HK1183790B - Composition for regenerating follicle which contains cd36-expressing connective tissue sheath cells - Google Patents

Description

Composition for regenerating hair follicle comprising connective tissue sheath cell expressing CD36

Technical Field

The present invention provides compositions for regenerating hair follicles containing connective tissue sheath ("DS": dermal sheath) cells (hereinafter also referred to as "CD-expressing DSc") expressing CD36 antigen (thrombospondin receptor) and optionally hair papilla ("DP": dermal papilla) cells (hereinafter also referred to as "DPc"), methods of regenerating hair follicles using the compositions, and further provides animals or 3-dimensional skin models loaded with hair follicles regenerated by the methods.

Background

It is extremely important that hair be viewed as aesthetically appealing. Thus, depilation for congenital or acquired reasons is a profound annoyance for many people. Particularly in modern society such as an aging society and a stress society, there are increasing chances that head hair will suffer from a crisis of depilation for various acquired reasons. In response to this, various attempts have been made to provide a hair growth agent which exhibits more excellent hair growth effects including promotion of hair growth and hair coarsening (hair frizziness).

Hair follicles are an exceptional organ in mature organisms that repeatedly regenerate themselves almost throughout life. It is expected that elucidation of the mechanism of self-regeneration is associated with highly demanded clinical applications such as depilatory treatment by tissue or cell transplantation, and construction of a skin sheet containing hair follicles and sebaceous glands and having an excellent function similar to natural skin. In recent years, with the growing interest in stem cell research, research on hair follicle epithelial stem cells (epithelial cells) has been rapidly progressing, and properties of hair follicle papilla cells, which are cells of a hair follicle-specific mesenchymal system, have been known. Hair papilla cells function as what is called a command tower that delivers an activation signal to hair follicle epithelial stem cells for self-regeneration of hair follicles, and are judged as cells indispensable together with hair follicle epithelial stem cells in a hair follicle reconstruction evaluation system (Kishimoto et al, proc. Natl. Acadsci. USA (1999), Vol.96, pp. 7336-.

Unlike the epithelial system cells that constitute most of the hair follicle, DP and DS surrounding the hair follicle are collectively constituted by a mesenchymal system-derived cell population. With respect to DS, many findings indicating its importance for hair follicle formation have been reported in recent years. It has also been reported that DP is regenerated from DS in hair bulb section-cutting hair follicle transplantation experiments in rat beard of papillary hair (1), and that DS transplantation in mice induces hair follicle regeneration in hair follicles of which the lower half is cut (2). Jahoda et al also reported (development.1992 Apr:114(4): 887-97; non-patent document 2) that transplantation of DS to humans could induce reconstruction of hair follicles (Horne KA and Jahoda CA. development.1992 Nov:116(3): 563-71; non-patent document 3). Further, the group of Tobin, Paus et al reported that cell movement occurred between DS and DP in the mouse hair cycle, and that proliferation of connective tissue sheath cells (DSc) began before DPc where the hair growth phase began to proliferate (Tobin DJ et al, J. invest. Dermatol., 120:895-904, 2003; non-patent document 4).

Thus, although DS has a high possibility of playing an important role in hair follicle formation, its mechanism of action is almost unknown, and the properties of DSC are not known. We therefore investigated the gene expression profiles characterizing DSCs, which were analyzed in order to elucidate the mechanism of action on hair follicle formation.

Documents of the prior art

Non-patent document

Non-patent document 1: kishimoto et al, Proc. Natl. AcaDSci. USA (1999), Vol.96, pp. 7336-

Non-patent document 2: jahoda CA et al, development.1992 Apr, 114(4):887-97.

Non-patent document 3: horne KA and Jahoda CA. development.1992 Nov;116(3):563-71.

Non-patent document 4: tobin DJ et al, J.Invest.Dermatol.120: 895-904, 2003

Non-patent document 5: linder et al, Federation of American society for Experimental Biology, 14(2), 319 (2000).

Disclosure of Invention

Problems to be solved by the invention

The present invention addresses the problem of providing a novel hair follicle regeneration system.

Means for solving the problems

The present inventors investigated the gene expression profile of DSc using a microarray, and as a result, identified 304 genes, which are DPc genes having an expression rate 2 times or more higher than that of FBc (fibroblast). The genes were functionally classified by GeneOntology of GeneSpring, and as a result, most of them were vascular-related factors, indicating the relationship between DS and blood vessels. Next, CD36 was found to be highly expressed in DSc, and it was found that expression of CD36 in DSc is interlocked with expression of HGF (hepatocyte growth factor) showing a hair growth promoting effect (J. Linder et al, department of American society for Experimental Biology, 14(2), 319 (2000): non-patent document 5).

Accordingly, the present application encompasses the following inventions:

[1] a composition for regenerating hair follicles, comprising CD 36-expressing connective tissue sheath ([ DS ]: dermal sheath) cells.

[2] [ 1 ] the composition for regenerating hair follicles, which further contains hair papilla ("DP": dermal papilla) cells.

[3] [ 2 ] the composition wherein the ratio of CD 36-expressing DSc to the cell number of DPc is about 10: 1-1: 10.

[4] the composition of [ 2 ] or [ 3 ], wherein the CD 36-expressing DSc and DPc are derived together from a mouse, or from a rat, or from a human.

[5] The composition of [ 2 ] or [ 3 ], wherein the CD 36-expressing DSc and DPc are xenogeneic cells, respectively, from mouse, rat or human.

[6] A method for regenerating hair follicles, which comprises transplanting the composition according to any one of [ 1 ] to [ 5 ] to a human.

[7] A method for regenerating hair follicles, which comprises transplanting the composition according to any one of [ 1 ] to [ 5 ] to a recipient animal.

[8] [ 7 ] the method wherein the recipient animal is an immune system-suppressed animal.

[9] The method of [ 7 ] or [ 8 ], wherein the recipient animal is an animal whose immune system is suppressed selected from the group consisting of a nude mouse, a severe combined immunodeficiency mouse (スきッドマウス), and a nude rat.

[10] A method for regenerating hair follicles, wherein a three-dimensional model of skin containing the composition according to any one of [ 2 ] to [ 5 ] is prepared.

[11] A chimeric animal formed as follows: transplanting the composition according to any one of [ 1 ] to [ 5 ] to a recipient animal, and loading the hair follicle thus reconstructed.

[12] [ 11 ] the chimeric animal, wherein the recipient animal is an immune system-suppressed animal.

[13] The chimeric animal according to [ 11 ] or [ 12 ], wherein the recipient animal is an animal whose immune system is suppressed, which is selected from the group consisting of a nude mouse, a severe combined immunodeficiency mouse (スきッドマウス), and a nude rat.

[14] A three-dimensional model of the skin formed by: a three-dimensional model of the skin containing the composition according to any one of [ 2 ] to [ 5 ] is prepared and loaded with the thus-reconstructed hair follicle.

Effects of the invention

The composition for hair follicle regeneration of the present invention can be used in transplantation surgery for hair follicle regeneration, research and development of hair follicle reconstruction.

Drawings

Fig. 1 is a schematic diagram showing the structure of hair follicle tissue.

FIG. 2 shows the expression of various vascular-associated factors in various cells.

FIG. 3 is a tissue staining pattern obtained by staining CD36 and CD 31.

FIG. 4 is a graph of whole-disc (whole-mount) staining of hair follicles stained with CD36 and CD 31.

FIG. 5 shows the co-culture of CD 36-positive DSc with vascular endothelial cells.

Fig. 6 HGF expression results for CD36 positive DSc.

Detailed Description

The present invention provides compositions containing DSc and optionally DPc for regenerating hair follicles, methods of regenerating hair follicles using said compositions, further providing an animal or 3-dimensional skin model loaded with hair follicles so regenerated.

The CD36 antigen is also known as the thrombospondin receptor. CD36 is an integral membrane protein seen on the surface of many cell types in vertebrates, also known as FAT, SCARB3, GP88, glycoprotein iv (gpiv), glycoprotein III b (gpIII b). CD36 is a member of the B class scavenger receptor family of cell surface proteins. CD36 binds to various ligands other than thrombospondin, such as collagen, red blood cells parasitizing plasmodium falciparum, oxidized low density lipoprotein, natural lipoprotein, oxidized phospholipid, and long-chain fatty acid. In recent studies using genetically modified rodents, the clear function of CD36 was confirmed in fatty acid and sugar metabolism, heart disease, taste, and intestinal food fat transport. CD36 may be associated with abnormal glucose tolerance, atherosclerosis, arterial hypertension, diabetes, cardiomyopathy, and alzheimer's disease.

The relationship of the CD36 antigen to hair growth is completely unknown.

DSc is a cell constituting a sheath portion surrounding DP in hair follicle, and is a mesenchymal system cell as in DPc. DP is derived from DS and DS is proliferated before DP is proliferated in the hair growth stage, so DS supply DPc is considered (Tobin DJ et al, J. invest. Dermatol., 120:895-904, 2003; non-patent document 4).

The DSc expressing CD36 is not particularly limited, and can be selected from DPc and the like by a commonly used cell sorting technique using an antibody, preferably a monoclonal antibody, against CD36, for example.

The DSc of the invention may be obtained from the epidermis of all mammals such as humans, chimpanzees, other primates, livestock animals such as dogs, cats, rabbits, horses, sheep, goats, cattle, pigs, other laboratory animals such as rats, mice, guinea pigs, more preferably nude mice, severe combined immunodeficiency mice (スきッドマウス), nude rats. The epidermal site may be a hairy site such as the scalp, or may be a hairless site such as the foreskin.

"DPc (hair papilla cells)" are located at the bottommost part of hair follicle as mesenchymal system cells, and serve as a command tower for delivering an activation signal to hair follicle epithelial stem cells for the self-regeneration of hair follicle. A hair papilla cell preparation containing only activated hair papilla cells can be prepared, for example, by the method described in Kishimoto et al, Proc. Natl. AcaDSci. USA (1999), Vol.96, pp. 7336-7341 using transgenic mice. However, from the viewpoint of yield and the like, it is preferable to prepare a dermal tissue fraction obtained by removing epidermal tissue from skin tissue, for example, by collagen treatment and preparing a cell suspension, and then freeze-preserving the cell suspension to kill hair follicle epithelial cells.

Specifically, the method of cryopreservation can be performed, for example, as follows.

1. Mammalian skin is prepared.

2. The skin is allowed to stand in a proteolytic enzyme solution such as a trypsin solution for an appropriate time such as evening, thereafter, the epidermal part is removed with tweezers or the like, and the remaining dermis is treated with collagenase to prepare a cell suspension.

3. The suspension was filtered through Cell strain as needed and left to remove the precipitate.

4. Measuring the number of cells at an appropriate cell density, preferably 1 × 10⁵～1x10⁸Resuspending the cells in a cryoprotectant solution at a cell density of about/ml, packaging in small quantities as required, and cryopreservation according to conventional cell preservation methods.

5. Melting and using after storing for a proper period.

The freezing method is not particularly limited, and the frozen food is stored in an ultra-low temperature freezer or liquid nitrogen at-20 ℃ or lower, preferably-50 ℃ or lower, more preferably-80 ℃ or lower. The period of cryopreservation is also not particularly limited, and epithelial cells can be killed, and for example, the period is 1 day or more, preferably 3 days or more, and more preferably 1 week or more. Even if the cells were stored in liquid nitrogen for 4 months, it was confirmed that the dermal papilla cells continued to survive. The cryoprotective solution may be any of the usual storage solutions used for Cell storage, for example, Cell Bank 2 Cell cryopreservation solution (catalog number BLC-2) (manufactured by Japan pharmaceutical Industrial Co., Ltd.).

The measurement of the number of cells can be carried out by methods known to the skilled worker. For example, the cell count can be measured by providing a cell suspension diluted with an equal amount of 0.4% trypan blue staining solution (No. 15250-.

DPc of the present invention can be derived from the skin of any mammal such as a human, chimpanzee, other primate, livestock animal such as dog, cat, rabbit, horse, sheep, goat, cow, pig, other laboratory animal such as rat, mouse, guinea pig, more preferably nude mouse, severe combined immunodeficiency mouse (スきッドマウス), nude rat, as with DSc.

Preferably, the composition for hair follicle regeneration of the present invention may further contain "epithelial system cells". "epithelial system cells" are cells that make up the epidermis or most of the epithelium of the skin, and are produced by 1 layer of basal cells connected to the dermis. In the case of a mouse, the epithelial cells may be preferably cells derived from a newborn (or fetus), mature skin, for example, epithelium of a dormant hair or epithelium of a growing hair, or 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 the skilled person.

In a suitable protocol, epithelial system cells may be prepared as follows.

1. Mammalian skin is prepared.

2. The epidermis was trypsinized at 4 ℃ overnight in 0.25% trypsin/PBS as required.

3. Only the epidermal part was peeled off with forceps or the like, and after chopping, the resultant was suspended in an appropriate culture medium (for example, a culture medium for keratinocytes) at 4 ℃ for about 1 hour.

4. The suspension was passed through a Cell separator with appropriate pore size, followed by recovery of epithelial system cells via a centrifugal separator.

5. The cell preparation is suspended with KGM or SFM medium at the desired cell density and left to stand on ice until use.

The epithelial cells of the present invention may be derived from the epidermis of any mammal such as human, chimpanzee, other primate, livestock animal such as dog, cat, rabbit, horse, sheep, goat, cow, pig, other laboratory animal such as rat, mouse, guinea pig, more preferably nude mouse, severe combined immunodeficiency mouse (スきッドマウス), nude rat, as in the case of DSc and DPs. The epidermal site may be a hairy site such as the scalp, or may be a hairless site such as the foreskin.

The ratio of DSc to DPc is not particularly limited, and the content ratio in the composition of the present invention is preferably 1: 10-10: 1. more preferably 1: 3-3: 1. further, for the total amount of DSc and DPs, the epithelial system cells may be present in a ratio of 1: 10-10: 1. more preferably 1: 1-10: 1. further preferably 1: 1-3: 1. most preferably 1: 1 contains.

The combination of DSc with DPc, further optionally epithelial system cells, may be of the same or different lineage. Thus, the composition for regenerating hair follicles of the present invention may be, for example, a combination in which all of DSc, DPc, and cells of the epithelial system are of human origin, a combination in which all of DSc, DPc, and cells of the epithelial system are of the same species as mammals other than humans (hereinafter referred to as "homogeneous system"), a combination in which DSc and DPc are of human origin and cells of the epithelial system are of mammals other than humans, a combination in which one of DSc and DPc is of human origin and the other and cells of the epithelial system are of the same species or different species as mammals other than humans, a combination in which one of DSc and DPc is of mammals other than humans and the cells of the epithelial system are of human origin (hereinafter referred to as "heterogeneous system"), and the like.

The method of transplanting the composition for hair follicle regeneration of the present invention to a recipient animal may be a method of transplanting known per se. For example, reference is made to Weinberg et al, J. invest. Dermatol. Vol.100(1993), pp.229-236. For example, when the cells are transplanted into a nude mouse, the prepared cells are mixed about 1 hour before transplantation, the culture solution is removed by centrifugation (9000 × g, 10 min.) to prepare about 50 to 100 μ L of cell mass, and then the cell mass rapidly flows into a silicone dome-shaped chamber embedded in the back skin of the nude mouse. After 2 weeks the chamber was removed carefully and after 3 weeks the presence of hair formation at the graft site was visually observed. Transplantation for hair growth in animals including humans can be similarly performed, and an appropriate method is appropriately determined by a physician or veterinarian.

When the composition is transplanted into a recipient animal, the transplantation may be an allogeneic transplantation, i.e., an autograft, an allogeneic transplant, an allogeneic xenogeneic transplant, or a xenogeneic transplant. In the case of allogeneic transplantation, both the dermal papilla cell preparation and the epithelial cells are of the same species as the recipient. In xenotransplantation, there are cases where either the dermal papilla cell preparation or the epithelial system cells are xenogeneic with the recipient, the other is syngeneic with the recipient, and both are xenogeneic with the recipient. The recipient animal includes all mammals, such as human, chimpanzee, other primates, livestock animals such as dog, cat, rabbit, horse, sheep, goat, cow, pig, other experimental animals such as rat, mouse, guinea pig, more preferably nude mouse, severe combined immunodeficiency mouse (スきッドマウス), and nude rat.

In addition, by transplanting the above-described composition of the present invention to an appropriate recipient animal, a chimeric animal loaded with a regenerative hair follicle can be provided. The chimeric animal can be used as a powerful animal model, for example, for studying and elucidating the mechanism of hair follicle regeneration, or for screening drugs and drug substances effective for hair follicle regeneration or hair growth or depilation. The recipient animal is not limited to the source of each cell contained in the system transplanted into the animal, and an animal whose immune system is suppressed is preferable. The animal species may be any animal that can be used as an experimental animal, and is preferably a mouse, a rat, or the like, for the purpose of the present invention. Of these animals, those with suppressed immune system include, for example, mice, such as nude mice, which have a thymus deficiency property. For the purpose of the present invention, particularly preferred recipient animals include commercially available nude mice (e.g., Balb-cnu/nu system), severe combined immunodeficiency mice (スきッドマウス) (e.g., Balb/c-SCID), and nude rats (e.g., F344/NJcl-rnu).

Further, by including the composition of the present invention in a three-dimensional skin model, a three-dimensional skin model loaded with regenerative hair follicles can be provided. However, in this case, papilla cells are necessary as a command tower for hair growth. Three-dimensional skin models can be prepared by methods known to the skilled worker (exp.cell res. Amano s. et al, (2001), vol.271, pp.249-362), for example as described below. Three-dimensional skin models are 1 x10 each⁶～10⁸Per cm²Preferably 1.0 to 1.5X 10⁷Per cm²More preferably about 1.27X 10⁷Per cm²Contains DSc and DPc.

Preparation method of three-dimensional skin model

Human fibroblasts were dispersed in appropriate amounts in 0.1% collagen solution/DMEM/10% FBS, dispensed in petri dishes, and rapidly CO at 37 deg.C₂And (5) standing in an incubator. After gelation, the gel was peeled from the side and bottom surfaces of the dish and allowed to float in the dish. The collagen gel was cultured while shaking, and the gel was contracted by about 5 times to prepare a dermal model. Placing the dermal model on a stainless steel grid, placing a glass ring thereon, and culturing human epidermal cells (1.0X 10) dispersed in KGM (culture medium for epidermal cell culture)⁶Cell count/ml) 0.4ml was injected into the glass ring for culture. DSc and DPc are now mixed and injected simultaneously. Mouse neonatal epidermal cells may also be used instead of human cultured epidermal cells.

Placing DMEM-KGM-5% FBS + Ca in a culture dish²⁺The culture medium was exposed to the air until the upper part of the dermal model was cultured, and the skin model was observed about one week later to judge the presence or absence of hair follicle germ formation and reproducibility.

The 3-dimensional skin model loaded with the reconstructed hair follicle can be used for studying and elucidating the mechanism of hair follicle regeneration, and screening agents and drug substances effective for hair growth and hair removal, as in the above-described chimeric animal loaded with the regenerated hair follicle.

The present invention will be described in further detail with reference to examples.

Example 1

(method)

Isolation and culture of cells

In DMEM (Gibco/invitrogen) containing 10% fetal bovine serum, the dermal portion of human scalp tissue was removed with a knife, and the hair follicle was removed from the section. The hair shaft containing ORS (cells of the follicular epithelial system) was removed from the hair follicle using precision forceps and DP and DS were removed. Isolated DP was cultured by standing on ニッスイ fibroblast-coated 35mm dishes (Iwaki) containing medium-1 (ニッスイ fibroblast-coated 35mm dishes containing 10% fetal calf serum, 10 ng/ml EGF, 20 ng/ml bFGF, 0.00075% beta-mercaptoethanol, 100 units/ml penicillin (titer), 0.1mg/ml streptomycin (titer), and 0.25ug/ml amphotericin B (titer)), and the isolated DS was subjected to collagenase treatment at 37 ℃ for 40 min and then similarly cultured by standing on the collagen-coated 35mm dishes. Both were confirmed to proliferate after 1 week, and then used as the test samples of DPc and DSc. Fibroblast cells (FBc) were obtained from commercially available cells (eastern ocean). The DSc, DPc and FBc were subjected to static culture for 7 to 10 days in the medium-1. Thereafter, the cells were passaged using trypsin. The culture conditions were 37 ℃ and 5% CO₂Collagen-coated shake flasks T-75(Iwaki) were used as culture vessels. In addition, the cells subjected to the experiment were applied to the cells subjected to passage 1 to 3.

Vascular endothelial cells were cultured using normal adult skin microvascular endothelial cells (hereinafter referred to as HMVEC) (Kurabo) in a culture medium Humedia-MvG (Kurabo) for low serum proliferation, and cells with a passage number of 5 were used in the experiment.

Comparison of Gene expression profiles of DSc, DPc, FBc Using microarray method

Total RNA containing mRNA was recovered from DPc, DSc, and FBc using RNeasy Micro kit (Qiagen). The collected RNA was synthesized into double-stranded cDNA using the protocol of agilent, and cRNA labeled with cyanin 3 and 5 was further synthesized. The labeled cRNA was hybridized by two-color method at 65 ℃ for 17 hours on a microarray slide (Agilent, whole human genome (4X 44K), G4110) of Agilent. Comparison of gene expression levels between 2 types of DSc and DPc, between DPc and FBc, and between 2 types of FBc and DSc was performed on each chip using 2 types of DS-derived RNA (4 types in total), 2 types of DP-derived RNA (2 types in total), and 2 types of FB-derived RNA (2 types in total) for 2 persons. After washing the slide, the fluorescent signal (cyanines 3, 5) of the cRNA on the chip was imaged with a dual laser microarray scanner (Agilent). The data of the image was quantified by Feature Extraction Software 9.1, and the abnormal value and a value almost equal to or lower than the background were marked and analyzed. For each comparison of expression levels (tagged), quantitative values of the obtained signals were compared among 2 persons.

(analysis of microarray data)

Each Gene expression level was analyzed by bioinformatics, and Gene Spring GX 7.3.1 software (Agilent) was used for more detailed analysis. The analysis was carried out by using Feature Extraction Software 9.1, labeling the abnormal value and a value almost equal to or lower than the background, and analyzing the gene other than the labeled gene. Genes with different expression levels among 2 patients were extracted and functionally classified by Gene ontology. (http:// www.geneontology.org /) additionally at this point, it was verified by Fisher's test how much predominance statistically accounted for.

Cell staining

For cell staining using the CD36 antibody, after the DSc was seeded on a 4-well chamber slide (Nalgene nunc) whose collagen surface was treated with an acidic collagen solution (Koken), the resultant culture was cultured for 1 to 2 days, and the resultant culture was used. After the culture was washed with PBS, fixed with 4% PFA for 30 minutes, washed with PBS, and treated in a PBS solution containing 0.1% TritonX-100 for 10 minutes. Subsequently, blocking treatment was performed for 30 minutes using 3% BSA-containing PBS, and the reaction was performed for 1 hour using a primary antibody solution prepared by 50-fold dilution of CD36 antibody (Abcam, ab17044) in 1% BSA-containing PBS. After washing 4 times with PBS, a secondary antibody solution prepared by 200-fold dilution of Alexa 594-labeled anti-mouse IgG antibody (Invitrogen) in PBS containing 1% BSA was reacted for 1 hour. After reaction in DAPI solution for nuclear staining, washed 4 times with PBS, enclosed by anti-fading Reagent Prolong Gold antibody Reagent (Invitrogen) and cover slip. Observations were performed using a fluorescence microscope (Olympus).

Tissue staining

Human scalp tissue was embedded with an OTC compound (Sakura Finetek) as a frozen tissue embedding medium, and frozen sections were prepared by a cryosection preparation apparatus, a cryostat (Leica). After the sections were fixed with 4% PFA for 15 minutes, they were washed with PBS, and 5% skim milk, 1% monkey serum, and 0.1% triton-X100 blocking solution were added to PBS to react for 1 hour. Subsequently, the mixture was reacted at room temperature for 1 hour or at 4 ℃ overnight using primary antibody solutions obtained by diluting 50-fold or 100-fold each of the CD36 antibody solution (Abcam, ab17044) and the CD31 antibody solution (R & D, AF806) with the blocking solution. The CD31 antibody is used to label CD31 for use as a marker of intravascular cells. After washing with PBS 3 times, secondary antibody solutions obtained by diluting Alexa 594-labeled anti-mouse IgG antibody (Invitrogen) and Alexa 488-labeled anti-rabbit IgG antibody (Invitrogen) 200-fold each with a blocking solution were applied and reacted at room temperature for 1 hour. After reaction with DAPI solution, washed 3 times with PBS, enclosed by a discoloration inhibitor Reagent and a cover slip. Observations were performed using a fluorescence microscope (Olympus).

Hair follicle whole-sheet dyeing

Hair follicles isolated from human tissue were fixed in 4% PFA for 2 hours at 4 ℃ with shaking. Dehydration treatment was performed by applying 0.1% tween PBS (hereinafter referred to as PBST) containing 25%, 50%, and 75% ethanol for 5 minutes and 100% ethanol for 5 minutes, respectively. The treated samples were stored in ethanol at-20 ℃. When used, rehydrated in the same ethanol series of PBST, treated in PBS containing 5% triton X-100 for 10 minutes. Thereafter, a blocking solution used for tissue staining, a primary antibody solution containing CD36 antibody (Abcam, ab17044) and CD31 antibody (R & D, AF806), a secondary antibody solution containing Alexa 594-labeled anti-mouse IgG antibody and Alexa 488-labeled anti-rabbit IgG antibody, and DAPI solution were sequentially reacted. During the reaction procedure for the antibody and after staining, each wash 8 times with 0.1% triton X-100 PBS. The reaction conditions are that the primary antibody solution is kept overnight at 4 ℃ and the secondary antibody is kept for 2-3 hours at 4 ℃. After sealing the Reagent with a coverslip using a discoloration inhibitor Gold antipide Reagent, observation was performed using a fluorescence microscope (Olympus).

RT-PCR

RNA was recovered from cells using the protocol provided using trizol (invitrogen). The concentration of the recovered RNA was determined by a nucleic acid quantifying apparatus nanodrop (thermo scientific). The RNA concentrations of the comparison subjects were made uniform, and cDNA was synthesized from RNA using oligo (dT) primers by reverse transcriptase Superscript III (Invitrogen) using the protocol of Invitrogen. Quantitative RT-PCR was carried out using the reaction reagent LightCycler (registered trade name) FastStart DNA MasterPLUS SYBR Green (Roche) and the reaction apparatus LightCycler (Roche) using the synthesized cDNA as a template. The composition conditions were performed according to the protocol of Roche. The PCR conditions were initial denaturation at 95 ℃ for 10 minutes, denaturation at 95 ℃ for 10 seconds, annealing at 60 ℃ for 10 seconds, and elongation at 72 ℃ for 10 seconds. The information of the primers used is described below.

G3PDH：

A forward primer: 5'-GCACCGTCAAGGCTGAGAAC-3' (SEQ ID NO: 1)

Reverse primer: 5'-ATGGTGGTGAAGACGCCAGT-3' (SEQ ID NO: 2)

CD36：

A forward primer: 5'-GAGGAACTATATTGTGCCTATTCTTTGGC-3' (SEQ ID NO: 3)

Reverse primer: 5'-CATAAAAGCAACAAACATCACCACACCAAC-3' (SEQ ID NO: 4)

CD31：

A forward primer: 5'-ATGCCGTGGAAAGCAGATACTCTAG-3' (SEQ ID NO: 5)

Reverse primer: 5'-AATTGCTGTGTTCTGTGGGAGCAG-3' (SEQ ID NO: 6)

HGF：

A forward primer: 5'-GAGGGAAGGTGACTCTGAATGAG-3' (SEQ ID NO: 7)

Reverse primer: 5'-AATACCAGGACGATTTGGAATGGCAC-3' (SEQ ID NO: 8).

The expression level of each gene was measured by using the attached software. G3PDH was used as an internal standard, and it was used to correct the cDNA amount of the control group when each gene was quantified.

Sorting of cells

The cells were fractionated using Cell Separation Magnet (BD Bioscience). The operating conditions were carried out according to the protocol suggested by BD Bioscience. After stripping the cells with trypsin solution, the cell suspension was passed through a 70um rack (falcon) and the number of cells was counted. 500 to 1000 ten thousand cells were suspended in 500ml of 3% fetal bovine serum-containing PBS, diluted 50-fold with the addition of CD36 antibody (Abcam, ab17044), and reacted on ice for approximately 15 minutes. After washing with 1 XIMag Buffer (BD Bioscience), the cells were recovered by centrifugation, resuspended in 30ul of anti-mouse IgG1 Magnetic Particles (BD Bioscience), and then kept on ice for 30 minutes. 500ul of 1 XMMAG Buffer (BD Bioscience) was added, and the mixture was placed in a Cell Separation Magnet (BD Bioscience), and allowed to stand for 8 minutes. The supernatant was collected without removing the cells adhered to the side of the magnet, and was designated as CD 36-negative DSc. Then 500ul of 1 × IMag Buffer (b) was added againBD Bioscience), the side-adhered cells were suspended, left to stand in a Cell Separation Magnet for 4 minutes, and then the supernatant was removed. This procedure was repeated 1 additional time, with the side-adherent cells as CD36 positive DSc. CD36 positive and negative DSc recovered by suspension with Medium-1, 5% CO at 37 deg.C₂The culture vessels were incubated for 2-4 days using collagen-coated shake flasks T-25(Iwaki), and the resulting cultures were used for subsequent experiments.

Co-culture experiments

Experiments were performed with N =3,4 each using CD36 positive and negative dscs from each sample source. The fractionated CD36 positive and negative dscs were each grown in 30 ten thousand collagen-coated T-25 shake flasks. Thereafter, after 2 days of culture with Medium-1, 40 million HMVECs were added and cultured for 1 day with Humedia-MvG (Kurabo). Thereafter, the medium was replaced with a medium containing 100 units/ml of penicillin (titer), 0.1mg/ml of streptomycin (titer), and 0.25ug/ml of amphotericin B (titer) 0.1% BSA (Sigma) in the vascular endothelial cell basal medium Humedia-EB2 (Kurabo). After further 1 day of co-culture, the cells were detached with trypsin solution and analyzed by FACS. In the same manner as for sorting of CD36 positive cells, the cells were treated with 70um of sealer (falcon), suspended in PBS containing 3% fetal bovine serum, and reacted on ice for 20 minutes using a CD31 antibody solution (R & D, AF806) as a primary antibody. After washing the cells with a PBS solution containing 3% fetal bovine serum, Alexa 488-labeled anti-rabbit IgG antibody (Invitrogen) as a secondary antibody was reacted on ice for 20 minutes, and the cells were resuspended in 0.5ml of PBS solution and analyzed using Cell Lab Quanta SC (BeckmanCoulter). Settings including laser quality management were performed using protocols and operating reagents specified by BeckmanCoulter. The number of CD31 positive cells was determined using FL1 channel (525 nm). Correction for omission of autofluorescence was performed using endothelial cells that did not react with the CD31 antibody. After the measurement, the number of CD31 positive cells was calculated based on the obtained whole cell number and the proportion of CD31 positive cells.

(results)

The results of expression of a part of the vascular-associated factors are shown in table 1. Vascular associated factors are known to be highly expressed in DS, and among them, CD36 and HGF are known to be specifically highly expressed in DS. The expression of various vascular associated factors in DS, DP, ORS, VEC (vascular endothelial cells) is shown in figure 2. CD36 and HGF were known to be expressed very specifically in DS. In the cell staining results, it was also confirmed that CD 36-positive cells were present only in the isolated DS cultured cells, and CD 36-positive cells were not present in DP and FB (data not shown).

[ TABLE 1 ]

The tissue staining results of CD36 and CD31 also confirmed specific staining of CD36 in DS, which is the sheath of the hair follicle (fig. 3). Further, as a result of staining the whole section of the hair follicle, DS showed a dense part of a part of the blood vessels, and it was found that CD 36-positive DSc cells were localized in the dense part. Thus, CD36 was shown to be closely related to blood vessels. It is also known that although CD 36-positive DSc cells were almost co-localized with blood vessels, they were not present near a portion of the blood vessels (fig. 4).

Thus, it was shown that the CD 36-positive DSc cells promote the formation of blood vessels, for example, the proliferation of vascular endothelial cells.

In an experiment in which CD 36-positive DSc isolated by cell sorting was co-cultured with vascular endothelial cells, CD 36-positive DSc showed a significant promotion of vascular endothelial cell proliferation compared to CD 36-negative DSc cells (fig. 5). Further, the isolated CD36 positive DSc also showed high expression of HGF compared to CD36 negative DSc cells (fig. 6). As described at the beginning of the present application, HGF is known as a factor that promotes hair growth and hair growth (non-patent document 5). It is therefore evident that if CD 36-positive DSc is transplanted in hair follicles, it is effective for hair growth.

Sequence listing

<110> SHISEIDO COMPANY, LTD.

<120> composition for regenerating hair follicle comprising connective tissue sheath cell expressing CD36

<130> Y742-PCT

<160> 8

<170> PatentIn version 3.5

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<223> CD36 Forward primer

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Claims (14)

1. A composition for regenerating hair follicles comprising dermal sheath cells (DSc) which are all CD 36-expressing.

2. The composition for regenerating hair follicles of claim 1, further comprising hair papilla cells (DPc).

3. The composition of claim 2, wherein the ratio of CD 36-expressing DSc to the cell number of DPc is 10: 1-1: 10.

4. the composition of claim 2 or 3, wherein CD 36-expressing DSc and DPc are derived together from a mouse, or together from a rat, or together from a human.

5. The composition of claim 2 or 3, wherein CD 36-expressing DSc and DPc are xenogeneic cells from mouse, rat, or human, respectively.

6. Use of a composition according to any one of claims 1 to 5 in the manufacture of a medicament for regenerating hair follicles in a human.

7. Use of a composition according to any one of claims 1 to 5 in the manufacture of a medicament for regenerating hair follicles in a recipient animal.

8. The use of claim 7, wherein the recipient animal is an immune system suppressed animal.

9. The use of claim 7 or 8, wherein the recipient animal is an immune system suppressed animal selected from the group consisting of a nude mouse, a severe combined immunodeficiency mouse, and a nude rat.

10. Use of a three-dimensional model of skin comprising a composition according to any one of claims 2 to 5 in the preparation of a device for regenerating hair follicles.

11. A method of producing a chimeric animal comprising: transplanting a composition according to any one of claims 1 to 5 to a recipient animal and loading the hair follicle so reconstituted.

12. The method of claim 11 wherein the recipient animal is an immune system suppressed animal.

13. The method of claim 11 or 12, wherein the recipient animal is an immune system suppressed animal selected from the group consisting of a nude mouse, a severe combined immunodeficiency mouse, and a nude rat.

14. A three-dimensional model of the skin formed by: preparing a three-dimensional model of skin containing a composition according to any one of claims 2 to 5 and loading the hair follicle so reconstructed.