TW202119028A - Method for identifying dermal sheath cup cells - Google Patents

Abstract

There is need for a method for allowing management of the quality of a cellular composition containing dermal sheath cup cells when providing the cellular composition in a hair regenerative therapy. The management of the quality of a cellular composition containing dermal sheath cup cells has so far been performed indirectly by conducting a keratinocyte negative test or a melanocyte negative test. There is a demand for a simpler, easier, and more direct method. According to the present invention, the use of CD13, CD90, CD10, CD29, CD105, CD49b, and NG2, which are cell surface markers enabling identification of dermal sheath cup cells through discrimination from melanocytes and keratinocytes, allows identification of dermal sheath cup cells, whereby the quality of a cellular composition containing dermal sheath cup cells can be managed.

Description

Method for identifying hair root sheath cells of hair bulb

The present invention relates to the technical field of cell therapy for hair regeneration. More specifically, the present invention relates to a method for identifying hair bulb root sheath cells used in cell therapy for hair regeneration.

As a treatment for alopecia or hair thinning, drug administration is mainly carried out, but continuous administration is required. On the other hand, there are cases where sufficient effects cannot be obtained depending on the target. On the other hand, it is expected that the cells taken from the scalp tissue of the subject can be proliferated and transferred back to the scalp of the subject by autologous transplantation, so as to regenerate the hair more safely and effectively. As the cells used for hair regeneration, dermal papilla cells, hair bulb root sheath cells, etc. can be used.

The practical application of hair regrowth therapy using autologous transplantation technology is approaching. Along with this, in order to further improve safety and effectiveness and achieve stable hair regeneration, the quality control of cell cultures has become important. When the hair bulb root sheath cells are used for transplantation, the melanocytes and keratinocytes existing near the harvesting site may be mixed. Therefore, the cell composition obtained by culturing and proliferating hair root sheath cells of the hair bulb is examined for the incorporation of melanocytes and keratinocytes before transplantation into the subject. Prior to this, in order to detect the incorporation of keratinocytes, the following test method was carried out. The test method was to deny the presence of keratinocytes by analysis by flow cytometry, and by the reaction of tyrosinase and matrix Sex and deny the existence of melanocytes.

In order to identify cell types, cell surface markers are generally used. The main cell surface markers are determined by the CD (Cluster of Differentiation) classification as an international classification. Cell surface markers help identify cell types. However, even if it is expressed in a certain cell type, as long as it is also expressed in the mixed cell type, it cannot be used for identification. In addition, since there are also cases where the cell surface markers change depending on the cell state, so far, no markers have been identified that distinguish the hair bulb root sheath cells before transplantation and distinguish them from other cell types. [Prior Technical Literature] [Non-Patent Literature]

[Non-Patent Document 1] Lee et al., J. DERMATOL 2006; 155: 858-860 [Non-Patent Document 2] Poblet et al., J. DERMATOL 2008; 159: 646-652 [Non-Patent Document 3] Sorrel et al., Exp Dermatol 2003; 12: 315 [Non-Patent Document 4] Park et al., J Cutan Pathol. 2017; 44: 909-914. [Non-Patent Document 5] Ernst et al., PLoS One. 2013; 8 (12) [Non-Patent Document 6] Huang et al., Sci Rep. 2016; 6 [Non-Patent Document 7] Dominici et al., Cytotherapy 2006; 8: 315-317 [Non-Patent Document 8] Wang et al., J Invest Dermatol. 2014; 134: 736-745

[The problem to be solved by the invention]

In the previous negative test of melanocytes and keratinocytes, the preparation of the positive control or the implementation of the test requires a lot of labor and time. In addition, this negative test is at best a method to deny the incorporation of keratinocytes and melanocytes, and there is a problem that it does not directly identify the root sheath cells (Dermal Sheath Cup Cells (DSCC)) of the hair bulb. Therefore, the industry seeks a method that can more easily control the quality of a cell composition containing hair bulb root sheath cells. [Technical means to solve the problem]

The present inventors conducted intensive research on the method of identifying hair bulb root sheath cells in a cell composition containing hair bulb root sheath cells, and found a method capable of identifying melanocytes and/or keratinocytes, and hair bulb root sheath cells. The cell surface marker of sheath cells has completed the present invention. Therefore, the present invention relates to the following: [1] A method for identifying hair bulb root sheath cells, which is a method of identifying hair bulb root sheath cells in a cell composition comprising hair bulb root sheath cells, the method comprising selecting from CD13, CD90, CD10 At least one cell surface marker in the group consisting of CD29, CD105, CD49b and NG2 can be detected. [2] The method according to item 1, wherein the cell surface marker is selected from the group consisting of CD13, CD90, CD10, CD29, CD105, and NG2, and the hair bulb root sheath cells are identified and combined with the above cells The keratinocytes contained in the product are distinguished from each other. [3] The method according to item 1, wherein the cell surface marker is selected from the group consisting of CD13, CD90, CD29, and CD49b, and the hair bulb root sheath cells are recognized and are included in the cell composition The melanocytes are distinguished. [4] The method according to item 1, wherein the cell surface marker is selected from the group consisting of CD13, CD90, and CD29, and the hair bulb root sheath cell is recognized and the melanin contained in the cell composition Cells and keratinocytes are distinguished. [5] The method according to any one of items 1 to 4, wherein the cell surface marker is selected from the group consisting of CD13 and CD90. [6] The method according to any one of items 1 to 5, wherein the cell surface marker is detected by flow cytometry. [7] The method according to any one of items 1 to 6, wherein the above-mentioned cell composition is a cell composition for hair regeneration. [8] The method according to any one of items 1 to 7, wherein the cell composition includes cultured hair bulb root sheath cells. [9] The method according to any one of items 1 to 8, wherein the cell composition is a cell composition before or after cryopreservation. [10] The method according to item 9, wherein the cell composition is a cell composition after cryopreservation. [11] A method for determining the quality of a cell composition by identifying hair bulb root sheath cells in a cell composition containing hair bulb root sheath cells by the method described in any one of items 1 to 10 To determine the quality of the cell composition. [12] A method for manufacturing a cell composition with managed quality, which includes the following steps: Determine the quality of the cell composition containing hair bulb root sheath cells by the method for determining the quality of the cell composition as described in item 11, and Remove the cell composition below the specified quality. [Effects of Invention]

With the present invention, the quality control of the cell composition containing the hair bulb root sheath cells can be realized in a shorter time and less labor.

The present invention relates to a method for identifying hair bulb root sheath cells in a cell composition containing hair bulb root sheath cells. More specifically, the detection of at least one cell surface marker selected from the group consisting of CD13, CD90, CD10, CD29, CD105, CD49b, and NG2 is included in a cell composition containing hair bulb root sheath cells . These cell surface markers are expressed in the hair root sheath cells of the hair bulb. These cell surface markers can be used alone or in combination.

Among the above-mentioned cell surface markers, keratinocytes have CD49b, and CD49b is not suitable for the separation of keratinocytes and DSCCs. Therefore, from the viewpoint of identifying hair bulb root sheath cells and keratinocytes in a cell composition containing hair bulb root sheath cells, use selected from the group consisting of CD13, CD90, CD10, CD29, CD105, and NG2 At least one cell surface marker in the population. On the other hand, the performance of CD29 and NG2 in DSCC is slightly lower (about 90%). On the other hand, the performance of CD29 and NG2 is seen in keratinocytes (15.7%, 5.34%). Although CD105 has a lower performance in keratinocytes (0.56%), it has a lower performance in DSCC (84.4%). Therefore, in a better aspect, at least one cell surface marker selected from the group consisting of CD13, CD90, and CD10 is used. These cell surface markers can be used alone or in combination.

Among the above-mentioned cell surface markers, melanocytes have CD10, CD105, and NG2, and these markers are not suitable for the separation of melanocytes from DSCC. Therefore, in terms of identifying hair bulb root sheath cells and melanocytes in a cell composition containing hair bulb root sheath cells, at least one selected from the group consisting of CD13, CD90, CD29, and CD49b is used Cell surface markers. On the other hand, the performance of CD29 and CD49b in DSCC is relatively low (92.4%, 96.7%), and a certain degree of performance (17.9%, 1.81%) can also be seen in melanocytes. Therefore, in a better aspect, at least one cell surface marker selected from the group consisting of CD13 and CD90 is used. These cell surface markers can be used alone or in combination.

In the cell composition containing hair bulb root sheath cells, in terms of identifying hair bulb root sheath cells and distinguishing them from melanocytes and keratinocytes, a composition selected from CD13, CD90, and CD29 is used Cell surface markers in the group. It is more preferable to use at least one cell surface marker selected from the group consisting of CD13 and CD90.

In the present invention, a cell surface marker refers to an antigen present on the surface of a cell. In particular, it can be represented by the CD number, which is classified and determined by the CD classification as an international classification. The CD classification was originally the classification of monoclonal antibodies, but it can also be used to name the surface antigens recognized by monoclonal antibodies. Antibodies that specifically bind to the required cell surface markers can be obtained from various distributors.

Therefore, in another aspect of the present invention, the present invention can also relate to the use of at least one monoclonal antibody selected from the group consisting of CD13, CD90, CD10, CD29, CD105, CD49b and NG2 to identify hair bulbs The method of hair root sheath cells. These monoclonal antibodies can be used and detected by any immunization technique.

The cell surface markers mentioned in the present invention can be used in any combination, for example, two, three, four, or more than four cell surface markers can also be used in combination. As an example, from the viewpoint of combining cell surface markers with higher recognition ability, a combination of CD13 and CD90, CD13 and CD29, and CD29 and CD90 can be used. In terms of using one monoclonal antibody to recognize keratinocytes with high precision and another monoclonal antibody to recognize melanocytes with high precision, CD10 and CD13, CD10 and CD90, CD10 and CD29, CD10 and CD49b, and CD13 can be used. And CD29, CD13 and CD49b, CD90 and CD29, CD90 and CD49b, CD29 and CD49b, CD105 and CD13, CD105 and CD90, CD105 and CD29, CD105 and CD49b, NG2 and CD10, NG2 and CD13, NG2 and CD90, NG2 and CD29 , NG2 and CD49b combination. As a combination of 3 types, CD13, CD90 and CD29 can be used.

The cell surface markers can be detected using any method known in the art. As an example, detection can be performed by flow cytometry using labeled antibodies that bind to various cell surface markers. In the case of detecting combinations, detection can be achieved by using monoclonal antibodies, which are labeled with fluorescent labels with different absorption wavelengths and excitation wavelengths. Such a combination of fluorescent markers is well known to the industry and can be selected appropriately. As an example, a combination of FITC (Fluorescein Isothiocyanate) and PE (Phycoerythrin) can be used. In the case of the flow cytometry method, the cells are sorted based on the detected fluorescent markers, thereby obtaining a cell composition containing substantially only the root sheath cells of the hair bulb.

In the present invention, a cell composition refers to any composition containing cells. The cell composition of the present invention is particularly a cell composition for hair regeneration or a cell composition for scalp injection. The cell composition of the present invention mainly contains hair bulb root sheath cells, and further contains a small amount of keratinocytes, melanocytes, and may also contain other cells. The cell composition for hair regeneration of the present invention can be obtained by cutting a human hair bulb-containing sample to distinguish it from hair papilla cells, obtaining hair bulb root sheath cells, and proceeding according to the situation bring up. Therefore, the cell composition for hair regeneration does not substantially contain dermal papilla cells. On the other hand, since keratinocytes and melanocytes exist in the vicinity of the hair bulb root sheath cells of the sample including the hair bulb portion, the cell composition containing hair bulb root sheath cells may include keratinocytes and melanocytes . It is not specifically limited, but the cell composition used in the identification method of the present invention is the cell composition after proliferating (or condensing) hair bulb root sheath cells, and usually contains more than 30% of the cell population, preferably 50% Above, more preferably more than 70% of hair bulb root sheath cells. For such a cell composition containing hair bulb root sheath cells, the identification method or quality determination method of the present invention can be used. By this, it can be determined that the hair bulb root sheath cells contained in the cell composition are more than the ratio required for cell clusters. As an example, they are more than 90%, and the quality of the cell composition can be guaranteed. The cell composition of the present invention may be a cell composition obtained by culturing cells. The cell composition may include components other than the cells, such as a culture medium, etc., and may also include a cryoprotectant or a cell activator according to the situation.

In another aspect, the present invention may also be related to a method for manufacturing a cell composition containing hair bulb root small cells whose quality is managed by determining the quality of the cell composition containing hair bulb root sheath cells. Produces a cell composition containing hair root small cells of the hair bulb with controlled quality. The manufacturing method specifically involves performing at least one cell surface marker selected from the group consisting of CD13, CD90, CD10, CD29, CD105, CD49b, and NG2 in a cell composition containing hair bulb root sheath cells. Detect and determine the quality of the cell composition and remove the cell composition below the prescribed quality, thereby obtaining the cell composition containing the hair root small cells of the hair bulb with controlled quality. Regarding quality, as an example, it can be determined by comparing the presence ratio of hair root small cells in the hair bulb with a predetermined threshold. As the predetermined threshold value, for example, 90%, preferably 95%, more preferably 97%, and still more preferably 99% can be used.

In the present invention, hair bulb root sheath cells can be identified in the cell composition containing hair bulb root sheath cells. The identification of hair bulb root sheath cells refers to identifying hair bulb root sheath cells to distinguish them from other cell types contained in the cell composition. Therefore, as long as the hair bulb root sheath cells can be distinguished from the undesired cells that may be included in the cell composition containing the hair bulb root sheath cells, such as keratinocytes and/or melanocytes.

In cell therapy for hair regeneration, cells are sometimes processed and cultured in a different setting from the clinical site where the tissue is obtained and the cultured cells are transplanted. As an example, the processing and culturing of cells is carried out in a separate cell processing and culturing facility. The composition containing the hair bulb root sheath cells produced in the cell processing and culture facility is sent to the clinical site after quality inspection. The method for identifying hair bulb root sheath cells in the cell composition comprising hair bulb root sheath cells of the present invention is usually carried out after cell processing and culture and before shipment. It can also be performed after cell processing, that is, after cells are separated from tissues and Carry out before cryopreservation and after cryopreservation. Generally speaking, the metabolism of cells in the cell composition after cryopreservation will change, so the cell surface markers may also change. On the other hand, the cell surface markers specified in the present invention, especially CD90 and CD13, showed no difference in the discrimination of DSCC even after cryopreservation (data not published), and can be used before cryopreservation. And any cell composition after cryopreservation.

When selecting the cell surface markers used in the present invention, eight surface antigens (CD10, CD13, CD29, CD49b, CD90, CD105, CD274, and NG2) were used as the research objects. The performance of the eight markers studied in the hair bulb root sheath cells has not been reported before. The findings on these markers are as follows.

CD10 is expressed in various tissues such as nervous system and hematopoietic system cells. It has been reported that CD10 in hair follicles is expressed in Dermal Sheath (Non-Patent Document 1: Lee et al., J. DERMATOL 2006; 155: 858-860 and Non-Patent Document 2: Poblet et al., J. DERMATOL 2008; 159: 646-652). In addition, this CD10 is a common antigen of human acute lymphoblastic leukemia and is known as human membrane-bound neutral endopeptidase (NEP).

CD13 is an aminopeptidase-N that exists in granulocytes, bone marrow precursor cells, endothelial cells, epithelial cells, etc., and is also widely expressed in other tissues such as the proximal tubule of the kidney, intestine, and placenta. It has been reported that CD13 is present in the hair follicle in the dermal hair root sheath (Non-Patent Document 3: Sorrel et al., Exp Dermatol 2003; 12: 315 and Non-Patent Document 4: Park et al., J Cutan Pathol. 2017; 44: 909 -914.).

CD29 is known as the integrin β1 chain. CD29 can be used as a marker for mesenchymal stem cells, but it is also expressed in dermal hair root sheaths (Non-Patent Document 5: Ernst et al., PLoS One. 2013; 8 (12)).

CD49b is an integrin α2 chain, which forms the VLA-2 receptor together with integrin β1. Because VLA-2 binds to collagen, fibrinogen, laminin, etc., it plays a role in keeping various cells in the extracellular matrix. It is well known in the literature that CD49b is expressed in lymphocytes, activated T cell monocytes, epithelial cells, and platelets.

CD90 belongs to the GPI (Glycosyl-phosphatidylinositol) anchoring protein of the immunoglobulin superfamily and is widely expressed in mesenchymal cells.

CD105, also known as endoglin, is known to act as a receptor for TGF-β1 and TGF-β3, and is expressed in adipose-derived stem cells (Non-Patent Document 6: Huang et al., Sci Rep. 2016; 6). The International Society for Cell Therapy uses CD90 positive and CD105 positive in the definition of human mesenchymal stem cells (Non-Patent Document 7: Dominici et al., Cytotherapy 2006; 8: 315-317).

CD274 is known as programmed cell death ligand 1, and is one of the ligands of PD-1. The interaction between PD-1 and PD-L1 plays an important role in the activation and tolerance of T cells, which is called immune checkpoint. Nivolumab (trade name: Opdivo) approved in 2014 is a human type of anti-human PD-1 monoclonal antibody and one of the immune checkpoint inhibitors. By inhibiting the binding of PD-1 and PD-L1 and PD-L2, T cells are activated, showing an anti-cancer effect. In addition, in an existing report, it is suggested that it is expressed in the hair bulb root sheath cell (Dermal Sheath Cup Cell: DSCC) (Non-Patent Document 9: Wang et al., J Invest Dermatol. 2014; 134: 736- 745).

NG2 is a kind of neuroglycan which is also called Chondroitin sulfate proteoglycan 4 (Chondroitin sulfate proteoglycan 4). The NG2 exhibits the following effects: controlling the interaction of the cell matrix when the melanoma cells differentiate and metastasize in the basement membrane of the endothelium. In addition, it is suggested that the above-mentioned NG2 is a molecule that inhibits neurite outgrowth and growth cone collapse in axon regeneration. [Example]

Example 1 : Identification of hair bulb root sheath cells A flow cytometer (Guava 8HT-2L (MERCK MILLIPORE)) was used to identify the cell composition containing hair bulb root sheath cells. The antibodies in Table 1 below were used for flow cytometric analysis. [Table 1] Antibody mark Dealer CD10 PE BD Bioscience CD13 PE BD Bioscience CD29 PE BD Bioscience CD49b FITC BD Bioscience CD90 FITC BD Bioscience CD105 FITC BD Bioscience CD274 FITC/PE BD Bioscience NG2 PE BD Bioscience IgG1 k PE BD Bioscience IgG1 k FITC BD Bioscience IgG2a PE BD Bioscience

Separation of hair root sheath cells and keratinocytes in the hair bulb Using 3 types of hair bulb root sheath cells (DSCC), keratinocytes, an equal mixture of DSCC and keratinocytes, the cells were suspended in each antibody (CD10, CD13, CD29, CD49b, CD90, CD105, CD274, NG2 and the corresponding isotype control (IgG1K antibody or IgG2a)) in the dilution solution, and react at 4°C for 30 minutes. It was washed with PBS (Phosphate Buffered Saline), resuspended in PBS, and detected by flow cytometry. The results are shown in Figure 1. In the case of using CD10, CD13, and CD90, keratinocytes and DSCC are significantly separated and can be recognized. In the case of using CD29, CD105, and NG2, although keratinocytes and DSCC overlap to a certain extent, they are separated to a moderate degree and can be roughly identified. In the case of using CD49b and CD274, keratinocytes and DSCC were not separated.

Separation of hair root sheath cells and melanocytes in hair bulb Using 3 kinds of hair bulb hair root sheath cells (DSCC), melanocytes, DSCC and melanocytes, the cells were suspended in each antibody (CD10, CD13, CD29, CD49b, CD90, CD105, CD274, NG2 and Corresponding isotype control) diluted solution, and react at 4°C for 30 minutes. After washing with PBS, resuspend in PBS, and perform detection by flow cytometer. The results are shown in Figure 2. In the case of using CD13 and CD90, melanocytes and DSCC are significantly separated and can be recognized. In the case of using CD29 and CD49b, although there is a certain degree of overlap between melanocytes and DSCC, the two are separated to a moderate degree and can be roughly identified. When using CD10, CD105, CD274 and NG2, melanocytes and DSCC were not separated.

Example 2: Confirmation of reproducibility Using 3 types of DSCC, keratinocytes, and melanocytes, the cells were suspended in the dilutions of each antibody of CD13, CD29, CD90 and the corresponding isotype control, and reacted at 4°C for 30 minutes. After washing with PBS, resuspend in PBS, and use flow cytometer for detection. The results are shown in FIG. 3A, and the graph formed by overlapping the histograms is shown in FIG. 3B. All antibodies against CD13, CD29 and CD90 can be detected by separating DSCC from keratinocytes and melanocytes.

Example 3: Study of double staining Use 2 batches of DSCC to suspend the cells in the dilutions of CD13 antibody (PE labeled), CD90 antibody (FITC labeled), and CD13 antibody (PE labeled) + CD90 antibody (FITC labeled) at 4°C React for 30 minutes for single staining and double staining. After washing with PBS, resuspend in PBS, and perform detection by flow cytometer. After implementing the flow cytometer, use the single staining data to correct the leakage of FITC to the PE detection system. The results are shown in Figures 4 and 5. It shows the ratio of cells judged to be DSCC by CD13 single staining (A), D90 single staining (B), and CD13 and CD90 double staining (C). In addition, the previous negative test of keratinocytes was performed on the same batch of DSCC. In the negative test of keratinocytes, the ratio of keratinocytes was determined to be 0.16% and 0.05%. Therefore, assuming that the keratinocytes other than keratinocytes are DSCC, the ratio of DSCC will become 99.84% and 99.95%. The results of single staining and double staining show that the ratio of DSCC is approximately the same as in the previous experiment.

Example 4: Verification of the detection power of DSCC Use 3 batches of DSCC and mix 5%, 10%, and 20% of keratinocytes to obtain a cell composition. The resulting cell composition is suspended in a dilution of CD90 antibody (FITC labeled) for 4 React for 30 minutes at °C for single dyeing. After washing with PBS, it was resuspended in PBS, and detected by flow cytometry. The results of the detection are shown in FIG. 6. Figure 6E shows the results of flow cytometry when the keratinocyte incorporation rate is 20% (A), 10% (B), 5% (C), and 0% (D), and the CD90-positive cell rate is shown in Figure 6E.

Figure 1 shows the results of flow cytometry analysis using cell surface markers to study the possibility of identifying hair bulb root sheath cells (DSCC) and keratinocytes (KC). Mix represents the result of a sample that mixes DSCC and KC at a ratio of 1:1. The solid line represents the distribution when each antibody is used, and the dotted line represents the distribution when the corresponding isotype control antibody is used. Figure 2 shows the results of flow cytometry analysis using cell surface markers to study the possibility of identifying hair bulb root sheath cells (DSCC) and melanocytes (MC). Mix represents the result of a sample made by mixing DSCC and MC at a ratio of 1:1. The solid line represents the distribution when each antibody is used, and the dotted line represents the distribution when the corresponding isotype control antibody is used. Figure 3 shows the results of flow cytometry analysis (A). The results of the flow cytometry analysis show that cell surface markers (CD13, CD29, and CD90) respectively identify hair bulb root sheath cells (DSCC) and keratinocytes (KC) and melanocytes (MC). Fig. 3B is a graph obtained by superimposing the DSCC, KC, and MC of Fig. 3A. The solid line represents the distribution when each antibody is used, and the dotted line represents the distribution when the corresponding isotype control antibody is used. Figure 4 shows the results (A and B) of flow cytometric analysis of hair bulb root sheath cells (DSCC) double-stained for CD90 and CD13. The solid line represents the distribution when each antibody is used, and the dotted line represents the distribution when the corresponding isotype control antibody is used. Figure 4C shows a collection of CD13 and CD90 double positive cells. Figure 5 shows the results (A-C) obtained by the same method as Figure 4 but using different batches of DSCC. The solid line represents the distribution when each antibody is used, and the dotted line represents the distribution when the corresponding isotype control antibody is used. Figure 6 shows the results of flow cytometric analysis using CD90 for 20% (A), 10% (B), 5% (C), and 0% (D) of DSCC mixed with keratinocytes. The solid line represents the distribution when each antibody is used, and the dotted line represents the distribution when the corresponding isotype control antibody is used. Figure 6E shows the rate of CD90-positive cells.

Claims (12)

A method for identifying hair bulb root sheath cells, which is a method for identifying hair bulb root sheath cells in a cell composition comprising hair bulb root sheath cells. The method includes selecting from the group consisting of CD13, CD90, CD10, CD29, At least one cell surface marker in the group consisting of CD105, CD49b and NG2 is tested. The method of claim 1, wherein the cell surface marker is selected from the group consisting of CD13, CD90, CD10, CD29, CD105, and NG2, and the hair bulb root sheath cell is recognized and is included in the cell composition The keratinocytes are distinguished. The method of claim 1, wherein the cell surface marker is selected from the group consisting of CD13, CD90, CD29, and CD49b, and the hair bulb root sheath cells are distinguished from the melanocytes contained in the cell composition . The method according to claim 1, wherein the cell surface marker is selected from the group consisting of CD13, CD90, and CD29, and the hair bulb root sheath cells are recognized to form the melanocytes and keratinocytes contained in the cell composition. Cell differentiation. The method according to any one of claims 1 to 4, wherein the cell surface marker is selected from the group consisting of CD13 and CD90. The method according to any one of claims 1 to 5, wherein the above-mentioned cell surface marker is detected by flow cytometry. The method according to any one of claims 1 to 6, wherein the above-mentioned cell composition is a cell composition for hair regeneration. The method according to any one of claims 1 to 7, wherein the above-mentioned cell composition comprises cultured hair bulb root sheath cells. The method according to any one of claims 1 to 8, wherein the cell composition is a cell composition before or after cryopreservation. The method of claim 9, wherein the cell composition is a cell composition after cryopreservation. A method for determining the quality of a cell composition by identifying hair bulb root sheath cells in a cell composition containing hair bulb root sheath cells by the method according to any one of claims 1 to 10, thereby determining The quality of the cell composition. A method for manufacturing a cell composition with managed quality, which includes the following steps: The quality of the cell composition containing the hair bulb root sheath cells is determined by the method for determining the quality of the cell composition as in claim 11, and Remove the cell composition below the specified quality.

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