Method for evaluating hair bulb sheath cells

Abstract

To provide a method for evaluating a dermal sheath cup cell in a more simplified manner.SOLUTION: A dermal sheath cup cell is evaluated by measuring the expression level of at least one gene selected from the group consisting of IGF1R, FGFR2, FGFRL1, CD36, DDR1, DDR2, EGFR, EDAA2R, G2A, cMet, PDGFR1, S1P2, S1P3, TLR2, and VEGFR2 and determining its migration ability on the basis of the expression level.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the technical field of cell therapy for hair regeneration. More specifically, the present invention relates to a method for evaluating hair bulb sheath cells used in cell therapy for hair regeneration. [Background technology]

[0002] Medication has been the primary treatment for alopecia and thinning hair, but it requires continuous administration and may not be sufficiently effective in some patients. Meanwhile, it is expected that hair regeneration can be achieved more safely and effectively by growing cells that can become hair harvested from the subject's scalp tissue and then autotransplanting them back into the subject's scalp. Cells used for hair regeneration include dermal papilla cells (DP cells) and dermal sheath cup cells (DSC cells) (Patent Document 1: JP 2011-101648 A).

[0003] The practical application of hair regeneration treatment using autologous transplantation of hair bulb sheath cells is approaching, and it is expected that this will improve efficacy and achieve stable hair regeneration. To improve efficacy, it is important to control the quality of the cells to be transplanted, and it is also important to obtain high-quality hair bulb sheath cells before transplantation. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2011-101648 Summary of the Invention [Problem to be solved by the invention]

[0005] Quality control of compositions containing hair bulb sheath cells is performed by confirming that the composition is free of contaminating cells such as keratinocytes and melanocytes. Furthermore, methods for evaluating the migration ability of hair bulb sheath cells contained in a composition have been developed with the aim of evaluating the cellular activity and / or hair regeneration ability based on cellular activity of the hair bulb sheath cells contained in the composition. Migration ability can be evaluated by seeding cells, incubating for a predetermined period of time, and then confirming the cells that have migrated. However, performing such experiments on cells before transplantation places a heavy burden on businesses. Therefore, the present inventors conceived the challenge of developing a method for more easily evaluating hair bulb sheath cells. [Means for solving the problem]

[0006] The present inventors have been working to develop a simple method for evaluating the migration ability of a cell composition containing hair bulb sheath cells, and have conducted extensive research focusing on migration-related genes. By comparing the expression of migration-related genes with the migration ability determined by actual migration tests, they have identified migration-related genes that are highly correlated with migration ability, leading to the present invention. Therefore, the present invention relates to the following: [1] In hair bulb sheath cells, the following: Measuring the expression level of at least one gene selected from the group consisting of IGF1R, FGFR2, FGFRL1, CD36, DDR1, DDR2, EGFR, EDAA2R, cMet, PDGFR1, S1P2, S1P3, TLR2, and VEGFR2; and Determining the migration ability based on the expression level A method for evaluating hair bulb sheath cells, comprising: [2] The evaluation method according to Item 1, wherein the migration ability is determined by comparing the expression level with a predetermined threshold. [3] The method according to item 1 or 2, wherein the hair bulb sheath cells are cultured cells. [4] The method according to Item 3, wherein the hair bulb sheath cells are cells that have been cryopreserved, thawed, and cultured. [5] The method according to item 3 or 4, wherein the culture is an adherent culture. [6] The method according to any one of items 3 to 5, wherein the expression level of at least one gene selected from the group consisting of IGF1R, FGFR2, DDR1, DDR2, CD36, EGFR, EDAA2R, cMet, S1P2, S1P3, TLR2, and VEGFR2 is measured. [7] The method according to item 1 or 2, wherein the hair bulb sheath cells are cells that have been cryopreserved after culture. [8] The method of item 7, wherein the antibody is selected from the group consisting of IGF1R, FGFRL1, FGFR2, CD36, and PDGFR1. [9] A method for determining transplant eligibility, comprising evaluating hair bulb sheath cells by the method according to any one of items 1 to 8. [Effects of the Invention]

[0007] According to the present invention, hair bulb sheath cells can be evaluated in a shorter time and with less effort. [Brief explanation of the drawings]

[0008] [Figure 1] Figure 1A shows a photograph of fluorescently stained nuclei of cells that migrated to the underside of the membrane chamber in the migration assay, and Figure 1B is a graph showing the number of cells that migrated to the underside of the membrane chamber in the migration assay. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention relates to a method for evaluating hair bulb sheath cells.

[0010] Hair bulb sheath cells are derived from the sheath cells surrounding the hair bulb (dermal sheath cells: DS cells) that surround the hair root. The hair bulb is located at the deepest part of the hair follicle and refers to the swollen region of the hair root, and is composed mainly of dermal papilla and hair matrix cells. Hair bulb sheath cells can be collected by incising the hair follicle of a scalp sample, inverting the bulb sheath to remove the dermal papilla, and culturing the bulb sheath from which the dermal papilla has been removed. By subculturing the bulb sheath cells, they can be expanded to the number of cells required for transplantation, and can be used to prepare a cell composition for transplantation. Hair bulb sheath cells in a cell composition for transplantation can be identified using cell surface markers. For example, they are characterized by the expression of at least one cell surface marker selected from the group consisting of CD10, CD13, CD29, CD49b, CD90, CD105, CD274, and NG2. During primary culture of hair bulb sheath cells, melanocytes and keratinocytes may become contaminated, and the quality of the cell composition for transplantation can be controlled so that the melanocytes and keratinocytes are contained in a certain proportion or less.

[0011] Without intending to be limited by theory, it is believed that when bulb sheath cells are injected into the scalp, they migrate and localize around the hair follicle, and after migration, differentiate into dermal papillae, thereby contributing to hair regeneration. On the other hand, bulb sheath cells that fail to migrate to the appropriate location are thought to be naturally eliminated. Therefore, the migration ability of bulb sheath cells contributes to the quality of bulb sheath cells, i.e., the hair regeneration ability of bulb sheath cells when transplanted. The higher the migration ability of bulb sheath cells, the higher the quality of bulb sheath cells and the higher the hair regeneration ability can be evaluated.

[0012] Migration ability refers to the ability of cells to migrate in tissues or culture environments. Cell migration ability can be broadly classified into chemotaxis, haptotaxis, wound healing, and cell infiltration. Of these, chemotaxis refers to the migration of cells driven by the concentration gradient of chemotactic factors such as chemokines, and hair bulb sheath cells are thought to migrate based on chemotaxis. The method and assay kit for measuring migration ability can be selected depending on the type of migration ability to be measured.

[0013] More specifically, the method for evaluating hair bulb sheath cells of the present invention includes the steps of measuring the expression level of at least one gene selected from the group consisting of IGF1R, FGFR2, FGFRL1, CD36, DDR1, DDR2, EGFR, EDAA2R, cMet, PDGFR1, S1P2, S1P3, TLR2, and VEGFR2 in hair bulb sheath cells; and Determining the migration ability based on the expression level Includes:

[0014] The expression levels of these genes may be measured by measuring the amount of mRNA of these genes, or by measuring the amount of protein produced by these genes. Methods well known in the art can be used to measure the amount of mRNA, but PCR-based measurements such as real-time PCR are typically used. Protein expression levels can be measured primarily by immunological techniques using antibodies, such as Western blotting and flow cytometry.

[0015] The migration ability can be determined by comparing the expression level of the gene with a predetermined threshold. The predetermined threshold is determined in advance for each gene. For example, the threshold can be arbitrarily determined between the expression level of a gene in bulb sheath cells with high migration ability and the expression level of a gene in bulb sheath cells with low migration ability. A more appropriate threshold can be selected by using multiple samples.

[0016] In the present invention, the migration ability of hair bulb sheath cells can be determined by a migration assay, measuring the number of cells that migrate to the lower surface of a membrane chamber. The migration assay is performed according to standard methods using a commercially available migration plate. A medium containing a substance capable of inducing migration, such as dermal papilla cell (DP) conditioned medium, can be introduced into the lower chamber of the migration assay for experiments. DP conditioned medium refers to the supernatant of a culture obtained by previously culturing dermal papilla cells. Any medium used for culturing dermal papilla cells can be used as the medium for culturing dermal papilla cells, including, for example, serum-free Follicle Dermal Papilla Cell Growth Medium (PromoCell), AmnioMAX (Thermo Fisher Scientific), and Follicle Dermal Papilla Cell Basal Medium (Takara-bio). Dermal papilla cells are seeded in a medium at any density, for example, at 70% confluence, and cultured at 37°C in a CO2 atmosphere for at least 1 hour, for example, 24 to 72 hours, and the culture supernatant can be used. Such dermal papilla cell culture supernatant contains various factors secreted by dermal papilla cells, such as a variety of proteins, including chemokines, cytokines, and enzymes, and these factors can attract hair bulb sheath cells, either alone or in combination. In the present invention, the dermal papilla cell culture supernatant can be used in a migration assay to evaluate the migration ability of hair bulb sheath cells. Alternatively, a medium containing a migration factor for hair bulb sheath cells can be used instead of the dermal papilla cell culture supernatant.

[0017] The expression levels of genes selected from the group consisting of IGF1R, FGFR2, FGFRL1, CD36, DDR1, DDR2, EGFR, EDAA2R, cMet, PDGFR1, S1P2, S1P3, TLR2, and VEGFR2 show a positive correlation with the migration ability of hair bulb sheath cells. Therefore, the higher the expression levels of these genes, the higher the migration ability of hair bulb sheath cells.

[0018] Gene expression in hair bulb sheath cells can change due to culture or freeze-thawing. Cell compositions for scalp transplantation are prepared by collecting and cryopreserving hair bulb sheath cells that have been subcultured and expanded. The cells are transported in a frozen state and then thawed before transplantation. It is preferable that the gene expression measured in the evaluation method of the present invention can determine the migration ability even when the cells are affected by culture and / or freeze-thawing. Therefore, the hair bulb sheath cells evaluated in the present invention may be cells before or after culture, frozen cells, or cells that have been thawed after cryopreservation. Culture is performed in suspension or adherent culture, with adherent culture being preferred from the viewpoint of migratory ability. Cells cryopreserved after culture refer to frozen cells or thawed cells that have not been cultured after thawing.

[0019] Based on the correlation between gene expression levels and migration ability in frozen cells, the expression level of at least one gene selected from the group consisting of CD36, FGFR2, FGFRL1, IGF1R, and PDGFR1 can be measured, and migration ability can be determined based on the expression level. Due to the high correlation, at least one gene selected from the group consisting of CD36, FGFR2, and PDGFR1 can be used. From the perspective of focusing on the absolute value of the number of cells whose migration is promoted by the DP cell culture supernatant, at least one gene selected from the group consisting of FGFR2, FGFRL1, and IGF1R can be used. From the perspective of focusing on the relative value of the number of cells whose migration is promoted by the DP cell culture supernatant, at least one gene selected from the group consisting of CD36 and PDGFR1 can be used.

[0020] Based on the correlation between gene expression levels and migration ability in cultured cells, the expression level of at least one gene selected from the group consisting of DDR1, DDR2, CD36, EGFR, EDAA2R, FGFR2, IGF1R, c-Met, S1P2, S1P3, TLR2, and VEGFR2 can be measured, and migration ability can be determined based on the expression level. Due to the high correlation, at least one gene selected from the group consisting of CD36, EGFR, FGFR2, IGF1R, S1P2, S1P3, and TLR2 can be used. From the perspective of focusing on the absolute number of cells whose migration is promoted by the DP cell culture supernatant, at least one gene selected from the group consisting of DDR1, EGFR, EDAA2R, FGFR2, IGF1R, c-Met, S1P2, S1P3, TLR2, and VEGFR2 can be used. From the viewpoint of focusing on the relative value of the number of cells whose migration is promoted by the DP cell culture supernatant, at least one gene selected from the group consisting of DDR2 and CD36 can be used.

[0021] IGF1R is the insulin-like growth factor 1 receptor and belongs to the insulin-like growth factor family. IGF1R is a receptor protein present on the cell surface. The Gene ID for IGF1R is 3480. FGFR2 (fibroblast growth factor receptor 2) is a member of the fibroblast growth factor receptor family. FGFR2 is a receptor protein present on the cell surface. The gene ID for FGFR2 is 2263. FGFRL1 is a fibroblast growth factor receptor-like protein 1 (FGFRL1) and belongs to the fibroblast growth factor receptor family. FGFRL1 is a cell surface receptor protein. The gene ID for FGFRL1 is 53834. CD36 is a membrane glycoprotein also known as platelet glycoprotein 4, fatty acid translocase, and scavenger receptor class B member 3 (SCARB3). It can also be used as a surface marker for hematopoietic cells. The gene ID for CD36 is 948. DDR1 and DDR2 are discoidin domain receptor 1 and discoidin domain receptor 2, which belong to the tyrosine kinase receptor subfamily. DDR1 and DDR2 are receptor proteins present on the cell surface. The Gene IDs of DDR1 and DDR2 are 780 and 4921, respectively. EGFR is an epidermal growth factor receptor, a receptor protein present on the cell surface. The gene ID for EGFR is 1956. EDAA2R is an ectodysplasin A2 receptor, a receptor protein present on the cell surface. The Gene ID of EDAA2R is 60401. cMet, also known as the hepatocyte growth factor receptor, is a cell surface receptor protein. The Gene ID for cMet is 4233. PDGFR1 is the platelet-derived growth factor receptor 1, a receptor protein present on the cell surface. The Gene ID for PDGFR1 is 5159. S1P2 and S1P3 are sphingosine-1-phosphate receptor 2 and sphingosine-1-phosphate receptor 3, respectively, and are receptor proteins present on the cell surface. The Gene IDs of S1P2 and S1P3 are 9294 and 1903, respectively. TLR2 (Toll-like receptor 2) is a receptor protein present on the cell surface. The gene ID for TLR2 is 7097. VEGFR2 is vascular endothelial growth factor receptor 2, a receptor protein present on the cell surface. The gene ID for VEGFR2 is 3791.

[0022] The primer pair used to measure the expression of the gene according to the present invention can be arbitrarily designed based on the base sequence of each gene. As an example, the primer pair used in the Examples can be used. The antibody used to measure the protein expressed from the gene according to the present invention may be a commercially available antibody, or it can be produced by introducing an antigen.

[0023] In the present invention, migration ability may be determined based on the expression level of one gene selected from the group consisting of IGF1R, FGFR2, FGFRL1, CD36, DDR1, DDR2, EGFR, EDAA2R, cMet, PDGFR1, S1P2, S1P3, TLR2, and VEGFR2, or by combining the expression levels of multiple genes. Any combination may be used. By using a combination of highly correlated genes, migration ability can be determined more accurately. Those skilled in the art can appropriately determine such combinations through statistical analysis or machine learning.

[0024] In another embodiment of the present invention, instead of determining the migration ability based on the expression level, a step of directly determining the efficacy of hair regeneration can be performed. For such a method, the expression level of one gene selected from the group consisting of IGF1R, FGFR2, FGFRL1, CD36, DDR1, DDR2, EGFR, EDAA2R, cMet, PDGFR1, S1P2, S1P3, TLR2, and VEGFR2 in the hair bulb sheath cells of the cell composition for transplantation can be compared with the efficacy in preclinical studies to determine a correlation. The efficacy of hair regeneration can be determined based on the total hair density or cumulative hair diameter after transplantation of the cell composition.

[0025] All documents mentioned herein are incorporated by reference in their entirety.

[0026] The following examples of the present invention are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. The present invention may be modified, for example, by adding, deleting, or substituting components of the present invention, provided that the modifications do not depart from the spirit of the present invention. [Example]

[0027] Example 1: Detection of migratory activity of hair bulb sheath cells based on DP-conditioned medium Acquisition of hair bulb sheath cells The hair follicle of the scalp sample was incised, and the hair bulb root sheath was inverted to remove the dermal papilla. The hair bulb root sheath from which the dermal papilla had been removed was cultured in AmnioMAX medium to obtain hair bulb root sheath cells.

[0028] Obtaining conditioned medium 1 × 10 obtained from hair follicles obtained from human scalp 6 ~5×10 6 The dermal papilla cells were seeded in a flask (manufactured by Corning) containing 10 ml of AmnioMAX medium and cultured at 37°C in a 5% CO2 atmosphere for 2 days, and the culture supernatant was collected and used as a DP conditioned medium.

[0029] Measurement of migratory activity of hair bulb sheath cells Obtained 2 x 10 5 ~1×10 6 Hair bulb sheath cells were seeded into a flask (Corning) containing 10 ml of AmnioMAX medium and cultured for 2–5 days until subconfluent. The medium was replaced with serum-free medium and cultured overnight. 40,000 bulb sheath cells were then seeded into the membrane chamber of a migration plate (Corning). DP-conditioned medium was added to the bottom chamber and cultured for 5 hours at 37°C in a 5% CO2 atmosphere. Serum-free AmnioMax(-) (Thermo Fisher Scientific) was used as a control. After 5 hours of culture, the membrane chamber was removed and fixed with 4% paraformaldehyde. After fixation, the cells were stained with Hoechst (Thermo Fisher Scientific), and the underside of the membrane chamber was photographed under a fluorescence microscope (Olympus) (Figure 1A). Hoechst-positive cells were counted (Figure 1B). For hair bulb sheath cell samples from 10 donors, the number of hair bulb sheath cells that migrated to the underside of the chamber using DP-conditioned medium (DP value) and the number of hair bulb sheath cells that migrated using AmnioMAX medium (Amnio value) were calculated, and the difference between the DP value and the Amnio value (DP-Amnio value) and the quotient of the DP value and the Amnio value (DP / Amnio value) were calculated. The results are shown below: [Table 1]

[0030] Example 2: Gene expression analysis of hair bulb sheath cells Preparation of unfrozen samples Hair bulb sheath cells from 10 donors were seeded into flasks (Corning) containing 10 ml of AmnioMAX (Invitrogen) medium and cultured as adherent cells until subconfluent. After removing the medium, the cells were washed with phosphate buffered saline (PBS). 2–5 ml of TrypLE SELECT Enzyme (1X), no phenol red (Thermo Fisher Scientific) was added, and the cells were incubated in a 37°C CO2 incubator for 5 minutes to detach. 10 ml of AmnioMAX medium was added, and the cells were harvested. After centrifugation, the medium was removed, and a cell pellet was obtained. This was used as an unfrozen sample.

[0031] Preparation of frozen samples Hair bulb sheath cells from 10 donors were passaged, dissolved in a cell preservation solution, Cellbanker 2 (Nippon Zenyaku Kogyo Co., Ltd.), and cryopreserved in a liquid nitrogen vapor tank. Hair bulb sheath cells from 10 donors that had been stored in a cell preservation solution, Cellbanker 2 (Nippon Zenyaku Kogyo Co., Ltd.) under the liquid nitrogen vapor tank were thawed in a 37°C water bath. The thawed cell solution and 10 ml of AmnioMAX medium were uniformly suspended in a centrifuge tube, then centrifuged. After removing the medium, a cell pellet was obtained and used as a frozen sample.

[0032] RNA was extracted from cell pellets from both unfrozen and frozen samples using the RNeasy Mini Kit (QIAGEN) as follows: The cell pellet was dissolved in 350 μl of Buffer RLT (included in the kit) and mixed thoroughly. 350 μl of 70% ethanol was added to the cell lysate and mixed by pipetting. The cell lysate was transferred to an RNeasy mini column (included in the kit) and centrifuged at 13,000 rpm for 15 seconds. The column was then centrifuged at 13,000 rpm for 15 seconds. 350 μl of Buffer RW1 was added to the RNeasy column and centrifuged at 13,000 rpm for 15 seconds. The RNeasy column was placed in a new collection tube (included in the kit), and 500 μl of Buffer PRE (included in the kit) supplemented with 96-100% ethanol was added, followed by centrifugation at 13,000 rpm for 15 seconds. The liquid that passed through the column was discarded, and Buffer PRE was added again. The column was centrifuged at 15,000 rpm for 2 minutes. The RNeasy column was placed in a new 1.5 ml tube (included in the kit), and 30 μl of RNase-free water (included in the kit) was added. The RNA was eluted by centrifugation at 13,000 rpm for 1 minute. cDNA was synthesized from the purified RNA using the SuperScript III RT-PCR KIT (Invitrogen) as follows: 1 μg of RNA was used per cDNA synthesis reaction, and the OligoDT primers listed in the table below were used as primers. The reverse transcriptase reaction was performed at 50°C for 1 hour. The resulting cDNA was used as a template to amplify the genes for GAPDH and migration-related receptors, which are constitutively expressed endogenous controls, using Blend taq (Toyobo). PCR reactions were performed using a LightCycler 480 System II (Roche Diagnostics KK) at 95°C for 5 minutes (1 cycle), followed by 45 cycles of 95°C for 10 seconds, 60°C for 15 seconds, and 72°C for 45 seconds. Expression analysis was performed using the comparative Ct method (ΔΔCt method), where the difference in Ct value (ΔCt) between the gene of interest and the housekeeping gene GAPDH was compared for relative quantification. [Table 2]

[0033] Example 3: Correlation analysis between gene expression analysis results and migration test results Correlation analysis was performed on Microsoft Office Excel (Microsoft Corporation) between the difference between the DP value and Amnio value (DP-Amnio value) and the quotient of the DP value and Amnio value (DP / Amnio value) determined for each donor's cells in Example 1, and the gene expression level of each gene in the unfrozen or frozen samples. Genes showing a positive correlation between the DP-Amnio value or DP / Amnio value and any of the four migration test values described above, with a correlation coefficient of 0.6 or higher, were selected as migration-related genes.

[0034] [Table 3]

Claims

1. In hair bulb sheath cells, the following: Measuring the expression level of at least one gene selected from the group consisting of IGF1R, FGFR2, FGFRL1, CD36, DDR1, DDR2, EGFR, EDAA2R, cMet, PDGFR1, S1P2, S1P3, TLR2, and VEGFR2; and determining the migration ability of hair bulb sheath cells based on the expression level; A method for evaluating the migration ability of hair bulb sheath cells, comprising:

2. The method of claim 1 , wherein the migratory ability is determined by comparing the expression level with a predetermined threshold for each gene.

3. The method according to claim 1 or 2, wherein the hair bulb sheath cells are cultured cells.

4. The method according to claim 3 , wherein the hair bulb sheath cells are cells that have been cryopreserved, thawed, and cultured.

5. The method according to claim 3 or 4, wherein the culture is an adherent culture.

6. The method according to any one of claims 3 to 5, wherein the expression level of at least one gene selected from the group consisting of IGF1R, FGFR2, CD36, DDR1, DDR2, EGFR, EDAA2R, cMet, S1P2, S1P3, TLR2, and VEGFR2 is measured.

7. The method according to claim 1 or 2, wherein the hair bulb sheath cells are cells that have been cryopreserved after culture.

8. 8. The method of claim 7, wherein the target gene is selected from the group consisting of IGF1R, FGFRL1, FGFR2, CD36, and PDGFR1.

9. A method for determining eligibility for transplantation for hair regeneration, comprising assessing the migration ability of hair bulb sheath cells by the method according to any one of claims 1 to 8.

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