present invention is to provide a novel hair follicle regeneration system.
  • the present inventor examined the gene expression profile of DSc using a microarray, and as a result, identified 304 genes as genes having an expression rate more than twice as high as that of DPc or FBc (fibroblast). As a result of functional classification of GeneSpring in GeneOntology, many of these genes belong to blood vessel-related factors, suggesting a relationship between DS and blood vessels.
  • DSc was found to express CD36 at a high level, and the expression of CD36 in DSc was HGF (hepatocyte growth factor) (J. Linder et al., Federation of). It was found that this was linked to the expression of American Societies for Experimental Biology, 14 (2), 319 (2000): Non-patent document 5).
  • this application includes the following inventions: [1] A composition for regenerating a hair follicle containing a CD36-expressing connective tissue sheath ([DS]: dermal sheath) cell. [2] The composition for regenerating a hair follicle according to [1], further comprising hair papilla (“DP”: dermal papilla) cells. [3] The composition of [2], wherein the ratio of the number of cells of CD36-expressing DSc to DPc is about 10: 1 to 1:10. [4] The composition of [2] or [3], wherein the CD36-expressing DSc and DPc are both derived from a mouse, or both are derived from a rat, or both are derived from a human.
  • [6] A method for regenerating a hair follicle by transplanting the composition according to any one of [1] to [5] to a human.
  • [7] A method for regenerating a hair follicle by transplanting the composition of any one of [1] to [5] into a recipient animal.
  • the chimeric animal of [11] wherein the recipient animal is an animal with a suppressed immune system.
  • a three-dimensional skin model comprising a skin three-dimensional model containing the composition according to any one of [2] to [5] and thus carrying a reconstructed hair follicle.
  • the hair follicle regeneration meter composition of the present invention can be used for transplantation surgery for hair follicle regeneration and for research and development of hair follicle reconstruction.
  • the schematic diagram which shows the structure of a hair follicle structure
  • tissue staining figure which performed CD36 and CD31 dyeing
  • HGF expression result of CD36 positive DSc.
  • the present invention relates to a composition containing DSc and optionally DPc for regenerating hair follicles, a method for regenerating hair follicles using the composition, and an animal or 3 carrying such regenerated hair follicles.
  • a composition containing DSc and optionally DPc for regenerating hair follicles a method for regenerating hair follicles using the composition, and an animal or 3 carrying such regenerated hair follicles.
  • CD36 antigen is also referred to as the thrombospondin receptor.
  • CD36 is an integral membrane protein found on the surface of many vertebrate cell types and is also known as FAT, SCARB3, GP88, glycoprotein IV (gpIV) and glycoprotein IIIb (gpIIIb).
  • CD36 is a member of the class B scavenger receptor family of cell surface proteins.
  • CD36 binds to many ligands such as collagen, red blood cells parasitized with Plasmodium falciparum, oxidized low density lipoprotein, natural lipoprotein, oxidized phospholipid, and long chain fatty acid.
  • CD36 may be involved in impaired glucose tolerance, atherosclerosis, arterial hypertension, diabetes, cardiomyopathy, and Alzheimer's disease.
  • the relationship between CD36 antigen and hair growth is not known at all.
  • DSc is a cell constituting a sheath part surrounding the DP in the hair follicle, and is a mesenchymal cell like the DPc. Since DP is derived from DS, and DS grows prior to DP growth in the hair growth stage, it is considered that DS supplies DPc (Tobin DJ et al., J. Invest. Dermatol., 120 : 895-904, 2003; Non-Patent Document 4).
  • the DSc expressing CD36 is not particularly limited, but can be selected from DPc and the like by a conventional cell sorting technique using, for example, an antibody against CD36, preferably a monoclonal antibody.
  • the DSc of the present invention may be any mammal, such as humans, chimpanzees, other primates, livestock animals such as dogs, cats, rabbits, horses, sheep, goats, cows, pigs, and other laboratory animals such as rats and mice. It can be derived from the epidermis of guinea pigs, more preferably nude mice, skid mice, nude rats. Further, the epidermis part may be a hairy part, for example, a scalp, or a hairless part, for example, foreskin.
  • DPc is a mesenchymal cell located at the bottom of the hair follicle and sends an activation signal to the hair follicle epithelial stem cell for self-renewal of the hair follicle.
  • a dermal papilla cell preparation containing only activated dermal papilla cells is described in, for example, Kishimoto et al., Proc. Natl. AcaDSci. USA (1999), Vol.96, pp. 7336-7341 using transgenic mice. It can be prepared by a method.
  • a cell suspension is prepared by collagen treatment of a dermis tissue fraction obtained by removing epidermal tissue from skin tissue, and then the cell suspension is cryopreserved.
  • the method by cryopreservation can be performed, for example, as follows. 1. Prepare mammalian skin. 2. If necessary, leave this skin in a proteolytic enzyme solution such as a trypsin solution for an appropriate time, for example overnight, then remove the epidermis with tweezers, treat the remaining dermis with collagenase, and cell suspension. To prepare. 3. If necessary, the suspension is filtered with a cell strainer, and the precipitate is removed by standing. 4). Count the number of cells, resuspend with cryoprotective solution at an appropriate cell density, preferably about 1 ⁇ 10 5 to 1 ⁇ 10 8 / ml, aliquot if necessary, and store frozen according to normal cell storage methods. To do. 5). After storage for an appropriate period, melt and use.
  • the freezing method is not particularly limited, but it is stored in an ultra-low temperature freezer of ⁇ 20 ° C. or lower, preferably ⁇ 50 ° C. or lower, more preferably ⁇ 80 ° C. or lower, or in liquid nitrogen.
  • the cryopreservation period is not particularly limited, but is set to, for example, 1 day or longer, preferably 3 days or longer, more preferably 1 week or longer so that the epithelial cells are killed. It was confirmed that the dermal papilla cells continued to survive even after being stored in liquid nitrogen for 4 months.
  • a normal preservation solution used in cell preservation for example, Cell Banker 2 cell cryopreservation solution (catalog No. BLC-2) (manufactured by Nippon Zenyaku Kogyo Co., Ltd.) can be used.
  • Measurement of the number of cells can be performed by methods well known to those skilled in the art. For example, the cell count is measured by using a hemocytometer (SLGC, EOSINOPHIL COUNTER) with a cell suspension diluted with an equal volume of 0.4% trypan blue stain (No.15250-061, Invitrogen). It can be calculated according to the method described in the attached instruction manual.
  • SLGC hemocytometer
  • EOSINOPHIL COUNTER EOSINOPHIL COUNTER
  • trypan blue stain No.15250-061, Invitrogen
  • the DPc of the present invention can be any mammal, such as humans, chimpanzees, other primates, livestock animals such as dogs, cats, rabbits, horses, sheep, goats, cows, pigs, and other laboratory animals such as It can be derived from the skin of rats, mice, guinea pigs, more preferably nude mice, skid mice, nude rats.
  • the composition for regenerating hair follicles of the present invention may further contain “epithelial cells”.
  • Epithelial cells are cells that make up the majority of the epidermis or epithelium of the skin and arise from a single layer of basal cells that touch the dermis. Taking mouse as an example, epithelial cells derived from neonates (or fetuses) can be preferably used as epithelial cells, but even cells derived from mature skin, for example, epithelium of resting hair or epithelium of growing hair It may also be a culture of cells in the form of keratinocytes. Such cells can be prepared from the skin of the desired donor animal by methods well known to those skilled in the art.
  • epithelial cells can be prepared as follows. 1. Prepare mammalian skin. 2. The epidermis is trypsinized by standing overnight at 4 ° C. in 0.25% trypsin / PBS if necessary. 3. Only the epidermis part is peeled off with tweezers, etc., and after chopping, suspended in an appropriate culture solution (for example, keratinocyte culture solution) at 4 ° C. for about 1 hour. 4). The suspension is passed through a cell strainer with an appropriate pore size and then centrifuged to collect epithelial cells. 5). This cell preparation is suspended in KGM or SFM medium at a desired cell density and allowed to stand on ice until just before use.
  • an appropriate culture solution for example, keratinocyte culture solution
  • the epithelial cells of the present invention can be tested in any mammal, such as humans, chimpanzees, other primates, livestock animals such as dogs, cats, rabbits, horses, sheep, goats, cows, pigs, etc., as well as DSc and DPs. It can be derived from the epidermis of a working animal such as a rat, mouse, guinea pig, more preferably nude mouse, skid mouse, nude rat. Further, the epidermis part may be a hairy part, for example, a scalp, or a hairless part, for example, a foreskin.
  • the ratio of DSc to DPc used is not limited, but it is preferably included in the composition of the present invention at 1:10 to 10: 1, more preferably 1: 3 to 3: 1. Furthermore, the epithelial cells are from 1:10 to 10: 1, more preferably from 1: 1 to 10: 1, even more preferably from 1: 1 to 3: 1, most preferably 1: to the total amount of DSc and DPs. 1 may be included.
  • composition for regenerating the hair follicle of the present invention is, for example, a combination in which DSc, DPc, and epithelial cells are all derived from humans, and DSc, DPc, and epithelial cells are all non-human mammals.
  • a combination derived from the same species (hereinafter, allogeneic), a combination in which DSc and DPc are derived from human, an epithelial cell is derived from a mammal other than human, one of DSc and DPc is derived from human, and the other and A combination in which the epithelial cell is a mammal other than human and derived from the same or different species, a combination in which one of DSc and DPc is derived from a mammal other than human, and the other and epithelial cell is derived from a human (above) , Heterogeneous), and the like.
  • the method for transferring the composition for regenerating hair follicles according to the present invention to a recipient animal can be a transfer method known per se. See, for example, Weinberg et al, J. Invest. Dermatol. Vol.100 (1993), pp.229-236.
  • the prepared cells are mixed immediately before transplanting to 1 hour before removal, and the culture solution is removed by centrifugation (9000 ⁇ g, 10 min.) To make a cell mass of about 50 to 100 ⁇ L. Immediately pour into a silicone dome-shaped chamber embedded in the back skin of a nude mouse.
  • the chamber is carefully removed, and after the 3rd week, the presence or absence of hair formation at the transplanted site can be visually observed.
  • a similar method can be used for transplantation for hair growth in animals including humans, and an appropriate method will be appropriately determined by a doctor or veterinarian.
  • the transplant may be allotransplantation, that is, autotransplantation, allogeneic transplantation, allogeneic transplantation, or xenotransplantation.
  • allogeneic transplantation the dermal papilla cell preparation and epithelial cells are both allogeneic with the recipient.
  • xenotransplantation either the dermal papilla cell preparation or the epithelial cells may be xenogeneic with the recipient and the other may be xenogeneic with the recipient, or both may be xenogeneic with the recipient.
  • Recipient animals include any mammals such as humans, chimpanzees, other primates, livestock animals such as dogs, cats, rabbits, horses, sheep, goats, cows, pigs, and other laboratory animals such as rats, mice, Guinea pigs, more preferably nude mice, skid mice and nude rats are mentioned.
  • a chimeric animal carrying a regenerated hair follicle can be provided by transplanting the composition according to the present invention to a suitable recipient animal.
  • a suitable recipient animal can serve as an influential animal model, for example, to study and elucidate the mechanism of hair follicle regeneration, or to screen for drugs / herbicides effective for hair follicle regeneration or hair growth or hair loss.
  • the recipient animal is preferably an animal with a suppressed immune system, regardless of the origin of each cell contained in the system to be transplanted into the animal.
  • the animal species may be any animal as long as it can be used as an experimental animal and meets the purpose of the present invention, and preferred examples include mice and rats.
  • nude mice those whose immune system is suppressed include those having traits such as athymic deficiency, such as nude mice, in the case of mice.
  • particularly preferred recipient animals include commercially available nude mice (for example, Balb-c nu / nu strain), skid mice (for example, Balb / c-SCID), nude rats ( For example, F344 / N Jcl-rnu) can be mentioned.
  • a 3D skin model carrying a regenerated hair follicle can be provided by including the composition according to the present invention in a 3D skin model.
  • a hair papilla cell serving as a control tower for hair growth is essential.
  • a three-dimensional skin model can be prepared by a method well known to those skilled in the art (Exp. Cell Res. Amano S. et al., (2001), Vol. 271, pp. 249-362), for example, as follows. .
  • the three-dimensional skin model has DSc and DPc in an amount of 1 ⁇ 10 6 to 10 8 pieces / cm 2 , preferably 1.0 to 1.5 ⁇ 10 7 pieces / cm 2 , more preferably about 1.27 ⁇ 10 7 pieces / cm 2 . Including.
  • Three-dimensional skin model preparation method Human fibroblasts are dispersed in an appropriate amount in 0.1% collagen solution / DMEM / 10% FBS, dispensed into a petri dish, and immediately left in a CO 2 incubator at 37 ° C. After gelation, the gel is peeled off from the petri dish wall and bottom surface so that it floats in the petri dish. The collagen gel is cultured while being shaken, and the gel is contracted to about 1/5 to obtain a dermis model.
  • a stainless steel grid place a glass ring on it, 0.4 ml of human cultured epidermal cells (1.0X10 6 cells / ml) dispersed in KGM (epidermal cell culture medium), inside the glass ring Inject and incubate. At this time, DSc and DPc are mixed and injected at the same time.
  • human cultured epidermal cells mouse neonatal epidermal cells can also be used. Place the medium of DMEM-KGM-5% FBS + Ca 2+ in the petri dish so that the upper part of the dermis model is exposed to air, and observe the skin model after about one week. Determine reproducibility.
  • the three-dimensional skin model carrying reconstituted hair follicles is used for research and elucidation of the mechanism of hair follicle regeneration, as well as for the screening of drugs and herbal medicines that are effective for hair growth and hair removal, similar to the chimeric animal carrying the regenerated hair follicles. it can.
  • the isolated DP was medium-1 (10% fetal bovine serum, 10 ng / ml EGF, 20 ng / ml bFGF, 0.00075% ⁇ mercaptoethanol, penicillin 100 units / ml (titer), streptomycin 0.1 mg / ml (titer) ), Stationary culture on collagen-coated 35mm dish (Iwaki) containing amphotericin B 0.25ug / ml (titer) containing Nissui fibroblasts), isolated DS is treated with collagenase at 37 ° C for 40 minutes After the application, the cells were statically cultured on a collagen-coated 35 mm dish.
  • medium-1 10% fetal bovine serum, 10 ng / ml EGF, 20 ng / ml bFGF, 0.00075% ⁇ mercaptoethanol, penicillin 100 units / ml (titer), streptomycin 0.1 mg / ml (titer
  • DPc and DSc proliferation were confirmed after one week, and then used as experimental samples as DPc and DSc.
  • Commercially available cells (Toyobo) were used as fibroblasts (FBc).
  • FBc fibroblasts
  • DSc, DPc, and FBc were cultured in medium-1 for 7-10 days. Thereafter, the cells were passaged using trypsin.
  • the culture conditions were 37 ° C. and 5% CO 2 , and a collagen-coated flask T-75 (Iwaki) was used as a culture vessel. Each cell used in the experiment was used after passage 1 to 3 times.
  • HMVEC human adult skin microvascular endothelial cells
  • Humedia-MvG Humedia-MvG
  • RNA including mRNA was recovered from DPc, DSc and FBc using RNeasy Micro kit (Qiagen).
  • the recovered RNA was synthesized using double-stranded cDNA using agilent's protocol, followed by further synthesis of cRNA labeled with cyanines 3 and 5.
  • the labeled cRNA was hybridized to an Agilent microarray chip slide (Agilent, whole human genome (4 ⁇ 44K), G4110) using a two-color method at 65 ° C. for 17 hours.
  • DSC was seeded on a 4-well chamber slide (Nalgene nunc) that had been subjected to collagen surface treatment using an acidic collagen solution (Koken), and then cultured for 1 to 2 days. After washing with PBS, it was fixed with 4% PFA for 30 minutes, washed with PBS, and treated with 0.1% TritonX-100-containing PBS solution for 10 minutes. Then, blocking treatment was performed with 3% BSA-containing PBS for 30 minutes, and a CD36 antibody (Abcam, ab17044) was reacted for 1 hour with a primary antibody solution diluted 50-fold with 1% BSA-containing PBS.
  • Tissue staining Human scalp tissue was embedded with a frozen tissue embedding agent OTC compound (Sakura Finetek), and a frozen section slide was prepared with a cryosection preparation apparatus cryostat (Leica). After fixing with 4% PFA for 15 minutes, the cells were washed with PBS and reacted for 1 hour using a blocking solution obtained by adding 5% skim milk, 1% donkey serum, and 0.1% triton-X100 to PBS. Next, using a primary antibody solution obtained by diluting CD36 antibody solution (Abcam, ab17044) and CD31 antibody solution (R & D, AF806) 50 times and 100 times with a blocking solution, respectively, at room temperature for 1 hour or at 4 ° C overnight. And reacted.
  • OTC compound Sakura Finetek
  • Leica cryosection preparation apparatus cryostat
  • the CD31 antibody was used for labeling CD31 used as a marker for intravascular cells. After washing 3 times with PBS, Alexa 594-labeled anti-mouse IgG antibody (Invitrogen) and Alexa 488-labeled anti-rabbit IgG antibody (Invitrogen) were each diluted 200-fold with a blocking solution at room temperature. For 1 hour. After reacting with DAPI solution, it was washed 3 times with PBS, and sealed with anti-fading agent Prolong Gold Antifade Reagent and a cover glass. Observation was performed using a fluorescence microscope (Olympus).
  • Hair follicle whole-mount staining Hair follicles isolated from human tissues were fixed with 4% PFA at 4 ° C for 2 hours with shaking. Dehydration was sequentially performed for 5 minutes each using 0.1% tween PBS (hereinafter, PBST) containing 25%, 50%, and 75% ethanol, and for 5 minutes using 100% ethanol. The treated sample was stored in ethanol at -20 ° C. At the time of use, it was rehydrated with the same ethanol series PBST, and then treated with PBS containing 5% tritonX-100 for 10 minutes.
  • PBST 0.1% tween PBS
  • the blocking solution used for tissue staining CD36 antibody (Abcam, ab17044), primary antibody solution containing CD31 antibody (R & D, AF806), Alexa 594-labeled anti-mouse IgG antibody, Alexa 488-labeled anti-rabbit IgG antibody
  • the reaction was sequentially carried out using the secondary antibody solution and DAPI solution. Washing was performed 8 times with 0.1% tritonX-100 PBS during antibody reaction and after staining.
  • the reaction conditions are that the primary antibody solution is at 4 ° C. overnight and the secondary antibody is at 4 ° C. for 2 to 3 hours. After enclosing with the anti-fading agent Prolong Gold Antifade Reagent and a cover glass, observation was performed using a fluorescence microscope (Olympus).
  • RT-PCR RNA was recovered from the cells using TRIzol (Invitrogen) and the protocol provided. The concentration of the recovered RNA was measured with a nucleic acid quantification device Nanodrop (Thermo scientific). After aligning the RNA concentrations in the comparison group, cDNA was synthesized from RNA using oligo (dT) primers by reverse transcriptase Superscript III (Invitrogen) using the invitrogen protocol. Quantitative RT-PCR was performed using the synthesized cDNA as a template and the reaction reagent LightCycler (registered trademark) FastStart DNA MasterPLUS SYBR Green (Roche) and the reaction device LightCycler (Roche). Composition conditions were performed according to the Roche protocol.
  • the conditions for PCR are initial denaturation at 95 ° C. for 10 minutes, denaturation at 95 ° C. for 10 seconds, annealing at 60 ° C. for 10 seconds, and extension at 72 ° C. for 10 seconds.
  • the primer information used is described below.
  • G3PDH Forward primer: 5'-GCACCGTCAAGGCTGAGAAC-3 '(SEQ ID NO: 1) Reverse primer: 5'-ATGGTGGTGAAGACGCCAGT-3 '(SEQ ID NO: 2)
  • CD36 Forward primer: 5'-GAGGAACTATATTGTGCCTATTCTTTGGC-3 '(SEQ ID NO: 3) Reverse primer: 5'-CATAAAAGCAACAAACATCACCACACCAAC-3 '(SEQ ID NO: 4)
  • CD31 Forward primer: 5'-ATGCCGTGGAAAGCAGATACTCTAG-3 '(SEQ ID NO: 5) Reverse primer: 5'-AATTGCTGTGTTCTGTGGGAGCAG-3 '(SEQ ID NO: 6)
  • HGF Forward primer: 5'-GAGGGAAGGTGACTCTGAATGAG-3 '(SEQ ID NO: 7) Reverse primer: 5'-AATACCAGGACGATTTGGAATGGCAC-3 '(SEQ ID NO: 8) Using the
  • Cell sorting Cells were fractionated using a Cell Separation Magnet (BD Bioscience). The operating conditions were performed according to the protocol presented by BD Bioscience. After detaching the cells using a trypsin solution, the cell suspension was passed through a strainer (Falcon) having a hole of 70 ⁇ m, and the number of cells was counted. Suspend 5 to 10 million cells in 500 ml of 3% fetal bovine serum-containing PBS solution, add CD36 antibody (Abcam, ab17044) to the solution at a 50-fold dilution, and continue on ice for about 15 minutes. Reacted.
  • BD Bioscience Cell Sorting Cells were fractionated using a Cell Separation Magnet (BD Bioscience). The operating conditions were performed according to the protocol presented by BD Bioscience. After detaching the cells using a trypsin solution, the cell suspension was passed through a strainer (Falcon) having a hole of 70 ⁇ m, and the number of cells was counted. Suspend 5 to 10 million cells in
  • CD36 positive DSc The collected CD36 positive and negative DSc were suspended in medium-1 and cultured at 37 ° C, 5% CO 2 for 2-4 days using a collagen-coated flask T-25 (Iwaki) as a culture vessel. Was used in subsequent experiments.
  • the secondary antibody Alexa 488-labeled anti-rabbit IgG antibody (Invitrogen) was reacted on ice for 20 minutes, and the cells were resuspended in 0.5 ml PBS solution, We proceeded to analysis using Cell Lab Quanta SC (BeckmanCoulter). A setup including laser quality control was performed using the protocol and operating reagents specified by BeckmanCoulter. The number of CD31 positive cells was measured using the FL1 channel (525 nm). In addition, the correction
  • Table 1 shows the expression results for some of the blood vessel-related factors.
  • Vascular factors were found to be highly expressed in DS, among which CD36 and HGF were found to be specifically expressed in DS.
  • FIG. 2 shows the expression of various blood vessel-related factors in DS, DP, ORS, and VEC (vascular endothelial cells). It can be seen that CD36 and HGF are very specifically expressed in DS. The cell staining results also showed that CD36 positive cells were present only in the isolated DS cultured cells, and that CD36 positive cells were not present in DP or FB (data not shown).

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PCT/JP2010/066999 WO2012042618A1 (ja) 2010-09-29 2010-09-29 Cd36発現性結合組織鞘細胞を含有する毛包を再生するための組成物
US13/876,608 US20130212724A1 (en) 2010-09-29 2010-09-29 Composition for Regenerating Hair Follicles Comprising CD36-Expressing Dermal Sheath Cells
CN201080069392.7A CN103237554B (zh) 2010-09-29 2010-09-29 含有cd36表达性结缔组织鞘细胞的用于再生毛囊的组合物
EP10857831.1A EP2623106B1 (en) 2010-09-29 2010-09-29 Composition for regenerating follicle which contains cd36-expressing connective tissue sheath cells
HK13110994.0A HK1183790B (en) 2010-09-29 Composition for regenerating follicle which contains cd36-expressing connective tissue sheath cells
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JPWO2016181885A1 (ja) * 2015-05-14 2018-03-01 株式会社セルテクノロジー 凍結間葉系細胞の製造方法、及び、移植用治療材の製造方法
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WO2019039376A1 (ja) * 2017-08-22 2019-02-28 国立大学法人横浜国立大学 複数の再生毛包原基の製造方法、毛包組織含有シートの製造方法、毛髪再生用キット及び発毛促進又は抑制物質をスクリーニングする方法
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JPWO2019039376A1 (ja) * 2017-08-22 2020-10-29 国立大学法人横浜国立大学 複数の再生毛包原基の製造方法、毛包組織含有シートの製造方法、毛髪再生用キット及び発毛促進又は抑制物質をスクリーニングする方法
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JP2021047046A (ja) * 2019-09-17 2021-03-25 学校法人東京理科大学 観察方法、測定方法、解析方法、定量方法、およびキット
JP7385842B2 (ja) 2019-09-17 2023-11-24 学校法人東京理科大学 観察方法、測定方法、解析方法、定量方法、およびキット

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