US20130212724A1 - Composition for Regenerating Hair Follicles Comprising CD36-Expressing Dermal Sheath Cells - Google Patents

Composition for Regenerating Hair Follicles Comprising CD36-Expressing Dermal Sheath Cells Download PDF

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US20130212724A1
US20130212724A1 US13/876,608 US201013876608A US2013212724A1 US 20130212724 A1 US20130212724 A1 US 20130212724A1 US 201013876608 A US201013876608 A US 201013876608A US 2013212724 A1 US2013212724 A1 US 2013212724A1
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cells
dsc
hair follicles
animal
composition
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Yuzo Yoshida
Tsutomu Soma
Shigeyoshi Fujiwara
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Shiseido Co Ltd
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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
    • 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
    • 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
    • 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 a composition
  • a composition comprising dermal sheath (DS) cells expressing CD36 antigen (thrombospondin receptor) (to be referred to as “CD36-expressing DSc”) and arbitrarily, dermal papilla (DP) cells (to be referred to as “DPc”), a method for regenerating hair follicles using such a composition, and an animal or three-dimensional skin model having hair
  • Hair follicles are exceptional organs that continue to repeatedly self-regenerate throughout nearly the entire life of a mature living organism. Elucidation of the mechanisms of this self-regeneration are expected to lead to clinical applications for which there are considerable demand, such as hair loss treatment using tissue cell transplants, and the construction of natural and functionally superior skin sheets containing hair follicles and sebaceous glands.
  • follicular epithelial stem cells epithelial cells
  • dermal papilla cells which are follicle-specific mesenchymal cells
  • Dermal papilla cells fulfill the role of a so-called control tower that induces activation signals to follicular epithelial stem cells for self-regeneration of hair follicles, and have been determined, along with follicular epithelial stem cells, to be essential cells in follicle reconstruction evaluation systems (Kishimoto, et al., Proc. Natl. Acad. Sci. USA (1999), Vol. 96, pp. 7336-7341: Non-Patent Document 1).
  • Dermal papilla (DP) and dermal sheath (DS) surrounding the periphery of hair follicles both differ from epithelial cells composing the majority of hair follicles in that they are composed of mesenchymal-derived cell populations.
  • DS dermal sheath
  • numerous findings have been reported in recent years suggesting its importance with respect to hair follicle formation.
  • DS has been reported to her regenerated from DP in an experiment involving transplantation of hair bulbs—removed hair follicles of rat whiskers (1), and follicle regeneration has been reported to be induced in mice by transplanting DS of hair follicles from which the lower half has been severed (2).
  • Non-Patent Document 2 (Development, 1992, April:114(4), pp 887-897: Non-Patent Document 2) reported that follicle regeneration. can he induced by transplanting DS to humans (Horne, K. A. and Jahoda, C. A., Development, 1992, November:116(3), pp. 563-571: Non-Patent Document 3). Moreover, the Tobin, Paus et al. group reported that cells migration occur between DS and DP, and that proliferation of dermal sheath cells (DSc) begins prior to DPc that begin to proliferated during the hair growth phase (Tobin, D. J. et al., J. Invest. Dermatol., 120, pp. 895-904, 2003: Non-Patent Document 4).
  • DSc dermal sheath cells
  • Non-Patent Document 1 Kishimoto, et. al Proc. Natl. Acad. Sci. USA (1999), Vol. 96, pp. 7336-7341
  • Non-Patent Document 2 Jahoda, et. al., Development, 1992, April:114(4), pp, 887-897
  • Non-Patent Document 3 Horne, K. A. and Jahoda, Development, 1992, November:116(3), pp. 563-571
  • Non-Patent Document 4 Tobin, P. S. et al., J. Invest. Dermatol., 120, pp. 895-904, 2003
  • Non-Patent Document 5 Linder, S. at al., Federation of American Societies for Experimental Biology, 14(2), 319 (2000)
  • An object of the present invention is to provide a novel follicle regeneration system.
  • composition for regenerating heir comprising CD36-expressing dermal sheath (DS) cells.
  • DP dermal papilla
  • a method for regenerating hair follicles comprising transplanting the composition of any of [1] to [5] to a human.
  • [7] A method for regenerating hair follicles by transplanting the composition of any of [1] to [5] to a recipient animal.
  • a method for regenerating hair follicles comprising producing a three-dimensional skin model containing the composition of any of [2] to [5].
  • a three-dimensional skin model imparted with regenerated hair follicles by producing a three-dimensional skin model containing the composition of any of [2] to [5].
  • the hair follicle regeneration composition of the present invention can be used in transplant surgery regenerating hair follicles and in research and development on hair follicle reconstruction.
  • FIG. 1 is a schematic diagram showing the structure of it follicle tissue.
  • FIG. 2 indicates expression level of various types of vascular-related factors in various cells.
  • FIG. 3 is an image obtained by CD36 and CD31 immunohistochemical staining.
  • FIG. 4 is an image of whole mount stained hair. follicles obtained by CD36 and CD31 immunostaining.
  • FIG. 5 indicates the results of co-culturing CD36-positive DSc and vascular endothelial cells.
  • FIG. 6 indicates the results of HGF expression level in CD36-positive DSc.
  • the present invention provides a composition for regenerating hair follicles comprising DSc and arbitrarily comprising DPc, a method for regenerating hair follicles using such a composition, and an animal or three-dimensional skin model having hair follicles regenerated by such a method.
  • CD36 antigen is also referred to as thrombospondin receptor.
  • CD36 is an integral membrane protein found on the surfaces of numerous cell types of vertebra animals, and is also known au 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 also binds with numerous other ligands such as collagen, erythrocytes parasitized by falciparum malaria parasites, oxidized low density lipoproteins, naturally-occurring lipoproteins, oxidized phospholipids and long-chain fatty acids.
  • CD36 was able to be confirmed to fulfill a definite role in fatty acid and sugar metabolism, heart disease, sense of taste and the transport of vegetable fats and oils in the intestinal tract. CD36 can also be involved in impaired glucose tolerance, athersclerosis, arterial hypertension, diabetes, cardiomyopathy and Alzheimer's disease.
  • DSc are cells that compose the sheath portion that surrounds the periphery of DP in hair follicles, and are mesenchymal cells in the same manner as DPc.
  • DP are considered to be derived from DS, and since DS proliferates prior to the proliferation of DP during the hair growth phase, DS is thought to supply DPc (Tobin, D. J. et al., J. Invest. Dermatol., 120, pp. 895-904, 2003: Non-Patent Document. 4).
  • DSc that express CD36 can be sorted from DPc and other cells by, for example, commonly used cell sorting techniques using antibodies, and preferably monoclonal antibodies, to CD36.
  • the DSc of the present invention can be derived from the skin of all mammals, such as humans, chimpanzees, other primates, domestic animals such as dogs, cats, rabbits, horses, sheep, goats, cows or pigs, and experimental animals such as rats, mice or guinea pigs, and preferably nude mice, SCID mice or nude rats.
  • the epidermal site may be a site where there is hair growth such as the scalp, or a site where there is no hair growth such as the foreskin.
  • DPc skin papilla cells
  • dermal papilla cell preparations containing only activated dermal papilla cells can be prepared according to for example, the method described in Kishimoto, et al., Proc. Natl. Acad. Sci. USA (1999), Vol. 96, pp. 7336-7341 using transgenic mice.
  • these preparations are preferably prepared by, for example, preparing a cell suspension by treating a dermal tissue fraction, obtained by removing epidermal tissue from skin tissue, with collagenase, followed by destroying the follicular epithelial cells by subjecting the cell suspension to cryopreservation.
  • cryopreservation method can be specifically carried out, for example, in the manner indicated below.
  • the skin is then allowed to stand undisturbed for a suitable amount of time, such as overnight, in a protease solution such as a trypsin solution as necessary, the epidermal portion is subsequently removed with a forceps and the like, and the remaining dermis is treated with a collagenase to prepare a cell suspension.
  • a protease solution such as a trypsin solution
  • the cell suspension is then filtered with a cell strainer as necessary, and the sediment is removed by allowing to stand undisturbed.
  • the cells are re-suspended in a cryopreservation solution at a suitable cell density such as 1 ⁇ 10 5 /ml to 1 ⁇ 10 8 /ml, and the suspension is dispensed into small aliquots as necessary and then placed in cryopreservation in accordance with ordinary cell storage methods.
  • a suitable cell density such as 1 ⁇ 10 5 /ml to 1 ⁇ 10 8 /ml
  • the cells are stored for a suitable amount of time and used after thawing.
  • the freezing method consists of storing at a temperature of ⁇ 20°C. or lower, preferably ⁇ 50°C. or lower and more preferably ⁇ 80° C. or lower in an ultra-deep freezer, or in liquid nitrogen.
  • the duration of cryopreservation it is a period of, for example, 1 day or more, preferably 3 days or more and more preferably 1 week or more to ensure that the epithelial cells are killed.
  • dermal papilla cells have been confirmed to remain viable even after storing for 4 months in liquid nitrogen.
  • An ordinary cryopreservation solution used to store cells such as CellBanker 2 cryopreservation solution (Catalog No. BLC -2, Nippon Zenyaku Kogyo Co., Ltd.), can be used for the cryopreservation solution.
  • Measurement of the number of cells can be carried out by a method commonly known among persons with ordinary skill in the art.
  • the number of cells can be measured by placing a cell suspension obtained by diluting the cells with an equal volume of 0.4% Trypan blue stain (No. 15250-061, Invitrogen Corp.) on a hemocytometer (Eosinophil Counter, SLGC Co., Ltd.), and calculating the number of cells in accordance with the method described in the instruction manual provided with the hemocytometer.
  • the DPc of the present invention can be derived from the skin of all mammals, such as humans, chimpanzees, other primates, domestic animals such as dogs, cats, rabbits, horses, sheep, goats, cows or pigs, and experimental animals such as rats, mice or guinea pigs, and preferably nude mice, SCID mice or nude, rats.
  • the composition for regenerating hair follicles of the present invention further comprises epithelial cells.
  • Epithelial cells are cells that compose the majority of the dermis or epidermis of skin, and arise from a single layer of basal cells in contact with the dermis in using the example of mice, although epithelial cells derived from newborn mice (or fetuses) can be preferably used as epithelial cells, they may also be cells derived from mature skin, such as the epithelium of resting phase hair or epithelium of growth stage hair, or cultured cells in the form of keratinocytes. These cells can be prepared from the skin of a desired donor animal according to methods commonly known among persons with ordinary skill in the art.
  • the epithelial cells can be prepared in the manner described below.
  • the epidermal tissue is treated by suspending for about 1 hour at 4° C. in a suitable culture broth (such as keratinocyte culture broth).
  • a suitable culture broth such as keratinocyte culture broth
  • the suspension is passed through a cell strainer having a suitable pore size, followed by subjecting to centrifugal separation and recovering the epithelial cells.
  • the cell preparation is suspended in KGM or SFM medium at a desired cell density, and then allowed to stand undisturbed on ice until just before the time of use.
  • the epithelial cells of the present invention can be derived from the skin of all mammals, such as humans, chimpanzees, other primates, domestic animals such as dogs, cats, rabbits, horses, sheep, goats, cows or pigs, and experimental animals such as rats, mice or guinea pigs, and preferably nude mice, SCID mice or nude rats.
  • the epidermal site may be a site where there is hair growth such as the scalp, or a site where there is no hair growth such as the foreskin.
  • DSc and DPc are contained at a ratio of preferably 1:10 to 10:1, and more preferably 1:3 to 3:1.
  • epithelial cells are contained at a ratio to the total number of DSc and DPc of 1:10 to 10:1, preferably 1:1 to 10:1, more preferably 1:1 to 3:1 and most preferably 1:1.
  • the combination of DSc, DPc and arbitrarily epithelial cells may be from the same species or different species.
  • the composition for regenerating hair follicles of the present invention may be, for example, a combination in which all of the DSc, DPc and epithelial cells are derived from a human, a combination in which all of the DSc, DPc and epithelial cells are derived from the same species of mammal other than human (the above combinations are combinations of the same species), a combination in which the DSc and DPc are derived from a human while the epithelial cells are derived a mammal other than human, a combination in which one of either the DSc or DPc are derived from a human, and the other and the epithelial cells are derived from the same species or different species of mammal other than a human, and a combination in which one of either the DSc or DPc are derived a mammal other than a human and the other
  • the method used to transplant the composition for regenerating hair follicles of the present invention into a recipient animal can be in accordance with a known transplant method.
  • a known transplant method Reference can be made to, for example, Weinberg, et al., Invest. Dermatol., Vol. 100 (1993), pp. 229-236.
  • the cells that have been acquired are mixed just before or within 1 hour before transplant, the culture broth is removed by centrifugation (9000 ⁇ g, 10 min), and after forming a cell aggregate of about 50 ⁇ L to 100 ⁇ L, the cell aggregate is promptly poured into a silicone dome-shaped chamber embedded in the skin on the backs of the nude mice.
  • transplantation may be a homotransplantation, namely an autotransplantation, isotransplantation or allotransplantation, or may be a heterotransplantation.
  • the dermal papilla cell preparation and the epithelial cells are from the same species as the recipient.
  • the dermal papilla cell preparation or the epithelial cells are from a different species than the recipient while the other may be from the same species as the redolent, or both may be from a different species than the recipient.
  • recipient animals include all mammals, such as humans, chimpanzees, other primates, domestic animals such as dogs, cats, rabbits, horses, sheep, goats, cows or pigs, and experimental animals such as rats, mice or guinea pigs, and preferably nude mice, SCID mice or nude rats.
  • a chimeric animal having regenerated hair follicles can be provided by transplanting the aforementioned composition according to the present invention into a suitable recipient animal.
  • the resulting chimeric animal can serve as a useful animal model for, for example, researching and elucidating the mechanism of hair follicle regeneration, or screening drugs or herbs effective for regenerating hair follicles, growing hair or preventing hair loss.
  • the recipient animal is preferably an immunosuppressed animal regardless of the source of each of the cells contained in system transplanted into the recipient animal.
  • an animal species capable of being used as an experimental animal can be used for the animal species, and although any animal species may be used provided it coincides with the object of the present invention, preferable examples thereof include mice and rats.
  • examples of immunosuppressed animals when using the example of mice include those in having the trait of a missing thymus in the manner of nude mice.
  • particularly preferable examples of recipient animals include commercially available nude mice (such as Balb-c nu/nu strain), SCID mice (such as Balb/c-SCID), and nude rats (such as F344/N Jcl-run)
  • a three-dimensional skin, model can be provided that has regenerated hair follicles.
  • dermal papilla cells serving as a control tower. for hair growth are essential.
  • a three-dimensional skin model can be produced in the manner described below, for example, according to a method commonly known among persons with ordinary skill in the art (Amano, S. at al., Exp. Cell Res. (2001), Vol. 271, no. 249-362).
  • the tree -dimensional skin model respectively contains DSc and DPc at 1 ⁇ 10 6 cells/cm 2 to 1 ⁇ 10 8 cells/cm 2 , preferably at 1.0 ⁇ 10 7 cells/cm 2 to 1.5 ⁇ 10 7 cells/m 2 , and more preferably at about 1.0 ⁇ 10 7 cells/cm 2 .
  • a suitable number of human fibroblasts are dispersed in DMEM containing 0.1% collagen and 10% FBS followed by dispensing into a Petri dish and immediately allowing to stand undisturbed in a CO 2 incubator at 37° C. After the medium has gelled, the gel is scraped from the side walls and bottom of the Petri dish so as to suspend the gel in the Petri dish. The cells are then cultured while shaking the collagen gel to contract the gel to about one-fifth its original size and obtain a dermal model.
  • the dermal model is then placed on a stainless steel grid, a glass ring is placed thereon, and 0.4 ml of cultured human epithelial cells (1.0 ⁇ 10 6 cells/ml) dispersed in KGM (keratinocyte growth medium) are injected into the ring and cultured. At this time, DSc and DPc are simultaneously mixed and injected.
  • Mouse neonatal epithelial cells can also be used instead of cultured human epithelial cells.
  • a three-dimensional skin model having regenerated hair follicles can be used for researching and elucidating the mechanism of hair follicle regeneration, or screening drugs or herbs effective for growing hair or preventing hair loss.
  • the isolated DP ware static-cultured in a 35 mm collagen-coated tissue dish containing Medium-1 (Nissui low-serum fibroblast medium containing 10% fetal bovine serum, 10 ng/ml EGF, 20 ng/ml bFGF, 0.00075% ⁇ -mercaptoetbanol, 100 units/ml (titer) penicillin, 0.1 mg/ml (titer) streptomycin and 0.25 ⁇ g/ml (titer) amphotericin B), while the isolated DS were treated with collagenase for 40 minutes at 37° C. followed by similarly static- culturing in a 35 mm collagen-coated tissue dish.
  • Medium-1 Non-sui low-serum fibroblast medium containing 10% fetal bovine serum, 10 ng/ml EGF, 20 ng/ml bFGF, 0.00075% ⁇ -mercaptoetbanol, 100 units/ml (titer) penicillin
  • DPc and DSc were used as experimental samples.
  • Commercially available cells (Toyobo Co., Ltd.) were used for the fibroblasts (FBc).
  • the DSc, DPc and FBc were static-cultured for 7 to 10 days using Medium-1. Subsequently, the cells were subcultured using trypsin. Culturing conditions consisted of culturing in a collagen-coated flask (T-75, Iwaki Glass Co., Ltd.) at 37° C. and 5% CO 2 . En addition, each of the cells used in the experiment had been subcultured one to three times.
  • HMVEC human adult dermal microvascular endothelial cells
  • Humedia-MvG Human adult dermal microvascular endothelial cells
  • RNA containing mRNA was collected from the DSc, DPc and FBc using the RNeasy Micro Kit (Qiagen Corp.). The collected RNA was subjected to double-stranded cDNA synthesis using Agilent's protocol followed by synthesis of cRNA labeled with cyanine 3.5. The labeled cRNA was hybridized for 17 hours at 65° C. on a microarray chip slide (Agilent, Whole Human Genome (4 ⁇ 4.4K), G4110) since a two-color assay.
  • Comparisons of the gene expression levels of two types of cells each consisting of DSc and DPc, DPc and FBc and FBc and DSc were carried out on each chip slide using two types each of RNA derived from the DS of two individuals (total of 4 types), two types each of RNA derived from the DP of two individuals (total of 4 types), and two types each of RNA derived from the FB of one individual.
  • images of fluorescent signals (cyanine 3.5) of the cDNA on the chip were obtained with a dual-laser microarray scanner (Agilent Technologies Inc.).
  • the image data was quantified using Feature Extraction Software Ver. 9.1, and at that time, taggings were made (tagged) to indicate abnormal values and low values at about the same level as background noise followed by analyzing the data.
  • Each expression level was compared by comparing two sets of quantitative values of the acquired signals.
  • Gene Spring GX 7.3.1 software (Agilent Technologies Inc.) was used to analyze each gene expression level in greater detail using bioinformatics techniques. Abnormal values and low values at about the same level as background noise were tagged in a procedure using Feature Extraction Software Ver. 9.1, and analyses were conducted using those genes that were not tagged. The genes for which there was a difference in expression level between two types of cells were extracted and subjected to functional categorization by using GeneOntology (http://www.geneontology.org/). At that time, the degree of statistical significance was tested using Fisher's exact test.
  • Cell staining using CD36 antibody consisted of seeding DSc in a four well-chamber slide (Nalgene, Nalge Nunc International Corp.) subjected to collagen surface treatment using an acidic collagen solution (Koken Co., Ltd.) and using the cells after culturing for 1 to 2 days. After washing with PBS, the cells were fixed for 30 minutes with 4% PFA, washed with PBS and treated for 10 minutes with PBS solution containing 0.1% Triton. X-100. After blocking for 30 minutes with PBS containing 3% BSA, the cells were allowed to react for 1 hour with a primary antibody solution obtained by diluting CD36 antibody (ab17044, Abcam Inc.) 50-fold with PBS containing 1% BSA.
  • the cells After washing for times with PBS, the cells were allowed to react for 1 hour with a secondary antibody solution obtained by diluting Alexa 594-labeled anti-mouse IgG antibody (Invitrogen Corp.) 200-fold with PBS containing 1% BSA. After reacting with DAPI solution in order to carry out nuclear staining, the cells were washed four times with PBS and sealed with an anti-fade reagent (Prolong Gold Antifade Reagent, Invitrogen Corp.) and a cover glass. The cells were observed using a fluorescence microscope (Olympus Corp.).
  • Human scalp tissue was embedded in a frozen tissue embedding agent (OTC Compound, Sakura Finetek Japan Co., Ltd.), and frozen section slides were prepared with a frozen section production system (Cryostat, Leica Camera AG). After fixing for 15 minutes with 4% PFA, the tissue was washed with PBS and allowed to react for 1 hour using a blocking solution obtained by adding 5% skim milk, 1% donkey serum and 0.1% Triton X-100 to PBS. Next, the tissue was allowed to react for 1 hour at room temperature or overnight at 4° C. using primary antibody solution obtained by diluting CD36 antibody solution (ab17044, Abcam Inc.) or CD31 antibody solution.
  • OTC Compound Sakura Finetek Japan Co., Ltd.
  • CD31 antibody was used to label CD31 used as a vascular endothelial cell marker.
  • the tissue was allowed to react for 1 hour at room temperature using a secondary antibody solution obtained by diluting Alexa 594-labeled anti-mouse IgG antibody (Invitrogen Corp.) or Alexa 488-labeled anti-rabbit IgG antibody (Invitrogen Corp.) 200-fold each with blocking solution.
  • DAPI solution After reacting with DAPI solution, the tissue was washed 3 times with PBS and sealed with an anti-fade reagent (Prolong Gold Antifade Reagent) and a cover glass. The tissue was observed using a fluorescence microscope (Olympus Corp.).
  • Hair follicles isolated, from human tissue were fixed while shake-culturing for 2 hours. at 4° C. with 4% PFA, The follicles were sequentially subjected to dehydration. treatment consisting of treating for 5 minutes each using 0.1% Tween PBS containing 25%, 50% or 75% ethanol to be referred to as PEST) and treating for 5 minutes each using 100% ethanol. The treated samples were stored in ethanol at ⁇ 20° C. At the time of use, after rehydrating with the same ethanol series PBST, the follicles were treated for 10 minutes with PS containing 5% Triton X-100.
  • the follicles were sequentially reacted with the blocking solution used for tissue staining, a primary antibody solution containing CD36 antibody. (ab17044, Abcam Inc.) and CD31 antibody (AP806 R&D Co., Ltd.), a secondary antibody solution containing Alexa 594-labeled anti-mouse IgG antibody and Alexa 488-labeled anti-rabbit IgG antibody, and DAPI solution. Furthermore, the follicles were washed. 8 times each using PBS containing 0.1% Triton X-100 both between the antibody reaction procedures and after staining. Reaction conditions consisted of reacting overnight at 4° C.
  • Quantitative RT-PCR was then carried out using the synthesized cDNA as template and using LightCycler® FastStart DNA MasterPLUS SYPR Green (Roche Diagnostics GmbH) for the reaction reagent and LightCycler (Roche Diagnostics GmBH) for the reaction device.
  • Composition conditions were in accordance with the Roche protocol. PCR conditions consisted of initial denaturation for 10 minutes at 95° C., denaturation for 10 seconds at 95° C., annealing for 10 seconds at 60° C., and elongation for 10 seconds at 72°C.
  • the primer data used is as indicated below.
  • G3PDH Forward primer: (SEQ ID NO: 1) 5′-GCACCGTCAAGGCTGAGAAC-3′ Reverse primer: (SEQ ID NO: 2) 5′-ATGGTGGTGAAGACGCCAGT-3′
  • CD36 Forward primer: (SEQ ID NO: 3) 5′-GAGGAACTATATTGTGCCTATTCTTTGGC-3′ Reverse primer: (SEQ ID NO: 4) 5′-CATAAAAGCAACAAACATCACCACACCAAC-3′
  • CD31 Forward primer: (SEQ ID NO: 5) 5′-ATGCCGTGGAAAGCAGATACTCTAG-3′ Reverse primer: (SEQ ID NO: 6) 5′-AATTGCTGTGTTCTGTGGGAGCAG-3′
  • HGF Forward primer: (SEQ ID NO: 7) 5′-GAGGGAAGGTGACTCTGAATGAG-3′ Reverse primer: (SEQ ID NO: 8) 5′-AATACCAGGACGATTTGGAATGGCAC-3′
  • the expression levels of each gene were measured using the software provided. Furthermore, G3PDH was used as an internal standard, and this was used to correct the amount of cDNA of a control group when quantifying each gene.
  • the cells were sorted using the Cell Separation Magnet (BD Biosciences Inc.). Operating conditions were in accordance with the protocol provided by BD Biosciences Inc. After separating the cells using trypsin solution, the cell suspension was passed through a cell strainer having a pore size of 70 ⁇ m (Falcon Inc.) followed by counting the number of cells. 5 million to 10 million of cells were suspended in 500 ml of PBS solution containing 3% fetal bovine serum, followed by the addition of CD36 antibody (ab17044, Abcam Inc. so as to be diluted 50-fold and allowing to react for about 15 minutes on ice.
  • CD36 antibody ab17044, Abcam Inc.
  • the cells After recovering the cells by washing and centrifuging using 1 ⁇ Imag Buffer (BD Biosciences Inc.), the cells were re-suspended in 30 ⁇ l of anti-mouse IgG1 magnetic particles (BD Biosciences Inc.) and allowed to stand undisturbed for 30 minutes on ice. 500 ⁇ l of 1 ⁇ Imag Buffer (BD Biosciences Inc.) were added followed by placing in the Cell Separation Magnet (BD Biosciences Inc.) and allowing to stand undisturbed for 8 minutes. The supernatant was recovered while being careful so as not to dislodge cells adhered to the lateral surfaces by the magnet, and the resulting supernatant was used as CD36-negative DSc.
  • 1 ⁇ Imag Buffer BD Biosciences Inc.
  • CD36-positive and CD36-negative DSc were suspended in Medium-1 followed by culturing for 2 to 4 days. at 37° C. and 5% CO 2 using a collagen-coated flask. (T-25, Iwaki Glass Co., Ltd.) for the culture vessel, followed by use in the experiment.
  • the medium was replaced with medium obtained by adding 100 units/ml (titer) of penicillin, 0.1 mg/ml (titer) of streptomycin, 0.25 ⁇ g/ml (titer) of amphotericin B and 0.1% BSA (Sigma Corp.) to vascular endothelial cell basal medium (Humedia-EB2, Kurabo Industries Ltd.). After further co-culturing for 1 day, the cells were separated with trypsin solution followed by proceeding with analysis using FACS.
  • the cells were then passed through a 70 ⁇ m cell strainer (Falcon Inc.), suspended in PBS solution containing 3% fetal bovine serum, and allowed to react for 20 minutes on ice using a primary antibody solution, i.e., CD31 antibody solution (AF806, R&D Co., Ltd.). After washing the cells with PBS solution containing 3% fetal bovine serum, the cells were allowed to react for 20 minutes on ice with secondary antibody solution, i.e., Alexa 488-labeled anti-rabbit IgG antibody (Invitrogen Corp.) and re-suspended in 0.5 ml of PBS solution followed proceeding with analysis using Cell Lab Quanta 5C (Beckman Coulter Inc.).
  • secondary antibody solution i.e., Alexa 488-labeled anti-rabbit IgG antibody (Invitrogen Corp.) and re-suspended in 0.5 ml of PBS solution followed proceeding with analysis using Cell Lab Quanta 5C (Beckman Coulter
  • Preparations were made using the protocol and working reagent designated by Beckman Coulter.
  • the number CD31-positive cells was measured using the FL 1 channel (525 nm). Furthermore, correction was made to eliminate autofluorescence by using endothelial cells that did not react with CD31 antibody. Following measurement, the number of CD31-positive cells was calculated based on total number of cells obtained and the ratio of CD31 -positive cells.
  • Table 1 shows the expression level of some vascular-related factors. Although vascular-related factors were determined to be highly expressed in DSs, CD36 and HGF were determined to be specifically highly expressed in DSs. FIG. 2 indicates the expression level of various types of vascular-related factors in DSc, DPc, ORS cells and VEC (vascular endothelial cells). CD36 and HGF were determined to be expressed extremely specifically in DS. On the basis of cell staining results as well, CD36-positive cells were observed to be present only in isolated DS cultured cells, while CD36-positive cells were determined to be absent in DPc or FBc (data not shown).
  • CD36-positive DSc was suggested to be intimately involved with blood vessels.
  • CD36-positive DSc cells are nearly always co-localized with blood vessels, CD36-positive DSc are absent in the vicinity of some vessels ( FIG. 4 ).
  • CD36-positive DSc cells were suggested to promote vascularization by, for example, promoting the proliferation of vascular endothelial cells.
  • CD36-positive DSc isolated by cell sorting were co-cultured with vascular endothelial cells
  • the CD36-positive DSc were indicated to significantly promote the proliferation of vascular endothelial cells in comparison with CD36-negative DSc ( FIG. 5 ).
  • isolated CD36-positive DSc were also indicated to highly express HGF in comparison with CD36-negative DSc cells ( FIG. 6 ).
  • HGF is commonly known as a factor that promotes the growth of new hair and hair growth (Non-Patent Document 5).
  • transplantation of CD36-positive DSc into hair follicles is clearly effective for the growth of new hair and hair growth

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US10774312B2 (en) * 2015-05-14 2020-09-15 Advanced Cell Technology And Engineering Ltd. Method for producing frozen mesenchymal cells and method for producing implantable therapeutic member
WO2017082512A1 (ko) * 2015-11-12 2017-05-18 김동구 발모 조절 중추 세포인 모유두 세포의 증식과 생존 및 세포 활성화 촉진 작용을 지닌 기능성 조절 항체 및 이의 용도
US11174463B2 (en) * 2015-11-17 2021-11-16 Technische Universität Berlin Method for the production of hair follicles and de novo papillae, and use thereof for in vitro tests and in vivo implants

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