JP5686957B2 - Composition for regenerating hair follicles containing CD36-expressing connective tissue sheath cells - Google Patents

Description

  The present invention relates to connective tissue sheath (“DS”) skin 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.

  Hair is very important in terms of 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-renewal 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 loss treatment by tissue and cell transplantation, construction of skin sheets that are functionally superior in nature containing hair follicles and sebaceous glands. Be expected. In recent years, research on hair follicle epithelial stem cells (epithelial cells) has progressed rapidly with increasing interest in stem cell research, and the characteristics 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 of hair follicle reconstitution (Kishimoto et al., Proc. Natl. AcaDSci. USA (1999), Vol. 96, pp. 7336-7341; Non-Patent Document 1).

  Both the DP and the DS surrounding the hair follicle are composed of a group of cells derived from the mesenchymal system, unlike the epithelial cells constituting most of the hair follicle. Numerous findings have been reported in recent years that suggest the importance of DS for hair follicle formation. In hair follicle rat hair hair cut hair follicle transplantation experiments, 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). In addition, Jahoda et al. (Development. 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). In addition, Tobin, Paus et al.'S group showed that cell migration between DS and DP occurs in the mouse hair cycle, and connective tissue sheath cells (DSc) proliferate prior to 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).

  Thus, although DS is likely to play an important role in hair follicle formation, most of the mechanism of action is 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.

Kishimoto et al., Proc. Natl. AcaDSci. USA (1999), Vol.96, pp. 7336-7341 Jahoda CA et al., Development. 1992 Apr; 114 (4): 887-97. Horne KA and Jahoda CA.Development.1992 Nov; 116 (3): 563-71. Tobin DJ et al., J. Invest. Dermatol., 120: 895-904, 2003 J. Linder et al., Federation of American Societies for Experimental Biology, 14 (2), 319 (2000)

  An object of the present invention is to provide a novel hair follicle regeneration system.

  As a result of examining the gene expression profile of DSc using the microarray, the present inventor has identified 304 genes as genes whose expression rate is twice or more higher than 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. Among them, DSc was found to express CD36 at a high level, and the expression of such CD36 in DSc is HGF (hepatocyte growth factor) (J. Linder et al., Federation of It was found to be linked to the expression of American Societies for Experimental Biology, 14 (2), 319 (2000): Non-Patent Document 5).

Accordingly, 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 according to [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.
[5] The composition according to [2] or [3], wherein the CD36-expressing DSc and DPc are heterologous cells, each derived from a mouse, rat or human.
[6] A method for transplanting the composition according to any one of [1] to [5] to a human and regenerating a hair follicle.
[7] A method for regenerating a hair follicle by transplanting the composition of any one of [1] to [5] to a recipient animal.
[8] The method of [7], wherein the recipient animal is an animal with a suppressed immune system.
[9] The method according to [7] or [8], wherein the recipient animal is an animal with a suppressed immune system selected from the group consisting of nude mice, skid mice, and nude rats.
[10] A method for producing a three-dimensional skin model containing the composition according to any one of [2] to [5] and regenerating a hair follicle.
[11] A chimeric animal in which the composition according to any one of [1] to [5] is transplanted to a recipient animal and thus carries a reconstituted hair follicle.
[12] The chimeric animal of [11], wherein the recipient animal is an animal with a suppressed immune system.
[13] The chimeric animal according to [11] or [12], wherein the recipient animal is an animal with a suppressed immune system selected from the group consisting of a nude mouse, a skid mouse, and a nude rat.
[14] A three-dimensional skin model comprising a composition of 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. Expression of various blood vessel-related factors in various cells. The tissue staining figure which performed CD36 and CD31 dyeing | staining. Whole-mount staining diagram of hair follicles by CD36 and CD31 staining. Co-culture results of CD36 positive DSc and vascular endothelial cells. 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. Provide a dimensional skin model.

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. In addition to thrombospondin, 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. Recent studies using genetically modified rodents have identified a clear role for CD36 in fatty acid and sugar metabolism, heart disease, taste, and intestinal food fat transport. 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 thought 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 can be used in 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, a 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. Cell. 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. However, in terms of yield and the like, preferably, for example, 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. Can be prepared by killing hair follicle epithelial cells.

Specifically, 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 a suitable cell density, preferably about 1 × 10 ^{5 to} 1 × 10 ⁸ / ml, aliquot if necessary, and cryopreserve 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 at -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. As the cryoprotective solution, 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.

  The number of cells can be measured by a method well known to those skilled in the art. For example, the cell count is measured 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.

  The DPc of the present invention, like DSc, 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.

  Preferably, 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.

In a preferred embodiment, 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 is peeled off with tweezers, etc., and after chopping, suspended in an appropriate culture solution (for example, a culture solution for keratinocytes) 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.

  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.

  Although the ratio of DSc to DPc used is not limited, it is preferably included in the composition of the present invention at a ratio of 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.

  The combination of DSc and DPc, and optionally epithelial cells, may be homogeneous or heterogeneous. Therefore, the 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 regrowth of the hair follicle according to the present invention to a recipient animal may be a transfer method known per se. See, for example, Weinberg et al, J. Invest. Dermatol. Vol. 100 (1993), pp. 229-236. For example, when transplanting nude mice, the prepared cells are mixed immediately before transplantation to 1 hour before transplantation, and the culture solution is removed by centrifugation (9000 × g, 10 min.) To make a cell mass of about 50 to 100 μL. Immediately pour into a silicone dome-shaped chamber embedded in the back skin of a nude mouse. After 2 weeks, 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.

  When the composition is transplanted into a recipient animal, the transplant may be an allograft, that is, an autograft, an allogeneic transplant, an allogeneic transplant, or a xenotransplant. In the case of allogeneic transplantation, the dermal papilla cell preparation and epithelial cells are both allogeneic with the recipient. In 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.

  In addition, 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. Such a chimeric 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. I will. 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. Among such animals, those whose immune system is suppressed include those having traits such as athymic deficiency, such as nude mice, in the case of mice. In view of the object of the present invention, particularly preferable 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.

Furthermore, 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. However, in this case, 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. . In the three-dimensional skin model, DSc and DPc are each 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. Place the dermis model on 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. As an alternative to 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 follicle. it can.

  Hereinafter, the present invention will be described in more detail with reference to examples.

(Method)
Isolation and culture of cells Human scalp tissue was removed with 10% fetal bovine serum-containing DMEM (Gibco / invitrogen) with a scalpel, and the hair follicle was taken out from the cut surface. The hair shaft containing ORS (hair follicle epithelial cells) was removed from the hair follicle using precision tweezers, and DP and DS were taken out. The isolated DP was medium-1 (10% fetal bovine serum, 10 ng / ml EGF, 20 ng / ml bFGF, 0.00075% β 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. In both cases, proliferation was confirmed after one week, and then used as experimental samples as DPc and DSc. Commercially available cells (Toyobo) were used as fibroblasts (FBc). DSc, DPc, and FBc were subjected to static culture with medium-1 for 7 to 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. Moreover, each cell used for experiment used what was passaged 1-3 times.
Normal human adult skin microvascular endothelial cells (hereinafter referred to as HMVEC) (Kurabo) are used as vascular endothelial cells, cultured in a low serum growth medium Humedia-MvG (Kurabo), and those with 5 passages are used for experiments. It was.

Comparison of gene expression profiles of DSc, DPc, and FBc using microarray method
Total 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 and DPc, DPc and FBc using 2 types of 2 individual DS-derived RNAs (4 types in total) and 2 types of 2 individual DP-derived RNAs (4 types in total) and 1 type of 2 individual FB-derived RNAs Comparison of gene expression levels between FBc and DSc was performed on each chip slide. After washing the slide, the fluorescence signal of cRNA on the chip (cyanine 3, 5) was imaged with a dual-laser Microarray scanner (Agilent). The imaged data was quantified with Feature Extraction Software 9.1, and at that time, abnormal values and values as low as the background were marked and analyzed. (Tagging) Each expression level was compared by comparing the obtained signal quantitative values between the two.

(Analysis of microarray data)
Gene spring GX 7.3.1 software (Agilent) was used to analyze each gene expression level in more detail using a bioinformatics method. In the operation using Feature Extraction Software 9.1, abnormal values and values as low as background were marked, but analysis was performed using genes other than those marked. Genes with different expression levels between the two were extracted and functionally classified using Gene ontology. (http://www.geneontology.org/) At that time, the statistical significance was verified by the Fisher test.

Cell staining
For cell staining using the CD36 antibody, DSC was seeded on a 4-well chamber slide (Nalgene nunc) treated with collagen using an acidic collagen solution (Koken) and 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. After washing 4 times with PBS, the reaction was performed for 1 hour with a secondary antibody solution in which Alexa 594-labeled anti-mouse IgG antibody (Invitrogen) was diluted 200-fold with PBS containing 1% BSA. After reaction with DAPI solution for nuclear staining, it was washed 4 times with PBS, and sealed with Prolong Gold Antifade Reagent (Invitrogen) and a cover glass. Observation was performed using a fluorescence microscope (Olympus).

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. 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. Then, 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 4 ° C. overnight for the primary antibody solution and 2 to 3 hours at 4 ° C. for the secondary antibody. 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 attached software, the expression level of each gene was measured. G3PDH was used as an internal standard, and was used to correct the amount of cDNA in the control group when quantifying each gene.

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 μm, 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. After washing with 1 × IMag Buffer (BD Bioscience) and centrifugation, the cells were collected, resuspended in 30 ul Anti-mouse IgG1 Magnetic Particles (BD Bioscience), and left on ice for 30 minutes. 500 ul of 1 × IMag Buffer (BD Bioscience) was added, set in a Cell Separation Magnet (BD Bioscience), and allowed to stand for 8 minutes. The supernatant was collected with care not to peel off the cells adhered to the side surface with a magnet, and this was designated as CD36 negative DSc. Subsequently, 500 ul of 1 × IMag Buffer (BD Bioscience) was added again, the cells adhering to the side surface were suspended, set in a Cell Separation Magnet, allowed to stand for 4 minutes, and then the supernatant was removed. This was repeated once more, and the cells adhering to the side surface were designated as 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.

Co-culture experiment Using each sample-derived CD36 positive and negative DSc, an experiment was performed at N = 3,4. 300,000 fractionated CD36 positive and negative DSc were each seeded in a collagen-coated T-25 flask. Thereafter, after culturing in medium-1 for 2 days, 400,000 HMVECs were added and co-cultured for 1 day in Humedia-MvG (Kurabo). After that, penicillin 100units / ml (titer), streptomycin 0.1mg / ml (titer), amphotericin B 0.25ug / ml (titer) 0.1% BSA (Sigma) to vascular endothelial cell basal medium Humedia-EB2 (Kurabo) The medium was changed to the added medium. After further co-culture for 1 day, the cells were detached with a trypsin solution and proceeded to subsequent analysis using FACS. In the same manner as CD36 positive cell sorting, treatment with 70um strainer (Falcon), suspension in PBS solution containing 3% fetal bovine serum, and primary antibody CD31 antibody solution (R & D, AF806) on ice For 20 minutes. After washing the cells with 3% fetal bovine serum-containing PBS solution, 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 | amendment in order to omit autofluorescence was performed using the endothelial cell which was not made to react with CD31 antibody. After the measurement, the number of CD31 positive cells was calculated based on the obtained total cell count and the ratio of CD31 positive cells.

(result)
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).

As a result of tissue staining in which CD36 and CD31 were stained, specific staining of CD36 was observed in the sheath of the hair follicle, that is, DS (FIG. 3). Furthermore, the results of whole-mount staining of hair follicles showed that a dense part of blood vessels was shown in a part of DS, and CD36 positive DSc cells were localized in the dense part. Therefore, it was suggested that CD36 is deeply involved with blood vessels. It was also found that CD36-positive DSc cells co-localize with blood vessels, but do not exist near some blood vessels (FIG. 4).
Therefore, it was suggested that CD36 positive DSc cells promote the formation of blood vessels, for example, by promoting the proliferation of vascular endothelial cells.

  Experiments in which CD36 positive DSc isolated by cell sorting was co-cultured with vascular endothelial cells showed that CD36 positive DSc significantly promoted proliferation of vascular endothelial cells compared to CD36 negative DSc cells (FIG. 5). Furthermore, it was shown that the isolated CD36 positive DSc expressed HGF higher than the CD36 negative DSc cells (FIG. 6). As described at the beginning, HGF is known as a factor that promotes hair growth and hair growth (Non-patent Document 5). Therefore, it is clear that if CD36 positive DSc is transplanted into a hair follicle, it is effective for hair growth and hair growth.

Claims (15)

  A composition for regenerating a hair follicle, comprising CD36-expressing connective tissue sheath cells (DSc).   The composition for regenerating a hair follicle according to claim 1, further comprising hair papilla cells (DPc).   The composition according to claim 2, wherein the ratio of the number of CD36-expressing DSc to DPc is 10: 1 to 1:10.   The composition according to claim 2 or 3, wherein the CD36-expressing DSc and DPc are both derived from a mouse, or both from a rat, or both from a human.   The composition according to claim 2 or 3, wherein the CD36-expressing DSc and DPc are heterologous cells, each derived from a mouse, a rat or a human.   A method for producing a three-dimensional skin model comprising the composition according to any one of claims 2 to 5 and regenerating a hair follicle.   A chimeric animal, excluding humans, transplanted with a composition according to any one of claims 1 to 5 to a recipient animal, and thus carrying a reconstituted hair follicle. The chimeric animal according to claim 7 , wherein the recipient animal is an animal with a suppressed immune system. The chimeric animal according to claim 7 or 8 , wherein the recipient animal is an animal in which an immune system selected from the group consisting of a nude mouse, a skid mouse, and a nude rat is suppressed.   A skin three-dimensional model comprising the composition according to any one of claims 2 to 5 and thus carrying a reconstructed hair follicle.   Use of CD36 expression positive DSc in the manufacture of a composition for regenerating hair follicles. The use according to claim 11 , wherein the composition further comprises dermal papilla cells (DPc). 13. Use according to claim 12 , wherein the ratio of the number of CD36 expressing DSc to DPc cells is 10: 1 to 1:10. 14. Use according to claim 12 or 13 , wherein the CD36 expressing DSc and DPc are both derived from a mouse, or both from a rat, or both from a human. 14. Use according to claim 12 or 13 , wherein the CD36 expressing DSc and DPc are heterologous cells, each derived from a mouse, rat or human.