JP4689175B2 - Composition, method for regenerating hair follicle and animal carrying regenerated hair follicle - Google Patents

Description

  The present invention relates to a composition containing dermal papilla cells and epithelial cells for regenerating hair follicles, a method for regenerating hair follicles using the composition, and an animal carrying a hair follicle regenerated by such a method Alternatively, a three-dimensional skin model is provided.

  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. Acad. Sci. USA (1999), Vol. 96, pp. 7336-7341; Non-Patent Document 1).

  Aiming at the regeneration of hair follicles, hair follicle reconstruction experiments using animal models have been conducted in various ways. Weinberg et al., J. Invest. Dermatol. (1993), Vol. 100, pp. 229-236 (Non-Patent Document 2) describes a method for reconstructing hair follicles by cell transplantation. The transplantation system of Weinberg et al. Has a complicated structure in which mouse 3T3 cells are added in addition to dermal papilla cells and neonatal epithelial cells (including follicular epithelial stem cells). According to the method of Weinberg et al., Hair follicles regenerate without adding neonatal epithelial cells including hair follicle epithelial stem cells to the transplantation system. However, this is considered to be a phenomenon that occurred because it was difficult to completely remove undifferentiated epithelial cells (hair follicle epithelial stem cells) and hair follicle primordia from the hair papillary cell fraction. Subsequently, Kishimoto et al. (Supra) succeeded in isolating and purifying dermal papilla cells for the first time, and as a result of performing follicular reconstitution experiments using cell transplantation methods in animal models using isolated and purified dermal papilla cells. When a cell fraction containing a combination of cells and epithelial cells is transplanted, hair follicles are reconstituted and hair growth is observed, but when a cell fraction containing only hair papilla cells or epithelial cells is transplanted, I have found that no hair is found.

  However, the method for purification of hair papilla cells by Kishimoto et al. Uses the fact that hair papilla cells have the property of specifically expressing versican (chondroitin sulfate proteoglycans), and uses DNA in which a reporter gene is linked to a versican gene. Isolation / concentration is performed by using the produced transgenic mouse model with versican expression as an index. Therefore, this method requires preparation of transgenic mice and purification of dermal papilla cells using a cell sorter. In particular, a large amount of dermal papilla cells are required for the hair follicle reconstitution method to actually regenerate and cause hair growth (eg, 5 million cells per transplant), so this method requires a large amount. The production of a transgenic mouse and the use of a high-speed cell sorter for a long time required problems in terms of economy, working time and labor. For example, Prouty et al., American J. Pathol. (1996) Vol.148, No.6, pp.1871-1885 (Non-patent Document 3) describes the isolation of dermal papilla cells. This is a complicated, low purity and low yield method that repeats fractionation.

Therefore, it is difficult to obtain active hair papilla cells isolated and purified in a sufficient amount for, for example, transplantation by the conventional hair papilla cell purification methods, and the role of the active hair papilla cells in the hair follicle regeneration system Could not be fully elucidated. In particular, it has been virtually impossible to determine the appropriate ratio of dermal papilla cells to epithelial cells in the hair follicle reconstitution system that regenerates hair follicles with prior art purification methods.
Kishimoto et al., Proc. Natl. Acad. Sci. USA (1999), Vol. 96, pp. 7336-7341 Weinberg et al., J. Invest. Dermatol. (1993), Vol. 100, pp. 229-236 Prouty et al., American J. Pathol. (1996) Vol.148, No.6, pp.1871-1885

  An object of the present invention is to provide a hair follicle regeneration system containing active hair papilla cells and active epithelial cells in a ratio suitable for hair follicle regeneration.

  Surprisingly, the inventor of the present invention, when cryopreserving a cell suspension of a dermis tissue fraction collected from skin tissue, follicular epithelial cells contaminated in the suspension are specifically killed, and the hair papilla The cells were found to remain largely active. Since the cell preparation thus obtained contains only hair papilla cells as active cell components, it can be used to determine the proportion of hair papilla cells, vs. epithelial cells, effective in regenerating hair follicles. . As a result, the present inventor was able to determine the ratio of the dermal papilla cells to the epithelial cells in the hair follicle reconstitution system that is optimal for hair follicle regeneration.

  Accordingly, the present invention comprises a hair follicle cell and an epithelial cell, wherein the ratio of the number of cells of the hair papilla cell to the epithelial cell is 1:10 to 10: 1. A composition for regenerating is provided.

  More preferably, the present invention is a composition for regenerating hair follicles comprising hair papilla cells and epithelial cells, wherein the dermal tissue fraction obtained by removing epidermal tissue from the skin tissue is treated with collagen. A cell suspension, and then comprising a hair papilla cell preparation and epithelial cells prepared by killing hair follicle epithelial cells by cryopreserving the cell suspension, wherein Provided is a composition for regenerating a hair follicle, wherein the ratio of the number of cells of the dermal papilla cells to epithelial cells is 1:10 to 10: 1.

  Preferably, the ratio of the number of dermal papilla cells to epithelial cells is 1: 3 to 10: 1, more preferably 1: 1 to 10: 1, even more preferably 1: 1 to 3: 1. Most preferred is 1: 1.

  The present invention further provides a method for regenerating a hair follicle on an animal or a three-dimensional skin model using the above composition, and thus an animal or a three-dimensional skin model in which the hair follicle is regenerated.

  A method for preparing a hair papilla cell preparation containing only hair papilla cells as an active cell component in a simple manner without using a transgenic mouse was provided. This dermal papilla cell preparation can be used for transplantation surgery for hair follicle regeneration and for research and development of hair follicle reconstruction. In particular, since the hair papilla cell preparation is free of active epithelial stem cells, it is necessary to precisely adjust the ratio of the number of active hair papilla cells and active epithelial hair stem cells used for hair follicle regeneration. Moreover, it is advantageous in a situation where a large amount of cells are required.

  The present invention relates to a composition containing dermal papilla cells and epithelial cells for regenerating hair follicles, a method for regenerating hair follicles using the composition, and an animal or a carrier carrying such regenerated hair follicles. A three-dimensional skin model is provided.

  “Papilla cells” are cells located at the bottom of the hair follicle as mesenchymal cells and send activation signals to the hair follicle epithelial stem cells for the self-renewal of hair follicles. Say. A dermal papilla cell preparation containing only activated dermal papilla cells can be prepared, for example, by the method of Kishimoto (supra) using a transgenic mouse. 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 a mammalian epidermis.
2. If necessary, leave this epidermis 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 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 in a cryoprotective solution at an appropriate 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 cell suspension diluted with an equal volume of 0.4% trypan blue staining solution (No. 15250-061, Invitrogen) on a hemocytometer (SLGC, EOSINOPHIL COUNTER) It can be calculated according to the method described in the instruction manual attached to the calculator.

  The dermal papilla cells of the present invention can be any mammal, such as humans, chimpanzees, other primates, livestock animals such as dogs, cats, rabbits, horses, sheep, goats, cows, pigs, as well as laboratory animals such as rats, It can be derived from the epidermis of mice, guinea pigs, more preferably nude mice, skid mice, nude rats.

  “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 mice 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, the epidermis of resting hair or the 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 a mammalian epidermis.
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, for example, humans, chimpanzees, other primates, livestock animals such as dogs, cats, rabbits, horses, sheep, goats, cows, pigs, etc. It can be derived from the epidermis of a working animal such as a rat, mouse, guinea pig, more preferably nude mouse, skid mouse, nude rat. Further, the epidermis part may be a hairy part, for example, a scalp, or a hairless part, for example, a foreskin.

  The present inventor obtained only the dermal papilla cells as active cells, obtained by the above cryopreservation, and contained the dermal papilla cell preparation in which the epithelial cells were killed, and only the active epithelial cells from which the dermal papilla cells were removed. The epithelial cell preparations were mixed at various cell ratios and transplanted into recipient animals to study hair follicle regeneration. It has been found that there is a certain relationship between That is, when it is desired to regenerate more hair follicles, the ratio of the number of active hair papilla cells to active epithelial cells is 1: 3 to 10: 1, more preferably 1: 1 to 10: It has been found that it should be 1, even more preferably 1: 1 to 3: 1, most preferably 1: 1. In other words, hair follicle regeneration can be adjusted by appropriately adjusting the ratio of active hair papilla cells and active epithelial cells and transplanting the cells into a recipient animal.

  The combination of dermal papilla cells and epithelial cells may be the same or different. For example, when the dermal papilla cell preparation is derived from a mouse, the epithelial cells may be derived from a mouse (homogeneous) or from other species such as rats, humans (heterologous). Therefore, the composition for regenerating the hair follicle of the present invention may be, for example, a combination in which the dermal papilla cells and epithelial cells are both derived from a mouse, a combination that is both derived from a rat, or a combination that is both derived from a human. Allogeneic), or hair papilla cells derived from mice, epithelial cells derived from rats, hair papilla cells derived from rats, epithelial cells derived from mice, hair papilla cells derived from mice A combination in which epithelial cells are derived from human, a hair papilla cell is derived from rat, a combination in which epithelial cells are derived from human, a combination in which hair papilla cells are derived from human, and an epithelial cell is derived from mouse, A combination of the hair papilla cells derived from a human and the epithelial cells derived from a rat (hereinafter, different types) may be used.

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 into 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 silicon dome-shaped chamber embedded in the back of a nude mouse. After one week, the chamber is carefully removed, and after the second 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.
For transplantation, for example, with respect to a circle having a diameter of about 1 cm, the amount of hair papilla cells transplanted is 1 × 10 ⁶ to 10 ⁸ cells / cm ² , preferably 1.0 to 1.5 × 10 ⁷ cells / cm ² , more preferably. Is preferably transplanted at a transplantation rate of 1.27 × 10 ⁷ cells / cm ² .

  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. The 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.249-262), for example, as follows. . The three-dimensional skin model has 1 × 10 ⁶ to 10 ⁸ cells / cm ² , preferably 1.0 to 1.5 × 10 ⁷ cells / cm ² , more preferably about 1.27 × 10 ⁷ cells / cm ² . in an amount of cm ^2.

Method for preparing three-dimensional skin model 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, set a glass ring on it, 0.4 ml of human cultured epidermal cells (1.0 × 10 ⁶ cells / ml) dispersed in KGM (epidermal cell culture medium), glass Inject into the ring and incubate. At this time, the hair papilla cell fraction is 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 the air, and observe the skin model after about one week. Determine reproducibility.

  The three-dimensional skin model carrying reconstituted hair follicles is used for research and elucidation of the mechanism of hair follicle regeneration, as well as for the screening of drugs and herbal medicines that are effective for hair growth and hair removal, similar to the chimeric animal carrying the regenerated hair follicles. it can.

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

  In order to confirm that cryopreservation of the dermal cell fraction kills epithelial cells and obtains a cell preparation containing only dermal papilla cells as active cells, the marker protein LacZ, downstream of the Versican promoter Flow cytometric analysis of cell preparations obtained by cryopreserving cell fractions obtained from skin tissue collected from transgenic mice (hereinafter referred to as “Versican-LacZ TG mice”) into which an expression vector linked with a structural gene has been introduced. did.

(1) Preparation of “cryopreserved” hair papilla cell preparation from dermal cells of Versican-LacZ TG mice (1-1) Among newborns born from Versican-LacZ TG mice (used within 4 days of birth) LacZ positive individuals were selected. Versican-LacZ TG mice can be prepared, for example, by the method described in Kishimoto et al.
(1-2) After washing each individual with ethanol and phosphate buffered saline (hereinafter “PBS”), the dorsal skin was peeled off and allowed to stand overnight at 4 ° C. in 0.25% trypsin / PBS. .
(1-3) The next day, the epidermis and dermis are separated with tweezers, etc., and the dermis side is treated with 0.35% collagenase / DMEM (Dulbecco's modified Eagle's minimal medium; hereinafter “DMEM”) at 37 ° C. for about 1 hour. did.
(1-4) After carefully suspending the cells of (1-3), the cells were passed through a cell strainer and then collected by a centrifuge (900 g, 10 minutes).
(1-5) Count the number of cells, resuspend in a cell cryoprotectant (Selvanka-2 (BLC-2), manufactured by Nippon Zenyaku Kogyo) at a cell density of 1 × 10 ⁵ to 1 × 10 ⁸ / ml, and freeze tube In accordance with a normal cell storage method, the cells were stored in liquid nitrogen.
(1-6) Thawed after about one week, and used for the following flow cytometry analysis.

(2) Fluororeporter LacZ flow cytometry analysis materials / Fluororeporter LacZ flow cytometry kits Molecular Probe, Catalog No. F-1930 (for 50 reactions) / F-1931 (for 250 reactions))
・ Reagent preparation:
Reaction solution: FDG reagent (Component A) in the kit was diluted 1:10 with MiliQ water. 50 μL was used per sample.
Stop solution: The PI reagent (Component D) in the kit was diluted 1: 100 with the buffer supplied with the kit. 0.9 ml was used per sample. It was chilled to 4 ° C. in ice until use. As a staining medium, a CD49f monoclonal antibody (manufactured by Cellotech, MCA699), which is a specific antibody that specifically stains epithelial cells, and 7-ADD (manufactured by Beckman Coulter, PN-IM 3422) that specifically stains dead cells Was used.

(2-1) A suspension of dermal cells derived from versican-LacZ TG mice was adjusted to a cell number of up to ˜1 × 10 ⁷ cells, 750 ul of staining medium was added, and the suspension was transferred to a 1.5 ml Eppendorf tube.
(2-2) The mixture was centrifuged at 3000 rpm for 5 minutes, and the supernatant was discarded. The cell pellet was resuspended in 100 μL staining media. This suspension was preincubated for 10 minutes in a 37 ° C. thermostat.
(2-3) 50 μL of a reaction solution preincubated for 10 minutes in a 37 ° C. constant temperature bath was added, and the reaction was performed at 37 ° C. for exactly 1 minute.
(2-4) 0.9 ml of stop solution was added and stored in ice.
(2-5) After 10 minutes, 40 μL of 50 mM PETG (Component B) was added to completely inhibit the reaction.
(2-6) The fluorescence intensity was immediately measured by FACS. The operation method of the flow cytometer (FACS) was according to the manufacturer's instruction manual. As the FACS measuring apparatus, for example, XL-MCL manufactured by Beckman Coulter can be used. The distribution of the fluorescence intensity of the cells was measured with a detection setting suitable for the fluorescent dye Fluorescein used in this kit.

(3) Analysis results
FACS analysis based on LacZ and 7-AAD showed that the majority of LacZ ⁺ cells in freeze-thawed cells were viable cells even after freezing and thawing (FIG. 1). Therefore, it became clear that the hair papilla cells were not killed by freezing and thawing. In addition, FACS analysis based on CD-49 and 7-AAD shows that most of CD-49 ⁺ cells (epithelial cells) are present in the 7-AAD ⁺ fraction (dead fraction) after freezing and thawing. (FIG. 2). Therefore, the living cells in the frozen and thawed cell preparation are LacZ ⁺ , CD-49 ⁻ , 7-AAD ⁻ , ie, hair papilla cells (LacZ ⁺ ) and non-epithelial cells (CD-49 ⁻ ), a ^- living cells (7-AAD). Summarizing the above results, it was found that most of the living cells were not epithelial cells but dermal papilla cells. Thus, it was revealed that epithelial cells can be specifically killed by freezing and thawing, and a hair papilla cell preparation containing only hair papilla cells as active cells can be prepared.

We tried to regenerate hair follicles by mixed transplantation of frozen and thawed dermal papilla cells and epithelial cells.
I. Preparation of various cells (1) Mouse-derived epithelial cells (1-1) The day before surgery, each individual was washed from a newborn ICR mouse with ethanol and phosphate buffered saline (hereinafter “PBS”), and the back skin was removed. The skin was trypsinized by exfoliation and standing overnight at 4 ° C. in 0.25% trypsin / PBS.
(1-2) Only the epidermis part was peeled off with tweezers, and after chopping, suspension treatment was performed at 4 ° C. for about 1 hour in a culture solution for keratinocytes (hereinafter “KGM”).
(1-3) The suspension of (1-2) passed through a cell strainer was subjected to a centrifuge (× 900 g, 10 minutes) to recover epithelial cells.
(1-4) An amount of epithelial cells (about 1 × 10 ⁷ as the number of cells) corresponding to two newborns was used for surgery per recipient animal. A considerable amount of cells were suspended in KGM or SFM medium and left on ice until just before use. This is a “mouse-derived epithelial cell” preparation.

(2) Preparation of “prepared in use” mouse-derived dermal cell preparation (comparative example) (2-1) Same day as above (1-1) and (1-2) from the day before surgery, from newborn ICR mouse strain The skin was trypsinized by the method.
(2-2) The epidermis part was peeled off with tweezers, and the remaining dermis was cut into small pieces and then suspended in an appropriate culture solution DMEM + 10% FBS containing 0.35% collagenase at 37 ° C. for about 1 hour.
(2-3) Dermal cells were collected by centrifuging the suspension (2-2) passed through the cell strainer.
(2-4) About 1 × 10 ⁷ dermal cells were used for surgery per recipient animal. A considerable amount of cells were suspended in DMEM + 10% FBS or the like and left on ice until just before use. This is a “prepared at the time of use” mouse-derived dermal cell preparation.

(3) Preparation of “cryopreserved” mouse-derived dermis cell preparation containing hair follicle cell fraction (3-1) Newborn ICR strain mouse dorsal skin was peeled and epidermis was collected.
(3-2) The plate was allowed to stand overnight in a trypsin solution, and the epidermis was removed with tweezers the next day, and the remaining dermis was treated with collagenase to prepare a cell suspension.
(3-3) The suspension was filtered with a cell strainer, and the precipitate was removed by standing.
(3-4) The number of cells was counted, resuspended in a cryoprotective solution at a cell density of 1 × 10 ⁵ to 1 × 10 ⁸ / ml, dispensed into a freezing tube, and stored in liquid nitrogen according to a normal cell storage method.
(3-5) Thawed after about 1 week, and 1 × 10 ⁷ cells / transplant were used for transplantation experiments. This is a “cryopreserved” mouse-derived dermal cell preparation.

II. Hair follicle reconstruction method (implantation method to animals)
The mouse-derived epithelial cell preparation (1-4) was mixed with “prepared when used” mouse-derived dermal cell preparation (2-4) or “cryopreserved” mouse-derived dermal cell preparation (3-5). These mixtures were used as “cell suspension” in the following “reconstruction hair follicle preparation procedure”.

<Reconstruction hair follicle creation procedure>
Things to prepare:
Recipient animals (Balb-c nu / un strain nude mice, 5 weeks of age or older),
Silicone dome-shaped cap with a diameter of about 1 cm (hereinafter referred to as “bulb”),
Anesthetic,
Surgical scissors, tweezers, suture device,
Micropipetter Cell suspension (suspended in about 150 μl of culture medium DMEM + 10% FBS)

<Procedure>
(I) Nude mice were anesthetized.
(Ii) The back skin was cut to a diameter of less than 1 cm.
(Iii) A valve was inserted into the wound and fixed with a suture instrument.
(Iv) The cell suspension was injected into the valve using a pipettor.
(V) They were kept for about a week and the valve was removed.
(Vi) After removing the bulb, the growth of the reconstructed hair follicle was observed after 1-6 weeks (usually after 2 weeks), after the scab was removed.

The results of the hair follicle reconstruction experiment are shown in Table 1 below and FIG. Hair follicle regeneration is observed even when transplanted only with “prepared” mouse-derived dermal cell preparations, whereas hair follicle regeneration is observed when only “cryopreserved” mouse-derived dermal cell preparations are transplanted. There wasn't. This result is consistent with the result obtained by using dermal papilla cells purified by cell sorter from the dermal cell fraction derived from transgenic mice of Kishimoto et al. It was demonstrated that only hair papilla cells were included as cell components.

Effect of cell ratio of dermal papilla cell fraction and epithelial cell fraction on hair follicle reconstitution efficiency Prepared by treatment similar to that of "cryopreserved" mouse-derived dermal cell preparation and mouse-derived epithelial cells described in Example 2 The rat neonatal skin-derived epithelial cells were adjusted to 0, 1 × 10 ⁶ , 3 × 10 ⁶ , and 1 × 10 ⁷ cells, mixed, and transplanted to the back skin of nude mice according to the above-described reconstructed hair follicle preparation procedure. The presence or absence of hair follicle reconstruction was examined. The results are shown in Table 2 below.

  From the results of Table 2, hair follicle regeneration was observed when the mixing ratio of dermal papilla cells and epithelial cells was in the range of 1:10 to 10: 1, and 1: 1 to 3: 1, particularly about 1: 1. It has been found that hair follicle regeneration occurs markedly when the cell mixture is implanted.

Reconstruction of hair follicles when epithelial cells are derived from mature mouse skin Preparation of epithelial cells derived from mature mouse skin (after 10 weeks of age) It went according to. Epidermal cells derived from mature mouse skin prepared from the resting hair epidermis of mice after 10 weeks of age (containing a resting hair epithelial cell-containing system) and hair removal by applying wax on resting hair skin Two types prepared from the epidermis of mice that were mechanically stimulated by treatment to promote hair growth (growth-phase-induced hair epithelial cell-containing system) were used. These mature mouse skin-derived epithelial cells were mixed with a “cryopreserved” mouse-derived dermal cell preparation so that the cell number was 1: 1, and transplanted to the back of the mouse by the above-described reconstructed hair follicle preparation procedure. The results are shown in Table 3 below.

  The presence or absence of hair follicle primordium is determined by preparing a thin section from the transplanted tissue, and after general staining such as hematoxylin-eosin staining, by morphological observation, by the depression of the epidermis layer and the aggregation of the dermal fibroblasts directly under the epidermis did.

  As is apparent from these results, hair growth was observed when either the resting hair epithelial cell-containing system or the growth-phase induced hair epithelial cell-containing system was transplanted. Therefore, it was found that epithelial cells are effective for hair follicle regeneration not only in the neonatal epidermis but also in the mature epidermis, for example, the epidermis of resting or growing hair.

Reconstruction of hair follicles when dermal papilla cells and epithelial cells are made heterogeneous. Mix approximately 1 × 10 ⁷ each of “cryopreserved” mouse-derived dermal cell preparations and rat neonatal skin-derived epithelial cells. The hair follicle reconstruction was examined by transplanting it onto the skin of the back of nude mice by the procedure for making the constituent hair follicle. The results are also shown in Table 4 below and FIG.

  As is apparent from these results, it was found that even if the mixture of dermal papilla cells and epithelial cells is a heterogeneous system, it is effective for regenerating hair follicles, as in the same system.

Reconstruction of hair follicles when epithelial cells are derived from human neonatal foreskin Prepare epidermal cells from human neonatal foreskin obtained by circumcision, mix with cryopreserved mouse hair papilla cell preparation, and reconstruct hair follicles as described above According to the preparation procedure, transplantation was performed on the back skin of nude mice, and the presence or absence of hair follicle reconstruction was examined.

  Foreskin tissue should be stored refrigerated in general fibroblast culture medium (such as DMEM medium) and keratinocyte culture medium (keratinocyte-SFM medium; Invitrogen, etc.) for about 3 to 1 week. Can do.

Foreskin-derived cultured cells were prepared by the following method.
Foreskin tissue (available regardless of race) in the above medium for 30 minutes in a Petri dish filled with PBS (for tissue culture, calcium and magnesium free) and added with antibiotics such as streptomycin and penicillin , Reacted. The mixture was further transferred to a Petri dish filled with fresh PBS and antibiotics for 10 minutes and allowed to react for 10 minutes. After removing excess adipose tissue, it was cut into about 1 cm ² skin pieces and allowed to react at 4 ° C. for about 18 hours while suspended in a despase solution (manufactured by Godo Sake: concentration 1000 U to 5000 U). After the reaction, it was washed again with a PBS solution, and the epidermis portion was carefully removed with tweezers. The peeled skin sheet was suspended in 5 ml of 0.05% trypsin-0.5 mM EDTA solution and reacted at 37 ° C. for 15 minutes.

Trypsin inhibitor was added to stop the reaction, followed by centrifugation at 900 rpm for 10 minutes, and the supernatant was discarded. The cells were resuspended in 10 ml of keratinocyte-SFM medium (Invitrogen) and the number of cells was counted. About 1 × 10 ⁶ to 10 ⁷ cells were subjected to one transplantation experiment. When preparing subcultured cultured epidermal cells for transplantation, about 1 × 10 ⁶ epidermal cells are seeded in a 100 mm petri dish or 75 cm ² flask coated with type I or type IV collagen solution, and keratinocyte-SFM is seeded. Using the medium, the cells were cultured in a CO ₂ incubator according to a conventional method. When the cells reached confluence, they were detached with trypsin, collected, adjusted to a cell density of 1 × 10 ⁶ again, and cultured until the required number of cells and passage number were reached.

  The results of the transplantation are also shown in Table 5 below.

この結果から、上皮系細胞としてヒト由来のものをマウス毛乳頭細胞と組み合わせても、毛包の再生に有効であることがわかった。また、興味深いことに、包皮細胞は毛包や毛根を有しない皮膚部位由来の上皮系細胞であるにもかかわらず、毛乳頭細胞と組み合わさることで毛包を再生させた。従って、毛包を再生させる系において毛乳頭細胞と組み合わせることができる上皮系細胞は、有毛部位の皮膚に限らず、無毛部位の皮膚に由来してよいことも明らかとなった。

From these results, it was found that even when epithelial cells derived from humans were combined with mouse hair papilla cells, they were effective for hair follicle regeneration. Interestingly, although the foreskin cells are epithelial cells derived from the skin site without hair follicles or hair roots, the hair follicles were regenerated by combining with hair papilla cells. Therefore, it has also been clarified that epithelial cells that can be combined with dermal papilla cells in a system that regenerates hair follicles may be derived not only from the skin of the hair site but also from the skin of the hairless site.

Reconstruction of hair follicle when epithelial cells are derived from human adult foreskin Prepare epidermal cells from adult foreskin tissue (20's) obtained during surgery such as removal of foreskins, and mix with cryopreserved mouse hair papilla cell preparation Then, it was transplanted into the back skin of nude mice by the above-described reconstructed hair follicle preparation procedure, and the presence or absence of hair follicle reconstruction was examined.

  Adult foreskin tissue can be stored for about one week by refrigerated storage in general fibroblast culture medium (such as DMEM medium) and keratinocyte culture medium (keratinocyte-SFM medium; Invitrogen, etc.). .

  The preparation method of cultured cells derived from adult foreskin tissue can be performed according to the preparation method of cultured cells derived from neonatal foreskin of Example 6.

The results of the transplantation are shown in Table 6 below.

  From these results, it was found that even epithelial cells derived from adult humans were effective for hair follicle regeneration even in combination with mouse hair papilla. This revealed that epithelial cells that can be combined with dermal papilla cells in a system that regenerates hair follicles can be used regardless of the developmental process and mature tissue.

Effects of passage number of cultured cells derived from human foreskin on hair follicle reconstitution In Examples 6 and 7, epithelial cells of different races derived from human newborn or adult foreskin were combined with mouse hair papilla after different passage numbers and culture. Table 7 below shows the efficiency of the reconstruction when the hair follicle reconstruction experiment is performed.

  From this result, it became clear that as the epithelial cells, the follicle is reconstructed as the passage number is younger regardless of the race.

Examination of reconstituted hair follicles To confirm whether reconstituted hair follicles are composed of a combination of dermal papilla cells and epithelial cells, for example, methods using species-specific antibodies or species-specific Gene sequences (such as human Alu sequences) can be used. Most conveniently, a heterogeneous composition is used as a composition according to the present invention for hair follicle regeneration (eg, hair papilla cells are derived from mice and epithelial cells are derived from rats or humans, or vice versa). In the case of a heterologous mouse, a plurality of dots (dots) are clearly observed in a heterogeneous mouse by staining with Hoechst # 33258 reagent (Molecular Probe) used for nuclear staining. Since it is not observed in rats, it can be easily distinguished by histological observation (Miller GJ & Ferrara JA, Stain Technol. 63 (1): 15-21, 1988).

Hoechst # 33258 Nuclear Staining Method Place a paraffin section sliced from the tissue at the transplant site on a slide glass (preferably 4-6μm), and perform normal deparaffinization (xylene wash 2 times → 99.9% ethanol wash → 80% Ethanol wash → 70% ethanol wash → water wash) and transferred to PBS solution (can be left in this state for a while).
Hoechst # 33258 (Molecular Probes) 4 mg was dissolved in 1 mL of PBS solution (shielded with aluminum). 10 μL of the finished Hoechst # 33258 solution was diluted with 10 ml of PBS (diluted 1000 times, final concentration 4 μg / mL), and a few drops were added so as to completely cover the tissue section of the flat slide. Thereafter, the mixture was allowed to react for 15 minutes while being shielded from light at room temperature, washed with water for 5 minutes, and then sealed with GVA Mounting Solution (glycerol-based mounting agent, manufactured by Zymed Lab., Handled by Funakoshi Chemical) and a cover glass. The observation was performed under a fluorescence microscope capable of observing excitation in the UV region.
According to this method, the mouse cell nucleus is bright and a plurality of dots are observed, and other rat and human cells do not see the dots, so that the species of tissue origin can be identified.
FIG. 5 shows the results of fluorescence micrographs obtained by transplanting a hair follicle reconstitution system in which hair papilla cells are derived from mice and epithelial cells are derived from humans (derived from human newborn foreskin). -Shown in comparison with histological images stained with eosin (HE) ("All about staining", Ishiyaku Shuppan Co., Ltd., p2-7, 1988). From the figure, it was clarified that the hair follicle is composed of both dermal papilla cells (parts having many bright dots derived from mice) and epithelial cells (parts having no dots derived from humans).

Hair follicle primordium regeneration in a three-dimensional skin model Disperse an appropriate amount of human fibroblasts in 0.1% collagen solution / DMEM / 10% FBS, dispense into a petri dish, and immediately leave in a CO ₂ incubator at 37 ° C. did. After gelation, the gel was peeled off from the petri dish wall surface and bottom, and allowed to float in the petri dish. The collagen gel was cultured while shaking, and the gel was contracted to about 1/5 to obtain a dermis model. Place the dermis model on a stainless steel grid, set a glass ring on it, 0.4 ml of human cultured epidermis cells (1.0 × 10 ⁶ cells / ml) dispersed in KGM (medium for epidermal cell culture) It was poured into a glass ring and cultured. At this time, “cryopreserved” mouse-derived dermal cell preparations were simultaneously mixed and injected. 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 the air, and observe the skin model after about one week. Reproducibility was determined by hematoxylin-eosin staining and Hoechst staining. The result is shown in FIG.

  As is apparent from the results of FIG. 6, even when the hair follicle regeneration system according to the present invention was transplanted into a three-dimensional skin model, hair follicle primordium formation was observed.

  The composition for regenerating the hair follicle according to the present invention can be used for research and development of hair follicle transplantation and hair follicle reconstruction.

FACS analysis results based on LacZ and 7-AAD. FACS analysis results based on CD-49 and 7-AAD. Results of hair follicle regeneration by mixed transplantation of freeze-thawed dermal papilla cells and epithelial cells. Hair follicle reconstitution result when hair papilla cells and epithelial cells are heterogeneous (mouse-rat). Hoechst nuclear staining result of hair follicle reconstruction when hair papilla cells and epithelial cells are made heterogeneous (rat-human). The result which shows the hair follicle primordium formation at the time of transplanting the hair follicle reproduction | regeneration system of this invention to a three-dimensional skin model.

Claims (22)

The dermal tissue fraction obtained by removing epidermal tissue from skin tissue collagenase treatment to prepare a cell suspension, followed by kill hair follicle epithelial cells by cryopreserving the cell suspension Regenerating a hair follicle comprising a prepared hair papilla cell preparation and an epithelial cell, wherein the ratio of the number of cells of the papilla cell to the epithelial cell is 1:10 to 10: 1 Composition for. Dermal papilla cells, versus cell number ratio of epithelial cells is 1: 1, The composition of claim 1. The composition according to claim 1 or 2 , wherein the cell density of the cell suspension is adjusted to 1 x 10 ⁵ to 1 x 10 ⁸ / ml and cryopreserved. The composition according to any one of claims 1 to 3 , wherein the cryopreservation is performed at a temperature of -80 ° C or lower. Wherein the cryopreservation performed in liquid nitrogen, the composition of any one of claims 1-4. The composition according to any one of claims 1 to 5 , wherein the cryopreservation is performed over a period of one week or more. The composition according to any one of claims 1 to 6 , wherein the dermal papilla cells and epithelial cells are both derived from a mouse, or both from a rat, or both from a human. The composition according to any one of claims 1 to 6 , wherein the dermal papilla cells and epithelial cells are heterogeneous cells, each derived from a mouse, rat or human. The composition according to any one of claims 1 to 8 , wherein the epithelial cells are derived from human foreskin. A method for regenerating a hair follicle by transplanting the composition according to any one of claims 1 to 9 to a recipient animal other than a human . 11. The method of claim 10 , wherein the recipient animal is an animal with a suppressed immune system. The method according to claim 10 or 11 , 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. The method according to any one of claims 10 to 12 , wherein the composition is transplanted such that hair papilla cells are transplanted at a transplant amount of 1 x 10 ⁶ to 10 ⁸ cells / cm ² . The method according to any one of claims 10 to 13 , wherein the composition is transplanted so that the hair papilla cells are transplanted at a transplant amount of 1.0 to 1.5 x 10 ⁷ cells / cm ² . A chimeric animal excluding a human who has transplanted the composition according to any one of claims 1 to 9 into a recipient animal excluding a human and thus carried a reconstituted hair follicle. The chimeric animal according to claim 15 , wherein the recipient animal is an animal with a suppressed immune system. The chimeric animal according to claim 15 or 16 , 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. The chimeric animal according to any one of claims 15 to 17 , wherein the composition is transplanted so that hair papilla cells are transplanted at a transplantation amount of 1 x 10 ⁶ to 10 ⁸ cells / cm ² . The chimeric animal according to any one of claims 15 to 18 , wherein the composition is transplanted such that hair papilla cells are transplanted at a transplantation amount of 1.0 to 1.5 x 10 ⁷ cells / cm ² . A skin three-dimensional model comprising a skin three-dimensional model comprising the composition according to any one of claims 1 to 9 , and thus carrying a reconstructed hair follicle. The skin three-dimensional model according to claim 20 , comprising hair papilla cells in an amount of 1 x 10 ⁶ to 10 ⁸ cells / cm ² . The skin three-dimensional model according to claim 21 , comprising follicular papilla cells in an amount of 1.0 to 1.5 x 10 ⁷ cells / cm ² .

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