Method for culturing mesenchymal stem cells

Abstract

This invention provides a method for obtaining and expanding mesenchymal stem cells (MSCORS) from the outer root sheath of hair follicles. The invention further provides a method for differentiating the expanded stem cells into other cells and tissues, and provides various cell types obtainable through this method.

Description

Technical Field

[0001] This invention provides a method for obtaining and expanding mesenchymal stem cells (MSCORS) from the outer root sheath of hair follicles. The invention further provides a method for differentiating the expanded stem cells into other cells and tissues, and provides various cell types obtainable through this method. Background Technology

[0002] Mesenchymal stem cells (MSCs) are stem cells found in the stroma, possessing pluripotent differentiation capacity, structural support, immunosuppression, and paracrine functions. As the most important source of stem cells for adult tissues, MSCs maintain and repair the entire body through differentiation and paracrine functions. They have been found in various tissues, including bone marrow, adipose tissue, peripheral blood, lungs, hair follicles, and neonatal tissues such as the placenta and umbilical cord. MSCs are the most widely used stem cells in clinical practice, attracting significant attention due to their pluripotent differentiation, autologous application, and ease of acquisition.

[0003] Therefore, MSCs, especially human MSCs, are the strongest contenders for clinical regenerative therapy and medical aesthetics. They can be used for cosmetic purposes, to improve body structure or function, and to treat diseases of related cells, tissues, organs or systems, particularly for bone repair, cartilage repair and heart repair, as well as for further lesions of related tissues and organs.

[0004] Bone marrow mesenchymal stem cells (BMMSCs) are currently the most widely recognized and used adult stem cells, considered the standard for MSCs. BMMSCs are typically obtained from the iliac crest via femoral aspiration. This method requires surgical intervention under anesthesia and carries the potential risk of post-biopsy complications. As another source of MSCs, adipose-derived mesenchymal stem cells (ADMSCs) are usually obtained from subcutaneous fat through liposuction and fat removal. This requires invasive skin incisions and surgical intervention under anesthesia, but is slightly simpler and less invasive than BMMSC isolation. ADMSCs are also more abundant than BMMSCs. However, each adult-derived mesenchymal stem cell source has an inherent problem: cell extraction requires an invasive biopsy.

[0005] Umbilical cord mesenchymal stem cells (UCB-MSCs) are isolated from umbilical cord blood or spongy Wharton's jelly tissue rich in stem cells, possessing the most primitive characteristics of mesenchymal stem cells. Compared to bone marrow and adipose tissue, these UCB-MSCs are fewer in number and lack adipogenic capacity. Isolated UCB-MSCs can be expanded and cryopreserved for the treatment of autologous or allogeneic hematopoietic disorders. Other readily available sources include dental pulp mesenchymal stem cells (DPSCs) and fallopian tube mesenchymal stem cells (FTMSCs), but their availability and non-invasiveness are relatively limited. DPSCs can be obtained during tooth extraction, and FTMSCs can be obtained through hysterectomy or from samples obtained from fallopian tube ligation / resection, but have limitations in autologous application.

[0006] External root sheaths (ORS) of hair follicles offer a novel source of stem cells, providing completely non-invasive autologous MSCs. As a micro-organism and stem cell bank, ORS contains diverse and heterogeneous stem cell populations with varying developmental potentials, including MSCs. Compared to the sampling methods described above, ORS offers significant advantages as a source of MSCs: it is painless, discomfort-free, and carries no risk of infection; it can be obtained simply by plucking hairs from a donor using tweezers. The sampling process leaves no wounds or scars, and hair regrowth occurs at the extracted site.

[0007] US 20 / 0086513 A1, WO 20 / 060899 A2, and Savkovic et al., in (2012) ExperimentalDermatology 21: 948-976, describe in detail a method for isolating melanocytes from plucked donor hair follicles. After isolation, the hair follicles and their derived cells are immediately cultured in a medium that promotes melanocyte differentiation. US 20 / 0086513 A1, WO 2013 / 060899 A2, and Savkovic et al. did not aim to amplify undifferentiated MSCs, nor did they disclose a method for isolating MSCs.

[0008] Hoogduijn et al. (2006) described dermal stem cells and their differentiation in hair follicles in Stem Cells and Development 15:49-60. Hoogduijn et al. isolated rat MSCs from rat whisker follicles by establishing primary cell cultures under normoxic conditions using 15% fetal bovine serum (FBS) through mechanical and enzymatic tissue digestion. The method described by Hoogduijn et al. did not disclose the step of excising the follicle bulb (proximal tissue of the hair follicle), or the use of explantation to induce cell migration from the hair follicle, nor did it include the use of gas-liquid interface culture methods or hypoxic culture conditions. Simultaneously, residual skin tissue and even blood vessels attached to the whisker follicles could be clearly observed. The obtained cells were tested to be positive for CD44, CD73, and CD90, and negative for CD34. However, CD105, an important positive marker for ISCT-MSC characterization, was not detected in Hoogduijn et al.'s work. EP2956543 and WO1974 / 127047 A1 describe a method for treating and repairing tendons. The non-bulbar dermal sheath (NDBS) cells derived from hair follicles and their isolation method are further described. EP2956543 describes the isolation of non-bulbar dermal cells (NBDCs) from a scalp perforation biopsy. This isolation method relies on microdissection to separate the hair follicle and surrounding tissue from the skin perforation biopsy. The follicular bulb is partially removed to avoid carrying dermal fibroblasts. The target tissue is the dermal sheath, referred to as "skin-derived cells," which is separated from the hair follicle while preserving the intact inner and outer root sheaths. Meanwhile, in this work, ORS are described as primarily composed of keratinocytes. The different target tissues and dissection methods clearly distinguish the method of EP 2956543 from the method of this invention. Furthermore, the isolated dermal sheath tissue is enzymatically digested and subjected to primary culture. The described cell culture conditions are fibroblast growth conditions, i.e., normoxic, using a medium containing FBS and FGF. Cells cultured by this method express CD90, CD73, and CD49b, but not CD34, CD45, and KRT14. While this partially aligns with the ISCT MSC characterization group, it does not fully meet the ISCT minimum identification criteria for MSCs, namely that MSCs must express CD105, while NBDCs do not. Furthermore, EP 2956543 does not disclose the expression of other markers in these cells: CD13, CD71, Nestin, and N-Cadherin. Therefore, not only are the starting tissues different, but the resulting cells are also different. In conclusion, the cells isolated from the dermal sheath appear to be fibroblasts.

[0009] Yoo et al. (2009) in Cellular Immunology 259:150-156 compared the immunomodulatory properties of MSCs derived from various adult tissues. Yoo et al. described MSCs derived from bone marrow, adipose tissue, umbilical cord blood, and umbilical cord Wharton's jelly. Yoo did not disclose MSCs obtained from hair follicles.

[0010] Previously, an inefficient method for extracting MSCs from ORS cell suspensions had been disclosed (Ma D, Kua JE, Lim WK, Lee ST, Chua AW (1975) Cytotherapy 17(8):1036-51).

[0011] In (2015) Cell Tissue Res 362: 69-82, Li et al. investigated the feasibility of human hair follicle-derived mesenchymal stem cells / CultiSpher®-G constructs in regenerative medicine. The inventors have replicated the method described by Li et al. and found that the number of MSCs generated per hair follicle was insufficient (see Example 3 below). Furthermore, the cells isolated by Li et al. appeared to contain a large number of fibroblasts because Li et al. preserved dermal papilla tissue during isolation.

[0012] In (2013) International Journal of Molecular Medicine 31:913-921, Zhang et al. stated that growth factors can maintain the high proliferation and pluripotency of mesenchymal stem cells derived from human hair follicles. The inventors have replicated the method described by Zhang et al. and found that the number of MSCs produced per hair follicle was insufficient (see Example 3 below). Furthermore, the cells isolated by Zhang et al. appeared to contain a large number of fibroblasts because Zhang et al. preserved dermal papilla tissue during isolation.

[0013] Wang et al. investigated pluripotent stem cells induced from human hair follicle MSCs in (2013) Stem Cell Rev and Rep 9: 451-460. The inventors have replicated the method described by Wang et al. and found that the number of MSCs generated per hair follicle was insufficient (see Example 3 below). Furthermore, the cells isolated by Wang et al. appeared to contain a large number of fibroblasts because they preserved dermal papilla tissue during isolation.

[0014] Obtaining a sufficient number of MSCs is crucial; for clinical cell therapy, a single dose typically requires at least 10 million cells. For research purposes, millions more are needed for a range of experiments (e.g., cell viability, cell growth, flow cytometry, RT-PCR, differentiation into different lineages). On the other hand, obtaining a sufficient number of MSCs from non-invasively extracted hair follicle ORS is challenging.

[0015] Therefore, there is a need for readily available and efficiently proliferating sources of MSCs to generate large quantities of MSCs that can be used for cell and tissue differentiation, which can then be used in regenerative medicine. Summary of the Invention

[0016] The inventors unexpectedly discovered that highly proliferative MSCs could be obtained from hair follicles by removing the follicle bulb, treating them with collagenase, and culturing them on a permeabilized membrane. In particular, the isolation method provided by the inventors significantly increases the proportion of hair follicles from which MSCs can be successfully cultured. For example, using this method, more than 6 million MSCs can be expanded from 45 hair follicles within 4 to 6 weeks. Furthermore, the MSCs obtained by this method were found to possess unique properties, including but not limited to the following: (i) The expression of "steminess" markers increases or at least remains stable until passage 4 or 5. Unlike MSCs from other sources such as ORS, which typically show signs of senescence after passage 3 or 4. (ii) The MSCs of this invention can differentiate efficiently into osteoblasts. (iii) The MSCs of this invention can differentiate efficiently into endothelial cells. They possess high angiogenic potential. (iv) The MSCs of this invention can differentiate efficiently into smooth muscle cells. (v) The MSCs of this invention exhibit different cell motility than MSCs from adipose tissue or bone marrow (see Example 5). (vi) The MSCs of the present invention exhibit different expression patterns of CD44 and CD90 compared to MSCs derived from adipose tissue or bone marrow (see Example 6).

[0017] Therefore, the present invention relates to the subject matter as defined in the following [1] to

[49] items:

[0018] [1] A method for generating mesenchymal stem cells, comprising the following steps:

[0019] (i) Excise the hair follicle bulb from which the hair is to be plucked, preferably human hair, more preferably human hair in the growth phase;

[0020] (ii) The remaining portion of the hair was incubated with a collagen degrading agent to obtain hair follicles containing stem cells and partially degraded extracellular matrix;

[0021] (iii) The hair follicles obtained in step (ii) are cultured on a permeable membrane using the first culture medium to promote stem cell migration from the hair follicles under conditions that induce stem cell proliferation but do not induce differentiation; and

[0022] (iv) The stem cells obtained in step (iii) are further cultured on a solid support using a second culture medium that induces stem cell proliferation without inducing differentiation.

[0023] [2] The method described in [1] wherein multiple hairs are plucked to generate mesenchymal stem cells.

[0024] [3] The method described in [2] wherein at least 10 hairs are plucked for the generation of mesenchymal stem cells.

[0025] [4] The method described in [2] wherein at least 20 hairs are plucked for the generation of mesenchymal stem cells.

[0026] [5] The method described in [2] wherein at least 30 hairs are plucked for the generation of mesenchymal stem cells.

[0027] [6] The method described in [2] wherein at least 40 plucked hairs are used to generate mesenchymal stem cells.

[0028] [7] The method described in [2] wherein 10 to 100 hairs are plucked for the generation of mesenchymal stem cells.

[0029] [8] The method of any one of [2] to [7], wherein at least 50% of the plucked hairs can generate proliferating mesenchymal stem cells.

[0030] [9] The method of any one of [2] to [7], wherein at least 70% of the hair plucked can generate proliferating mesenchymal stem cells.

[0031]

[10] The method of any one of [2] to [7], wherein at least 90% of the hair plucked can generate proliferating mesenchymal stem cells.

[0032]

[11] The method of any of the preceding claims includes, prior to step (i), immersing the plucked hair in a culture medium containing L-glutamine and optionally at least one antibiotic for 1 to 10 hours, preferably 2 to 5 hours.

[0033]

[12] The method described in any of the preceding claims includes removing the hair follicle bulb portion from which the hair was plucked.

[0034]

[13] The method described in any of the preceding claims includes rinsing the remaining portion of the hair follicle with a culture medium containing a buffer, L-glutamine and optionally at least one antibiotic after step (i) and before step (ii).

[0035]

[14] The method of any of the preceding claims, wherein the collagen degrading agent is collagenase.

[0036]

[15] The method of

[14] wherein the collagenase is capable of degrading type X collagen or type V collagen or type IV collagen.

[0037]

[16] The method described in any of the preceding claims includes, after step (ii) and before step (iii), explanting the remaining portion of the hair follicle onto a permeable membrane, preferably onto a Transwell membrane with a pore size of 0.4 μm.

[0038]

[17] In any of the preceding methods, the cell chamber below the permeation membrane contains a fibroblast trophoblast layer inactivated by radiation or mitomycin, preferably inactivated human dermal fibroblasts, and the culture medium in the cell chamber is in contact with the bottom surface of the permeation membrane.

[0039]

[18] The method of any of the preceding claims, wherein the first culture medium comprises serum, preferably fetal bovine serum, and most preferably human serum.

[0040]

[19] The method of any of the preceding claims, wherein the first culture medium comprises bFGF and / or EGF; and optionally insulin, human transferrin and / or selenite.

[0041]

[20] The method described in any of the preceding claims, wherein step (iii) is performed for 14 to 25 days.

[0042]

[21] The method described in any of the preceding claims, wherein step (iii) is performed for 18 to 23 days.

[0043]

[22] The method described in any of the preceding claims includes transferring the stem cells obtained in step (iii) onto a solid matrix, preferably a liquid-impermeable solid matrix, after step (iii) and before step (iv).

[0044]

[23] The method of any of the preceding claims, wherein the solid matrix is ​​substantially composed of polystyrene and optionally coated with a polymer that promotes cell attachment and proliferation (e.g., Advanced TC polymer-treated polymer). TM Cell culture dish; Greiner Bio-One).

[0045]

[24] The method of any of the preceding claims, wherein the second culture medium comprises serum, preferably fetal bovine serum, and most preferably human serum.

[0046]

[25] The method of any of the preceding claims, wherein the second culture medium comprises bFGF and / or EGF; and optionally insulin, human transferrin and / or selenite.

[0047]

[26] The method of any of the preceding claims, wherein the second culture medium comprises IL-6.

[0048]

[27] The method of any of the preceding claims, wherein step (iv) comprises culturing isolated stem cells until the number of proliferating stem cells is at least 6 x 10⁻⁶. 6 .

[0049]

[28] The method described in any of the preceding claims, wherein

[0050] - Step (iv) is performed for 21 to 35 days;

[0051] - Step (iv) includes culturing stem cells for 8 to 20 days, preferably 10 to 16 days, excluding cell passage;

[0052] - Step (iv) includes passage the cells twice, wherein after the first passage, the culture time is optionally 16 to 26 days, preferably 18 to 24 days; and / or

[0053] - The stem cells obtained from step (iv) were passaged twice (P2).

[0054]

[29] The method described in any of the preceding claims, wherein step (iii) and / or (iv) includes culturing cells under hypoxic conditions.

[0055]

[30] The method described in any of the preceding claims, wherein the number of mesenchymal stem cells obtained from step (iv) is at least 10 per plucked hair. 5 indivual.

[0056]

[31] The method described in any of the preceding claims, wherein the MSCs obtained from step (iv) are positive for staining of markers CD73, CD90, CD105, CD44, CD133, CD13, CD71, Nestin, N-Cadherin, Fibronectin, Vimentin, Integrin α5 (CD49e) and Integrin αV (CD51), and negative for staining of markers CD34, CD45, CD11b, CD19 and HLA-DR.

[0057]

[32] A method for generating differentiated cells, comprising: (a) generating MSCs by any of the preceding methods, and (b) differentiating the MSCs or proliferating stem cells obtained in step (iv) of claim 1 into other differentiated cells.

[0058]

[33] The method of

[32] wherein the differentiated cells include cardiomyocytes, chondrocytes, osteoblasts, adipocytes, endothelial cells and smooth muscle cells.

[0059]

[34] The method described in

[31] , wherein the differentiated cells are chondrocytes.

[0060]

[35] The method described in

[31] , wherein the differentiated cells are cardiomyocytes.

[0061]

[36] The method described in

[31] , wherein the differentiated cells are osteoblasts.

[0062]

[37] The method described in

[31] , wherein the differentiated cell is an adipocyte.

[0063]

[38] The method described in

[31] , wherein the differentiated cells are endothelial cells.

[0064]

[39] The method described in

[31] , wherein the differentiated cells are smooth muscle cells.

[0065]

[40] Mesenchymal stem cell populations can be obtained by any of the methods described in [1] to

[31] above.

[0066]

[41] Use of mesenchymal stem cells obtained by any of the methods described in any of [1] to

[31] above for the purpose of generating other differentiated cells, preferably differentiated cells as defined in any of

[33] to

[39] .

[0067]

[42] Use of mesenchymal stem cells obtained by any of the methods in [1] to

[31] for the treatment of diseases of cells, tissues, organs or systems that are structurally or functionally related to mesenchymal cells and structures.

[0068]

[43] Use of mesenchymal stem cells as described in

[42] , wherein the condition is a bone disease.

[0069]

[44] Use of mesenchymal stem cells as described in

[43] , wherein the bone disease is bone injury.

[0070]

[45] Use of mesenchymal stem cells as described in

[42] , wherein the condition is a disease affecting cartilage.

[0071]

[46] Use of mesenchymal stem cells as described in

[45] , wherein the disease affecting cartilage is osteoarthritis.

[0072]

[47] Use of mesenchymal stem cells as described in

[42] , wherein the condition is cardiovascular disease.

[0073]

[48] ​​Use of mesenchymal stem cells as described in

[42] , wherein the condition is a skin wound of any thickness.

[0074]

[49] Use of mesenchymal stem cells as described in

[42] , wherein the condition is a condition that can be treated with MSCs or cells derived therefrom. Attached Figure Description

[0075] Figure 1 shows the expression of positive and negative biomarkers of MSCs in MECORS and ADMSCs analyzed by flow cytometry (surface biomarker expression of MECORS and ADMSCs was analyzed by flow cytometry according to the ISCT-defined MSCs classification criteria. Flow cytometry scatter plots and histograms with representative markers are shown here).

[0076] Figure 2 Gene expression profiles of MSCORS from generation 2 to generation 5 are shown in the data from successive passages (based on the ISCT classification criteria for MSCs).

[0077] Figure 3 shows the subcellular morphology results of αSMA fibers (semi-quantitative analysis of actin stress fibers immunostained with αSMA using ImageJ to study the number and length of filaments).

[0078] Figure 4 shows osteogenic differentiation and semi-quantitative analysis of MSCORS and ADMSCs (quantitative analysis by alizarin red staining and alkaline phosphatase activity assay, with osteogenic differentiation assessed using ImageJ).

[0079] Figure 5 Calcium deposition in MSCORS and ADMSCs is shown (extracellular calcium phosphate deposition in MSCORS and ADMSCs after osteogenic differentiation).

[0080] Figure 6 shows the results of a semi-quantitative analysis of angiogenesis assays of MSCORS and ADMSCs (showing neovascularization of endothelial differentiated MSCORS and ADMSCs, with quantitative analysis of angiogenesis performed using ImageJ software).

[0081] Figure 7 shows the cell yield of MSCORS cultured to passage 15 using the method described in this invention (comparison of MSCORS and D12 method: cell count at each passage).

[0082] Figure 8 The results of comparing the MSCORS culture method described in this invention with Wang's method for culturing MSCs from hair follicles are shown (comparison of MSCORS and D12: expected cell yield per passage).

[0083] Figure 9 Cell migration parameters (mean ± variance) of MSCOR, ADMSC, and BMMSC over 24 hours are shown as described in Example 5. (A) Cumulative distance (Kruskal-Wallis test, p = 0.0072), (B) Velocity (Kruskal-Wallis test, p = 0.0083), (C) Euclidean distance (Kruskal-Wallis test, p = 0.0131) and (D) Directionality (One-Way ANOVA test, p = 0.0037).

[0084] Figure 10 Immunofluorescence staining of CD44 in MSCOR, ADMSC, and BMMSC is shown (mean ± variance), as illustrated in Example 6. (A) Immunofluorescence staining images of CD44 in MSCOR, ADMSC, and BMMSC under the same staining method and imaging parameters. CD44 and CD90 showed different total intensities and different intracellular distributions in MSCOR, ADMSC, and BMMSC. (B) CD44 staining intensity (Kruskal-Wallis test, p < 0.0001), CD44 cell membrane / cytoplasm index (Kruskal-Wallis test, p < 0.0001), CD90 staining intensity (Kruskal-Wallis test, p < 0.0001), and CD90 cell membrane / cytoplasm index (Kruskal-Wallis test, p < 0.0001). Scale bar corresponds to 100 μM. Detailed Implementation

[0085] The term mesenchymal stem cells (MSCs) used in this invention refers to cells with the following properties: First, when cultured under standard conditions using tissue culture flasks, MSCs must adhere to a plastic surface and grow. Second, ≥95% of MSCs must express CD105, CD73, and CD90, as detected by flow cytometry. Furthermore, these cells must not express CD45, CD34, CD14 or CD11β, CD79α or CD19, or HLA class II markers (≤2% positive). Third, under standard in vitro differentiation conditions, these cells must be able to differentiate into osteoblasts, adipocytes, and chondrocytes.

[0086] This invention relates to a method for producing MSCs, comprising the following steps:

[0087] (i) Excise the hair follicle bulb from which the hair was plucked;

[0088] (ii) Incubate the remaining portion of the hair with a collagen degrading agent to obtain hair follicles containing stem cells and partially degraded extracellular matrix;

[0089] (iii) The hair follicles obtained in step (ii) are cultured on a permeable membrane using the first culture medium to promote stem cell migration from the hair follicles under conditions that induce stem cell proliferation but do not induce differentiation; and

[0090] (iv) Using a second culture medium, the stem cells obtained in step (iii) are further cultured on a solid support, which induces stem cell proliferation without inducing their differentiation.

[0091] The hair referred to in the method of this invention is preferably human hair, more preferably human hair in the growth phase. Preferred embodiments regarding the source of hair are applicable to any aspect of this invention.

[0092] The method of the present invention optionally includes, using the follicle-plucking step in step (i), plucking one or more hairs from the donor's scalp. The hairs can be plucked from any area of ​​the scalp, preferably from the temporal or occipital region. Up to five hairs are plucked using sterilized tweezers near the root, in the direction of hair growth. The plucked follicles can be soaked at room temperature (e.g., 25°C) in a culture medium (e.g., DMEM) selectively supplemented with antibiotics and / or L-glutamine, for example, for 2–4 hours. Alternatively, the hairs can be preserved in phosphate buffer (PBS) without added calcium or magnesium until the next step. Hereinafter, PBS refers to phosphate buffer without added calcium or magnesium.

[0093] Technicians can adjust the number of hairs plucked according to the required number of MSCs. In one embodiment, using step (i) of the present invention, 1-500 hairs, preferably 10-80 hairs, more preferably 30-60 hairs, and most preferably 15-20 hairs are plucked from a donor scalp.

[0094] The method of this invention includes removing the follicular bulb portion of the plucked hair follicle. In the text of this invention, the "follicular bulb" of the plucked hair refers to the proximal portion of the hair root, which mainly comprises phenotypic fibroblast-like cells, dermal papillary cells (DP cells) with limited differentiation and proliferation, and differentiated cells (such as melanocytes and keratinocytes). In a preferred embodiment, if not lost during plucking, the distal portion of the hair shaft, referred to as the "raised area," is also cut off. In a preferred embodiment, only the middle portion of the plucked hair follicle is used. In this invention, the middle portion of the plucked hair is preferably the follicular morphological unit between the follicular bulb (proximal portion) and the sebaceous gland. In the text of this invention, the terms "plucked hair" and "plucked hair follicle" are used interchangeably. Preferably, the proximal portion of the hair follicle and excess hair shaft are removed before rinsing.

[0095] Typically, after step (i) and before step (ii), the remaining portion of the plucked hair follicle is rinsed with a rinsing solution (e.g., PBS + antibiotics + L-glutamine), preferably at least 5 times, more preferably at least 10 times. Each rinsing step typically lasts 5 to 20 minutes, preferably 10-15 minutes. This step removes fungal / bacterial contamination from the culture. Preferably, a large volume of rinsing solution is used, for example, 10 ml in a 50 ml conical centrifuge tube. After each rinse, the hair quickly settles in the rinsing solution; all supernatant is carefully aspirated and discarded, ensuring no hair follicle is lost. The advantage of the rinsing step is that it reduces or eliminates the use of antibiotics in the culture medium for subsequent steps. In one specific embodiment, only antibiotic-free culture media are used in steps (ii), (iii), and (iv).

[0096] The treated hair follicles were co-incubated with a collagen degrading agent. According to the present invention, the collagen degrading agent is used to promote cell migration. Without being bound by any specific theory, it is believed that the collagen degrading agent can degrade collagen fiber complexes, thereby loosening the dense hair follicle ORS tissue, which facilitates the migration of MSCORS.

[0097] Those skilled in the art are familiar with collagen degrading agents; in the context of this invention, a collagen degrading agent is any agent that digests collagen into smaller subunits. Digestion of collagen degrades the extracellular matrix and releases cells, including stem cells, progenitor cells, and differentiated cells. In a preferred embodiment of the invention, the collagen degrading agent is capable of degrading type I collagen, type IV collagen, type V collagen, type X collagen, and combinations thereof. In a preferred embodiment, the collagen degrading agent is an enzyme, preferably a collagenase. The collagenase is preferably capable of degrading more than one type of collagen. In a particularly preferred embodiment, the collagen degrading agent is capable of degrading type X collagen. In another preferred embodiment, the collagen degrading agent is used at a concentration of about 5 mg / ml. For example, rinsed hair follicles can be carefully transferred to a solid carrier supporting the hair follicles (e.g., a sieve with a 100 µm pore size) and digested with a suitable reagent (e.g., digested with 5 mg / ml collagenase for about 12 minutes).

[0098] In a preferred embodiment of the invention, the method further includes the step between steps (ii) and (iii): rinsing the hair follicles with a rinsing solution after incubation with a collagen degrading agent.

[0099] Suitable rinsing solutions are recognized in the art and can be selected by those skilled in the art. In a preferred embodiment, the rinsing solution is Dulbecco's Modified Eagle Medium (DMEM). In another embodiment, the rinsing solution contains antibiotics, preferably from gentamicin, amphotericin B, penicillin, streptomycin, neomycin, carbenicillin, penicillin G, ampicillin, polymyxin-B, tetracycline, ciprofloxacin, lincomycin, spectinomycin, carbenicillin, thiostreptomycin, cefoxitin, apramycin, vancomycin, tobramycin, rifamycin, and hygromycin. The washing solution may contain antibiotics, more preferably from gentamicin, amphotericin B, penicillin, and streptomycin.

[0100] Technicians can determine the appropriate concentration of antibiotics used in the rinsing solution. Preferred antibiotics and concentrations are listed in parentheses as follows: penicillin, for example (50 U / ml to 150 U / ml, preferably 100 U / ml), streptomycin sulfate (50 pg / ml to 20 mg / ml, preferably 100 μg / ml), gentamicin sulfate (5 μg / ml to 3000 μg / ml, preferably 50 μg / ml), amphotericin B (0.25 μg / ml to 30 μg / ml, preferably 2.5 μg / ml), (25 μg / ml to 600 μg / ml, preferably 50 μg / ml), (50 pg / ml to 10000 μg / ml, preferably 100 μg / ml), (25 μg / ml to 3000 μg / ml, preferably 50 μg / ml). μg / ml), ampicillin trihydrate (50 U / ml to 200 U / ml, preferably 100 U / ml), carbenicillin (50 U / ml to 200 U / ml, preferably 100 U / ml), polymyxin B sulfate (25 μg / ml to 3000 μg / ml, preferably 100 μg / ml), (2 μg / ml to 80 μg / ml, preferably 10 μg / ml), 7-hydroxytetracycline (2 μg / ml to 25 μg / ml, preferably 5 μg / ml), (5 to 35 μg / ml, preferably 10 μg / ml), erythromycin (50 μg / ml to 300 μg / ml, preferably 100 μg / ml), (2 μg / ml to 300 μg / ml, preferably 10 μg / ml), (2 μg / ml) The following are listed: up to 30 μg / ml, preferably 5 μg / ml), ciprofloxacin (1 μg / ml to 10 μg / ml), lincomycin (about 50 μg / ml), spectinomycin (5 μg / ml to 50 μg / ml, preferably 10 μg / ml), carbenicillin (about 100 U / ml), thiostrepton (about 25 μg / ml), ceftazidime-hydrate (about 100 μg / ml), apramycin (2.5 μg / ml to 25 μg / ml), vancomycin hydrochloride (100 μg / ml), tobramycin (4 μg / ml to 100 μg / ml, preferably 80 μg / ml), rifamycin (about 400 μg / ml), and hygromycin (about 200 μg / ml).

[0101] In a preferred embodiment, the rinsing solution comprises penicillin at a concentration of 50 U / ml to 150 U / ml, preferably 100 U / ml, streptomycin sulfate (50 pg / ml to 20 mg / ml, preferably 100 μg / ml), and gentamicin at a preferred concentration of 10 μg / ml to 100 μg / ml, more preferably 25 μg / ml to 75 μg / ml, and even more preferably 50 μg / ml. In a further preferred embodiment, the rinsing solution comprises amphotericin B at a preferred concentration of 1 μg / ml to 20 μg / ml, more preferably 5 μg / ml to 15 μg / ml, and even more preferably 10 μg / ml. In the most preferred embodiment, the rinsing solution comprises four antibiotics, preferably penicillin, streptomycin, gentamicin, and amphotericin B.

[0102] In another preferred embodiment, the rinsing solution comprises DMEM, gentamicin at a concentration of approximately 50 μg / ml, and amphotericin B at a concentration of approximately 10 μg / ml. In the most preferred embodiment, the rinsing solution consists of DMEM, gentamicin at a concentration of 50 μg / ml, and amphotericin B at a concentration of 10 μg / ml.

[0103] In another preferred embodiment, the rinsing solution contains penicillin and streptomycin.

[0104] In yet another preferred embodiment, the rinsing solution does not contain antibiotics.

[0105] The hair follicles obtained in step (ii) are then cultured in a first culture medium (hereinafter referred to as the "first medium") that induces stem cell proliferation but does not induce their differentiation. This step preferably includes transferring the hair follicles onto a liquid-permeable matrix, such as a Transwell porous membrane (available from Corning). Preferably, the lower chamber of the permeable membrane is separated from the upper space, so that cells cannot pass through the membrane, but liquid can pass through. For example, hair follicles can be carefully transferred onto a Transwell membrane nested in a 6-well plate, approximately 10 hairs per well. The hair follicles are preferably carefully and evenly transferred onto the membrane, spaced apart to allow cell migration. During the transfer of hair follicles, it is preferable to allow only the hair shaft to be grasped with forceps so as not to damage the ORS tissue.

[0106] The first culture medium does not contain any components sufficient to promote the differentiation of MSCs into other cell types. Specifically, the first culture medium preferably does not contain any reagents that can be added alone to promote the differentiation of MSCs into cardiomyocytes, chondrocytes, osteoblasts, adipocytes, endothelial cells, and smooth muscle cells. Preferably, the first culture medium is substantially free of...

[0107] -β-adrenergic receptor ligands, such as adrenaline and its derivatives;

[0108] -Dexamethasone

[0109] -ascorbic acid,

[0110] -β-glycerophosphate,

[0111] - Bone morphogenetic protein 4 (BMP-4).

[0112] - Vascular endothelial growth factor (VEGF),

[0113] 2-Mercaptoethanol,

[0114] - Transforming growth factor β1 (TGF-β1).

[0115] -Sodium pyruvate,

[0116] -Non-essential amino acids,

[0117] -insulin,

[0118] -Human transferrin,

[0119] -Selenite,

[0120] 3-Isobutyl-1-methylxanthine (IBMX)

[0121] -Indomethacin,

[0122] -α-melanocyte-stimulating hormone (α-MSH).

[0123] - Hydrocortisone,

[0124] - Stem cell factor (SCF)

[0125] - Nerve growth factor β (NGF-β)

[0126] -Hepatocyte growth factor (HGF)

[0127] -5-azacyclobutane (5-Aza)

[0128] -Saturated succinyl aniline isohydroxyoxime (SAHA), and / or

[0129] - Bone morphogenetic protein-2 (BMP-2).

[0130] In another preferred embodiment, the first culture medium does not contain any of the above-mentioned differentiation inducers in sufficient quantities to induce cell differentiation. In another preferred embodiment, the first culture medium does not contain any of the differentiation inducers listed above.

[0131] The first culture medium contains basic nutrients, such as Dulbeccos Modified Eagle Medium (DMEM). It may further contain serum, preferably human serum. In another preferred embodiment, the first culture medium also contains fibroblast growth factor (bFGF) and epidermal growth factor (EGF). The first culture medium may also contain insulin, human transferrin, and / or selenite.

[0132] The inventors discovered that a liquid-air interface encapsulates hair follicles within a thin film of culture medium, preventing the follicles from floating and allowing them to adhere to the permeable membrane, thereby promoting cell growth and migration. For this purpose, 0.7 ml to 1.1 ml, preferably 0.8 ml to 1.0 ml, and most preferably about 0.9 ml of culture medium is typically used for the first two days to enhance adhesion. Subsequently, greater than 1.1 ml to 1.5 ml, preferably 1.2 ml to 1.4 ml, and most preferably about 1.3 ml, is typically used to expand the liquid area around the hair follicle to promote cell migration.

[0133] In the cell chamber at the bottom of the Transwell, i.e., the compartment beneath the membrane, trophoblast cells are preferably used. The trophoblast cells are preferably human fibroblasts, more preferably inactivated human dermal fibroblasts. There is no physical contact between the hair follicle and the trophoblast cells, but they are connected by a culture medium. The trophoblast cells release nutrients and growth factors into the culture medium, which are utilized by the hair follicle and migrating cells, thus mimicking to some extent the dermal mesenchymal environment conducive to cell growth.

[0134] Preferably, cell isolation and culture are performed under hypoxic conditions. For example, 5% O2 can be used during isolation and culture. In one embodiment, step (iii) is performed under hypoxic conditions. In another embodiment, step (iv) is performed under hypoxic conditions. In yet another embodiment, both steps (iii) and (iv) are performed under hypoxic conditions.

[0135] Preferably, cell isolation and culture are performed under hypoxic conditions. The term "hypoxic conditions" as used herein refers to an oxygen concentration below 20%. On one hand, hypoxic conditions are characterized by an oxygen concentration below about 15%, preferably below about 10%. On the other hand, hypoxic conditions are characterized by oxygen concentrations of about 1% to 10%, 2% to 9%, 3% to 8%, 4% to 9%, 4% to 6%, for example, about 5%. Hypoxic conditions can be generated and maintained using a culture apparatus that controls the concentration of ambient gases. For example, 5% O2 can be used during isolation and culture. In one embodiment, step (iii) is performed under hypoxic conditions. In another embodiment, step (iv) is performed under hypoxic conditions. In yet another embodiment, both steps (iii) and (iv) are performed under hypoxic conditions.

[0136] Approximately 7 days later, cells migrate from the hair follicle ORS to the permeate membrane and form a monocell layer. When the monocell layer is nearly confluent, a first culture medium or another suitable MSCORS separation medium is added to the upper layer of the permeate membrane, allowing the cell layer to regrow for about two days. The upper layer of the Transwell is then immersed in culture medium for two days to promote cell proliferation by providing rich nutrients, allowing cells to grow on the entire surface of the permeate membrane. MSCORS rapidly migrate out of the hair follicle ORS and form a cell layer, with a strong matrix support provided by the permeate membrane (e.g., a PET membrane). The method described in this invention enables the hair follicle to produce a migrating cell layer with a larger surface area.

[0137] The culture in step (iii) is typically carried out for 14 to 25 days, preferably 15 to 24 days, more preferably 16 to 23 days, even more preferably 17 to 22 days, and most preferably about 18, or about 19, or about 20, or about 21 days.

[0138] Preferably, the cell layer typically fuses within approximately 3 weeks of culture, requiring cell harvesting from the permeate membrane to obtain a cell suspension. A preferred harvesting method using multi-step trypsin digestion is as follows: Aspirate the culture medium from both sides of the permeate membrane (upper and lower chambers), and rinse with pre-warmed PBS; gently rinse the cell layer and hair follicles, repeating the rinsing step 3 times; add 0.5 ml of 0.04% / 0.03% trypsin / EDTA to the membrane, incubate for 6-8 minutes, and observe under a microscope; when many cells shrink into spherical shapes, gently tap the 6-well plate and gently rinse the cell layer with a pipette. Collect the supernatant and aspirate it into a 15 ml centrifuge tube containing 0.5 ml of FBS for neutralization. Add another 0.5 ml of trypsin / EDTA to the Transwell, and repeat the trypsin digestion process 2-3 times until all cells on the permeate membrane detach. Collect all supernatant along with the cells into the same 15 ml conical tube, and add 0.5 ml of FBS again for neutralization.

[0139] Preferably, multi-step trypsin digestion refers to digesting the cells 2 to 3 times until all cells are collected and placed in the same 15 ml tube with FBS. After 3 weeks of growth, the cell layers adhere very tightly, so a single trypsin digestion cannot harvest all cells. On the other hand, prolonged exposure to trypsin is harmful to the cells. Therefore, "multi-step trypsin digestion" is used to minimize cell damage while harvesting all cells.

[0140] Therefore, the inventors designed trypsin digestion into several short steps lasting 6-8 minutes. After trypsin digestion, the digested and separated cells are transferred to a tube containing FBS, the reaction is neutralized, and the process is stopped. Unseparated cells remain on the permeate membrane, and the digestion process continues by adding fresh trypsin until they are all finally separated.

[0141] This "multi-step digestion" method provides longer processing time for cells that require more digestion and protects them from prolonged trypsin damage.

[0142] The harvested cell suspension is transferred to a solid substrate, such as a polystyrene culture dish or flask. The solid substrate is preferably coated with a polymer that promotes cell attachment and proliferation. Suitable substrates include, for example, Advanced TC. TM Culture dishes and flasks. After cell plating, according to step (iv) of the method of the present invention, further cell proliferation is carried out in a second culture medium (hereinafter referred to as "second culture medium") that promotes MSC proliferation but not differentiation.

[0143] The preferred plating method after cell harvesting is as follows: Centrifuge the cells and resuspend them in a second culture medium, then plate them on Advanced TC. TM In 6-well plates, after cells have adhered for 24 hours, wash away any unattached cells and proceed with normal cell culture. Change the culture medium twice a week and passage the cells into standard T25 or T75 cell culture flasks.

[0144] The second culture medium contains basic nutrients, such as Dulbeccos Modified Eagle Medium (DMEM). It may further contain serum, preferably human serum. In another preferred embodiment, the second culture medium also contains fibroblast growth factor (bFGF) and epidermal growth factor (EGF). The second culture medium may also contain insulin, human transferrin, and / or selenite. Preferably, the second culture medium contains interleukin-6 (IL-6). In a particular embodiment, the first and second culture media are identical.

[0145] The culture in step (iv) typically lasts 21 to 35 days.

[0146] The method of this invention is highly efficient and can generate a large number of MSCs from a certain number of hair follicles within a few weeks. Typically, the number of MSCs obtained from step (iv) is at least 10 per hair. 4 Each hair should ideally be at least 2 x 10. 4 Each hair should ideally be at least 5x10. 4 Ideally, each hair should have at least 1x10 hairs. 4 One, or at least 1.5x10 per hair. 4 In one embodiment, no more than 100 hairs are treated using the method of the present invention, and the number of MSCs obtained from step (iv) is at least 10. 7 Preferably at least 1.5x10 7In another embodiment, no more than 60 hairs are treated using the method of the present invention, and the number of MSCs obtained from step (iv) is at least 6 x 102 6 Preferably at least 9x10 6 In yet another embodiment, 40 to 60 plucked hairs are processed using the method of the present invention, and the number of MSCs obtained from step (iv) is at least 4 x 102 6 Preferably at least 6x10 6 .

[0147] MSCs obtained by the method of this invention are positive for staining of the markers CD73, CD90, and CD105, but negative for staining of the markers CD34, CD45, CD11b, CD19, and HLA-DR. In a preferred embodiment, the MSCs of this invention are positive for staining of the markers CD44, CD133, CD13, CD71, Nestin, N-Cadherin, Fibronectin, Vimentin, Integrin α5 (CD49e), and Integrin αV (CD51). The MSCs obtained by this invention meet the MSC definition criteria provided by the International Society for Cell Therapy (ISCT) (Cytotherapy (2006) Vol. 8, No. 4, 315-317). Therefore, the MSCs of this invention have the following characteristics 1 to 3:

[0148]

[0149] On the other hand, the MSCs obtained from step (iv) of this invention can differentiate into various target cells. Examples of target cell differentiation include, but are not limited to, cardiomyocytes, chondrocytes, osteoblasts, adipocytes, endothelial cells, and smooth muscle cells. For inducing differentiation, the differentiation medium for MSCs contains one or more differentiation-promoting components. Suitable components of inducing factors that promote cell differentiation into various cell types are known to those skilled in the art.

[0150] For example, to differentiate into osteoblasts, MSCs can be induced using a medium containing dexamethasone, ascorbic acid, and β-glycerophosphate. The medium used for differentiation may also contain serum, such as human serum or fetal bovine serum, and / or L-glutamine.

[0151] To differentiate into chondrocytes, MSCs can be induced in high-cell-density cultures using an induction medium containing TGF-β1, BMP-4, dexamethasone, ascorbic acid, sodium pyruvate, insulin, transferrin, and selenite. The differentiation medium may also contain serum and / or non-essential amino acids, and / or L-glutamine.

[0152] To differentiate into endothelial cells, MSCs can be cultured in an induction medium containing VEGF. The differentiation medium may also contain serum, BMP-4, 2-mercaptoethanol, and / or L-glutamine.

[0153] To differentiate into smooth muscle cells, MSCs can be cultured in an induction medium containing TGF-β1. The differentiation medium may also contain L-glutamine and serum.

[0154] To differentiate into adipocytes, MSCs can be cultured in commercially available adipocyte differentiation media, such as the StemProAdipogenesis Differentiation Kit (Thermo Fisher Scientific). In addition to commercially available differentiation formulations, alternative media for adipocyte differentiation may include human insulin, human transferrin, selenite, 3-isobutyl-1-methylxanthine (IBMX), and indomethacin.

[0155] To differentiate into cardiomyocytes, MSCs can be cultured in an induction medium containing 5-azacytidine (5-aza), succinyl aniline isohydroxamic acid (SAHA), bone morphogenetic protein-2 (BMP-2), and 2-mercaptoethanol. The differentiation medium may also contain serum, such as human serum or fetal bovine serum, and / or L-glutamine.

[0156] The inventors unexpectedly discovered that, compared to MSCs derived from adipose tissue, the MSCs of the present invention can differentiate into osteoblasts, endothelial cells, and smooth muscle cells more effectively. For example, osteoblasts derived from MSCORS exhibit significantly higher mineral deposition and alkaline phosphatase activity; endothelial cells derived from MSCORS show significantly higher accumulated angiogenesis length in angiogenesis experiments; and smooth muscle cells differentiated from MSCORS exhibit significantly elongated cell morphology (see Example 4). Therefore, in a preferred embodiment, the method of the present invention includes differentiating MSCs obtainable from step (iv) into osteoblasts, endothelial cells, or smooth muscle cells.

[0157] On the other hand, the present invention relates to all species of MSCs that can be obtained by the method described in the present invention.

[0158] In another aspect, the present invention relates to the use of MSCs obtained by the method described herein for generating differentiated cells, preferably differentiated cells as defined above.

[0159] On another front, the present invention relates to mesenchymal stem cells obtainable through the methods described herein, for purposes such as cosmetic, structural, or functional improvement of healthy tissues, organs, or systems, or for treating conditions of cells, tissues, organs, or systems related to mesenchymal tissue, particularly in the following embodiments. In one embodiment, the disease is a bone disease, such as bone injury. In another embodiment, the condition is a condition affecting cartilage, such as osteoarthritis. In another embodiment, the condition is a cardiovascular condition. In yet another embodiment, the condition is a wound.

[0160] In a further aspect, the present invention relates to the following:

[0161] Mesenchymal stem cells obtained using the method described in this invention are used to produce conditioned medium containing MSCs synthetic products.

[0162] Mesenchymal stem cells obtained using the method described in this invention are used to produce and isolate exosome components containing MSCs synthetic products.

[0163] Mesenchymal stem cells can be obtained by the method described in this invention.

[0164] The conditioned medium as described above.

[0165] The exosome components as described above.

[0166] The conditioned media described above are used to treat degenerative, inflammatory, immune, autoimmune, malignant, acute or chronic diseases, as well as functional and structural damage to cells, tissues, organs or systems associated with the interstitium.

[0167] The conditioned media described above are used for the activation and regeneration of cells, organs, tissues, and systems.

[0168] The conditioned medium as described above is used to replace one or more differentiated tissues as defined above.

[0169] The exosome components described above are used to treat degenerative, inflammatory, immune, autoimmune, malignant, acute or chronic diseases, as well as functional and structural damage to cells, tissues, organs or systems associated with the interstitium.

[0170] The exosome components described above are used for the activation and regeneration of cells, organs, tissues, and systems.

[0171] The exosome components described above are used to replace one or more differentiated tissues as defined above.

[0172] Example

[0173] Example 1: Isolation, purification and proliferation of hMSCORS

[0174] Human hMSCORS isolation medium (“first medium”):

[0175] DMEM (low glucose) 88%

[0176] 10% human serum

[0177] ITS Premix 1%

[0178] Fibroblast growth factor 10 ng / ml

[0179] Recombinant human epidermal growth factor 20 ng / ml

[0180] L-Glutamine 2mM

[0181] Penicillin 100U / ml, Streptomycin 100μg / ml

[0182] 1% Sigma-Aldrich provides volumetric concentration

[0183] Human serum was used throughout the separation process.

[0184] Human hMSCORS culture medium (“second medium”):

[0185] DMEM (low glucose) 89%

[0186] 10% fetal bovine serum

[0187] Fibroblast growth factor 10 ng / ml

[0188] Recombinant human epidermal growth factor 20 ng / ml

[0189] L-Glutamine 2mM

[0190] IL-6 10ng / ml

[0191] Penicillin 100U / ml, Streptomycin 100μg / ml

[0192] 1% Sigma-Aldrich provides volumetric concentration

[0193] Sampling and processing

[0194] Donors did not wash their hair for at least 48 hours to prevent loss of sebum secreted by the sebaceous glands and shrinkage of the hair follicles, which would reduce the amount of ORS tissue extracted along with the hair shaft. Temporal hairs were grasped with tweezers at approximately 2 mm from the root and plucked in the direction of hair growth. The hair follicle was extracted along with the hair shaft, providing most of the native ORS tissue. This separation method was used on 45 hair follicles from the same donor (tissue weight 16,0875 ± 2.95 mg, individual variations exist).

[0195] The plucked hair follicles are soaked in DMEM containing antibiotics and L-glutamine and incubated at room temperature for 2–4 hours. This method removes scalp oil, dandruff, and other debris. After a rough rinse with antibiotic-containing PBS, the dermal papilla of the hair follicle is excised under a microscope. The hair follicle is then vigorously rinsed 10 times with antibiotic-containing PBS, 10 minutes each time, with vigorous shaking at 300 rpm.

[0196] The treated hair follicles were digested with 5 mg / ml collagenase X containing antibiotics at 37°C for 12 minutes.

[0197] MSCORS separation

[0198] After digestion, hair follicles were carefully rinsed with PBS and then seeded onto a Transwell porous membrane (Corning). The membrane was assembled into a Corning Cellstar 6-well plate, and human fibroblast trophoblasts were cultured in the lower chamber of the membrane. 0.9 ml of culture medium was filled below the Transwell membrane, and the treated hair follicles were cultured under hypoxic conditions of 37°C and 5% O2. Two days later, the culture medium was changed for the first time (1.3 ml), and changes in the hair follicles were recorded under a microscope. The culture medium was changed twice a week, with gentle washing with pre-warmed PBS before each change.

[0199] Seven days later, the cells begin to migrate from the hair follicle ORS to the Transwell permeation membrane. Within 2-3 weeks, the cells form a monocellular layer around the hair shaft on the membrane.

[0200] MSCORS Harvest

[0201] Three weeks later, MSCORS were collected by trypsin digestion: the culture medium was aspirated and the cell layer on the permeabilized membrane was gently rinsed three times with pre-warmed PBS.

[0202] Add 0.5 ml of 0.04% / 0.03% Trypsin / EDTA to the cell layer in the upper chamber of the Transwell and incubate at 37°C for 6 minutes for microscopic observation. When most cells become round, gently tap the culture dish and aspirate all cells into a 15 ml conical tube containing 0.5 ml FBS to neutralize the trypsin digestion reaction. Repeat this step several times and incubate for 2 minutes each time until all cells are separated from the Transwell membrane. Collect all cell suspensions into tubes containing FBS, centrifuge at 220 g for 3 minutes, resuspend in culture medium, and seed all cells into 6-well cell culture plates.

[0203] MSCORS culture:

[0204] After 24 hours, approximately 30% of the target cells adhered to the surface of the plastic culture vessel, while over 60% remained unattached. After rinsing with PBS, fresh culture medium (second medium) was added. Over the next 5 days, the cells divided and grew in colonies, beginning to expand on the 2D plastic culture surface. When cell confluence reached 80-90%, the cells were digested with trypsin and passaged in a standard cell culture vessel.

[0205] MSCORS cells have a doubling time of less than 8 days.

[0206] Example 2: Characterization of hMSCORS

[0207] Immunofluorescence staining was used to characterize MSCORS, demonstrating that they possessed the correct MSC phenotype and differentiation potential. Using adipose-derived MSCs as controls, MSCORS showed equal or higher protein expression of CD44, CD133, CD13, CD71, Nestin, and N-Cadherin.

[0208] Based on the minimum identification criteria for pluripotent MSCs defined by the International Society for Cell Therapy (ISCT), flow cytometry and MSC marker analysis revealed that MSCORs express MSC markers specified in the ISCT MSC criteria at the protein level. MSCORs expressed high levels of the MSC-positive markers CD105, CD73, CD90, and CD44, but not the negative markers CD19, CD11b, CD14, CD34, CD45, CD79a, or HLA-DR. This method successfully conferred the correct ISCT phenotype on the isolated MSCs, achieving the same level as adipose-derived MSCs. The summarized results are listed in the table below, and flow cytometry analysis figures are further provided below.

[0209]

[0210] Table 1. Flow cytometry analysis results of MSCs biomarker expression:

[0211] The expression of MSCs positive and negative markers in MSCORS (A) and ADMSCs (B) was analyzed by flow cytometry, as shown in Figure 1.

[0212] Gene expression profile

[0213] qRT-PCR analysis of mRNA expression levels revealed that MSCORS expressed CD44 and showed high levels of CD73, CD90, and CD105. Throughout the monitoring process, the expression levels of these biomarkers continuously increased with directed culture of MSCs (based on three biological experiments, with three technical replicates per experiment, and a single sample, during five consecutive passages of culture).

[0214] The results of gene expression data are as follows Figure 2 As shown.

[0215] Example 3: Comparison with existing technical methods

[0216] The following study describes a method for isolating stem cells from hair follicles:

[0217] -Li et al. (2015) Cell Tissue Res 362: 69-82 (hereinafter referred to as D10)

[0218] -Zhang et al. (2013) International Journal of Molecular Medicine 31:913-921 (hereinafter referred to as D11)

[0219] -Wang et al. (2013) Stem Cell Rev and Rep 9: 451-460 (hereinafter referred to as D12)

[0220] The method of the present invention differs from the method described in D10-D12 in that the hair follicle bulbs from which the hair was plucked are removed and subjected to collagen degradation treatment. The hair follicles are first cultured on a first culture medium on a permeable membrane and then cultured on a second culture medium on a solid matrix, as stated in the preceding claims.

[0221] The methods of D10-D12 are compared with the method of the present invention, and are briefly described below.

[0222] For each experimental method, a total of 45 hairs were collected from 3 donors, 15 from each donor. The hair follicles were treated exactly as described in D10, D11, D12, or Example 1 of this invention. The results showed that only a very small number of hair follicles treated according to the D10-D12 method showed cell migration. After 4 weeks of culture, the following results were obtained:

[0223] Table 2

[0224]

[0225] The interpretation of these results is as follows. To isolate and amplify MSCs, the D10, D11, and D12 methods placed hair follicles directly onto the surface of cell culture dishes (plastic). In the experiments described above, this method was less effective because cell migration directly from the hair follicles to the cell culture dish surface was observed in fewer cases. Therefore, the reports for D10, D11, and D12 appear to be based on only a few single successful cases (few hair follicles exhibiting cell migration), and no other hair follicles without cell migration were reported in the D10, D11, and D12 studies.

[0226] Infiltration culture atop the Transwell membrane provides ample culture medium and growth surface area, promoting hair follicle cell proliferation. This environment offers a robust growth substrate, liquid-gas interface stimulation, and nutrient supply from the culture medium and trophoblast cells, aiding in the rapid migration of MSCORS from the hair follicle ORS and the formation of a monocellular layer. Clearly, hair follicles treated using the method of this invention exhibit a larger area of ​​migrating cell layer.

[0227] After passage 0, the MSCORS cells isolated from three donors using the method of this invention could stably proliferate and be passaged, while in the D12 method, only one batch of cells was successfully isolated, and the cell count was significantly lower. Figure 7 and Figure 8 As shown, only one hair follicle cell emerged from D10 and D11. However, after passage to generation 0, these cells failed to proliferate, continued to age, and rapidly apoptoticated. Cell counts during successive passages of this invention and D12 are shown... Figure 7 In this study, the estimated cell yield from each passage cell count was shown in [the data]. Figure 8 middle.

[0228] Furthermore, the proximal portion of the hair was not removed in D10, D11, and D12. Images from the D10, D11, and D12 studies clearly show cells migrating from the hair follicle bulb to the surface of the culture dish. These dermal fibroblasts, also known as dermal papilla cells (DP cells), play a crucial role in hair growth and the hair matrix. Residual cells in the dermis are often removed along with the dermal papilla and the proximal portion of the hair follicle. These fibroblasts are known to express some MSCs markers, but their cell differentiation capacity is limited. Furthermore, given the incomplete expression lineage and low differentiation potential, the “hf-MSCs” cells in D10 are more likely dermal fibroblasts. Fibroblasts share very similar characteristics with MSCs in terms of marker expression and differentiation potential, and it is difficult to distinguish between these two cell populations, as reported in other literature: Hematti, P. (2012) Cytotherapy 14(5): 516-521. Denu, RA et al. (2016) ActaHaematol 136(2):85-97. Alt, E. et al. (2011) Biol Cell 103(4): 197-208.

[0229] It should also be noted that, according to the ISCT identification criteria (Cytotherapy (2006) Vol. 8, No. 4, 315-317), D10-D12 did not show a complete MSCs immunophenotype.

[0230] In summary, the methods used from D10 to D12 are insufficient to generate a sufficient number of MSCs from a quantitative sample of hair follicles. Furthermore, there is reasonable doubt as to whether the cells isolated and expanded during D10 to D12 are indeed MSCs; it is possible that the majority of the cells are fibroblasts.

[0231] Example 4: Differentiation of MSCORS

[0232] Cultured mecocells were differentiated into osteoblasts, chondrocytes, adipocytes, smooth muscle cells, and endothelial cells. Compared to painful bone marrow and synovial fluid biopsies and uncomfortable blood draws, adipose tissue-derived MSCs (ADMSCs) were considered less invasive and were therefore chosen as experimental controls. However, it must be acknowledged that liposuction requires the use of long, thick cannulas inserted deep into the subcutaneous tissue, constantly digesting and agitating the adipose tissue during the procedure, making it significantly more invasive and incomparable to non-invasive hair follicle extraction.

[0233] method

[0234] Osteogenesis

[0235] 6x10 4 Cells were seeded into 24-well plates and cultured in osteogenic MSC medium for 3 or 4 weeks. Differentiated cells were examined using alkaline phosphatase activity assays, alizarin red staining, and calcium phosphate assays.

[0236] MSCs osteogenic culture medium:

[0237] DMEM up to 90%

[0238] FBS 10%

[0239] Dexamethasone 2 x 10 -7 M

[0240] Ascorbic acid 50μg / ml

[0241] β-glycerophosphate 10mM

[0242] chondrogenesis

[0243] 2.5x10 5 Cells were centrifuged at 800g for 5 minutes in 15ml conical tubes and cultured in MSCs chondrogenesis medium for 3, 4, and 5 weeks. Differentiated cartilage tissue was examined using histological staining methods, including H&E, Alcian blue, Safranin O, collagen I, and collagen II.

[0244] MSCORS chondrogenesis medium:

[0245] DMEM / F12 up to 97%

[0246] Human serum 1%

[0247] 1% premix

[0248] TGF-β1 10ng / ml

[0249] BMP-410ng / ml

[0250] Dexamethasone 10 -7 M

[0251] Ascorbic acid 50μg / ml

[0252] Sodium pyruvate 50 μg / ml

[0253] 1% of non-essential amino acids

[0254] Adipogenic differentiation

[0255] 2x10 4Cells were seeded into 24-well plates and cultured for 3 or 4 weeks using commercially available StemPro™ Adipogenesis Differentiation Kit (Thermo Fisher Scientific) for MSC adipogenesis differentiation. Intracellular lipid vesicles in mature adipocytes were detected by Oil Red staining.

[0256] Optional (homemade adipose differentiation culture medium):

[0257] Adipogenic differentiation medium:

[0258] DMEM (low sugar) up to 88%

[0259] 10% fetal bovine serum

[0260] L-Glutamine 2mM

[0261] 1% of non-essential amino acids

[0262] Dexamethasone 1μM

[0263] 3-Isobutyl-1-methylxanthine (IBMX) 500μM

[0264] Indomethacin 100μM

[0265] Insulin 10μm / ml

[0266] Endothelial differentiation

[0267] 2.4x10 5 Cells were seeded in one well of a 6-well plate and cultured in MSCORS endothelial medium for 3 and 4 weeks. Cells were stained with anti-CD31 antibody, angiogenesis assays were performed using Matrigel, and the results were analyzed using ImageJ software.

[0268] MSCORS endothelial medium:

[0269] DMEM (low sugar) up to 94%

[0270] FBS5%

[0271] L-Glutamine 1% v / v 2mM

[0272] 2-Mercaptoethanol 0.5mM

[0273] Vascular endothelial growth factor 30 ng / ml

[0274] BMP-45ng / ml

[0275] Smooth muscle differentiation

[0276] MSCs prior to passage 4 were cultured in DMEM + 10% FBS medium until 80% confluence was achieved. Cells were digested with trypsin / EDTA and thawed at 1.5 x 10⁻⁶ ppm. 5 / cm 2 Density seeding was performed in new cell cultures for passage. From this point on, 3x10⁻⁶ cells were passaged. 5 One cell was seeded into one well of a 6-well plate and cultured for two days. The culture medium was then replaced with smooth muscle differentiation medium, and differentiation continued for 3 and 4 weeks. Upon reaching the time point, the differentiation-induced cells were harvested and immunofluorescence stained with anti-α-smooth muscle actin (αSMA) antibody.

[0277] MSCORS smooth muscle culture medium:

[0278] DMEM (low sugar) up to 89%

[0279] 10% fetal bovine serum

[0280] L-Glutamine 1% v / v 2mM

[0281] TGFβ-110ng / ml

[0282] Cardiac cell differentiation

[0283] Incubate with 5 μM 5-aza for 24 hours, then culture with 1 μM 5-aza for 7 days.

[0284] Incubate with 10 μM salicylanilide isohydroxamic acid (SAHA) for 24 hours, then incubate with 1 μM SAHA for 7 days.

[0285] Bone morphogenetic protein-2 (BMP-2) 10 ng / ml

[0286] 2-Mercaptoethanol 1mM

[0287] Experimental results

[0288] Cell count

[0289] The number of MSCORS cells isolated and enriched from plucked hair follicles is comparable to that of ADMSCs, but MSCORS requires the least amount of tissue. A single MSCORS isolation session requires 15-60 hairs, with a total weight of 5mg-22mg due to individual differences. ADMSC isolation requires at least 25 grams of adipose tissue to achieve the same isolation efficiency. Using MSCORS technology, 1 to 5 million MSCs can be generated within 4 weeks after isolation, which already meets the minimum cell number required for cell therapy. Within 40 days of cell harvesting, over 30 million cells can be obtained from P5 generation cells, with a cell doubling time of 7.5 days, which is sufficient for most MSC-based cell therapies.

[0290] MSCORS is a marker of human mesenchymal stem cell expression at levels comparable to or higher than those of adipose-derived ADMSCs.

[0291] Immunofluorescence staining was used to characterize MSCORS, and the expression of MSC markers and differentiation potential were investigated. Using adipose-derived ADMSCs as a control, MSCORS showed equal or higher levels of expression of CD44, CD133, CD13, CD71, nestin, and N-Cadherin. These results are described in detail in Example 2 above.

[0292] chondrogenesis

[0293] During differentiation, MSCORS cells form normally structured cell clusters. Histological staining and immunofluorescence staining revealed that these cells synthesize proteoglycans and type II collagen. At higher magnification, the extracellular matrix is ​​densely deposited. The amount and staining intensity of the extracellular matrix formed by MSCORS chondrocytes are significantly higher than those of ADMSCs, which exhibit more porous structures.

[0294] Adipogenic differentiation

[0295] MSCORS can differentiate into mature adipocytes, forming intracellular lipid vesicles. Lipid formation was detected by Oil Red staining. Compared with MSCORS, ADMSCs exhibited stronger adipogenic differentiation capacity.

[0296] Smooth muscle differentiation

[0297] Following smooth muscle differentiation, cells form spindle-shaped muscle cells, exhibiting commonalities but varying sizes. Compared to ADMSC-differentiated muscle cells, MSCORS-differentiated cells are more elongated, possessing a more distinctive morphology and higher α-smooth muscle actin (αSMA) expression. MSCORS-derived smooth muscle cells exhibit multinucleated fibrous structures, demonstrating myogenic fusion, while the commonalities of ADMSC-differentiated muscle cells are less pronounced. At the same exposure time, compared to ADMSC-differentiated cells, vascular smooth muscle cells (VSM) differentiated from MSCORS show stronger αSMA signaling and cell body elongation in fluorescence images. After smooth muscle differentiation, MSCORS-differentiated cells display myogenic fused syncytia with multinucleated fibrous structures, and are significantly longer than ADMSC-derived cells.

[0298] To investigate the degree of smooth muscle differentiation in MSCORS and ADMSCs, we developed a semi-quantitative method based on ImageJ to assess intracellular αSMA-bound actin stress fibers. At 3 and 4 weeks of differentiation, cells exhibited extreme elongation, abundant cytoskeleton and αSMA expression, but individual cells were difficult to distinguish and assess from photographs due to their overlapping and interconnected nature. Therefore, we extracted the fluorescence staining signal of αSMA actin stress fibers using ImageJ software and quantified the number and length of αSMA actin stress fibers in representative MSCORS and ADMSC photographs.

[0299] The fluorescence intensity of αSMA without DAPI signal (Alexa 594, red fluorescence) in fluorescence images was analyzed using the ImageJ plugins "Tubeness" and "Angiogenesis Analyzer". The number and length of αSMA fibers were measured in each image. A total of 11 ADMSC images and 74 MECORS images were analyzed (Carpentier G, Martinelli M, Courty J and Cascone I. Angiogenesis Analyzer for ImageJ. 4th ImageJ User and Developer Conference proceedings. Mondorf-les-Bains, Luxembourg. ISBN: 2-919941-18-6 : 198-201, 2012).

[0300] Semi-quantitative results from αSMA actin stress fiber analysis showed that smooth muscle cells differentiated from MSCORS had a greater number of fibers and a higher total fiber length compared to cells differentiated from ADMSCs (3 weeks of differentiation: 134% more fibers and 258% longer fibers; 4 weeks of differentiation: 19% more fibers and 60% longer fibers). This confirms the descriptive morphological analysis that smooth muscle cells differentiated from MSCORS are longer than those differentiated from ADMSCs.

[0301] Semi-quantitative results are shown Figure 3 .

[0302] Osteogenesis

[0303] MSCORS cells can differentiate into osteoblasts, exhibiting high levels of alkaline phosphatase activity and calcium deposition. Furthermore, compared to ADMSCs, MSCORS unexpectedly demonstrate highly efficient osteogenic differentiation capacity. MSCORS cells differentiate into osteoblasts faster and more efficiently, showing higher alkaline phosphatase activity and greater calcium deposition.

[0304] Semi-quantitative analysis

[0305] Using ImageJ software, semi-quantitative analysis was performed on bright-field images of osteogenic differentiation from MSCORS and ADMSCs stained with alkaline phosphatase (dark blue) and alizarin red (red). The staining intensity of each signal was measured and quantified as integrated optical density (IOD). A total of 36 ADMSC osteogenic images and 200 MSCORS osteogenic images were analyzed.

[0306] The staining signal is analyzed by measuring the area and average intensity of the target signal. The threshold for each image is determined independently by the non-positive stained background and excluded during the measurement process.

[0307] The semi-quantitative analysis results are shown in Figure 4.

[0308] Quantitative analysis

[0309] Quantitative analysis of calcium concentration in calcium deposits was performed using a calcium reaction chromogenic reagent—o-cresylphthalide complex (CPC-kit)—determined spectrophotometrically. Results showed that the calcium content in osteodifferentiated MSCORS was higher than that in osteodifferentiated adipose-derived MSCs. The difference in calcium content was highly significant after differentiation at week 4.

[0310] Quantitative analysis results as follows Figure 5 As shown.

[0311] Endothelial differentiation

[0312] Following endothelial differentiation, cells expressed high levels of the endothelial cell marker CD31. The results showed that CD31 expression was stronger in endothelial cells differentiated from MSCORS than in those differentiated from ADMSCs.

[0313] Semi-quantitative analysis of angiogenesis assay

[0314] The ability of endothelial cells to form vascular structures is inherent in vascular structures, which forms the basis of quantitative angiogenesis assays (Tube-Test). Endothelial cells differentiated from MSCORS showed a higher angiogenic capacity than those differentiated from ADMSCs.

[0315] Semi-quantitative analysis method for angiogenesis assay

[0316] Angiogenesis fluorescence images with calcein AM signal (green, live cells) were analyzed using the ImageJ plugin AngiogenesisAnalyzer, developed by Gilles Carpentier in 2012. Several important indices of angiogenesis were analyzed, including the number of connectors, number of segments, vessel branches, and total branch length. A total of 8 angiogenesis images from ADMSCs and 54 images from MSCORS were analyzed.

[0317] Image analysis and semi-quantitative results showed that, compared with ADMSC-differentiated cells, MECORS-differentiated endothelial cells exhibited superior angiogenic properties, with more junctions, branches, and segments. MECORS-differentiated cells also showed stronger calcein AM signaling compared to ADMSC-differentiated endothelial cells, indicating higher cell viability after differentiation.

[0318] The results of semi-quantitative analysis of angiogenesis assays in MSCORS and ADMSC are as follows: Figure 6 As shown.

[0319] Example 5: MSC live-cell imaging and cell tracking

[0320] Cell motility is related to cell viability, migration ability, and chemotaxis. To study cell motility in mesenchymal stem cells (MSCs) and quantitatively analyze the differences between MSCs from different sources, live-cell imaging and software-based cell tracking methods were used to distinguish and quantify the basic parameters of MSCOR cell motility and to test whether these parameters differ from those of ADMSCs (adipose-derived mesenchymal stem cells) and BMMSCs (bone marrow mesenchymal stem cells).

[0321] Based on our experimental experience, MSCOR showed higher cell motility (greater cumulative cell movement distance and higher speed) than other tested MSCs, such as ADMSCs and BMMSCs. At the same time, MSCOR showed more randomized movement than ADMSCs and was comparable to the randomness of BMMSCs.

[0322] method

[0323] Experiments were conducted using MSCORS (n=3 donors), ADMSCs (n=3 donors), and BMMSCs (n=3 donors), with two experimental replicates and three technical replicates per experiment (ne=2, nt=3). 6000 cells / well were seeded in 4-well slides and incubated at 37°C under hypoxic conditions (5% O2, 5% CO2) for 24 hours. After cell attachment, three images per well were captured at 10-minute intervals within a 37°C heated incubation chamber using a Keyence BZ-9000 live-cell imaging system over 24 hours (10X objective). This resulted in 145 images from three independent time-lapse experiments. These images were sequentially grouped and imported into ImageJVersion 53a software for analysis using a manual tracking plugin. To quantify cell movement, 15 single cells from each time frame of each image group were manually tracked by determining their position on each frame of each image group; each cell came from three stacks. Import the tracking file (labeled text results) into the ImageJ chemotaxis and migration tool for further analysis of cumulative and Euclidean distance, velocity, and directionality. Directionality is a measure of the straightness of the cell trajectory. Calculate and compare the Euclidean distance (the straight-line distance between the starting and ending points) and the cumulative distance between the starting and ending points of the migrating cells (the entire trajectory). Directionality values ​​approaching 0 indicate indirect, curvilinear cell migration, corresponding to random motion. Directionality values ​​approaching 1 indicate linear, directional migration from the starting point to the ending point, corresponding to directional, strategic motion.

[0324] Statistical analysis

[0325] Analysis of variance (ANOVA) or the nonparametric Kruskal-Wallis test was used to statistically analyze cumulative distance, Euclidean distance, velocity, and directionality. The normality and uniformity of the data differences were assessed using the Shapiro-Wilk normality test and Bartlett's test for equal variances. Bonferroni's post-test and Dunn's post-test were used for pairwise comparisons. A p-value <0.05 was considered statistically significant.

[0326] result

[0327] like Figure 9As shown, compared with ADMSCs and BMMSCs, MSCORS exhibit different cell motility characteristics in terms of cumulative distance, Euclidean distance, velocity, and directionality.

[0328] MSCORS showed the largest cumulative cell migration distance and highest velocity compared to ADMSCs and BMMSCs (p<0.05). Meanwhile, MSCORS exhibited Euclidean distance comparable to BMMSCs, but lower Euclidean distance compared to ADMSCs, and a significantly lower tendency towards directionality than ADMSCs. This suggests that MSCORSs exhibit more randomized movement than ADMSCs, comparable to BMMSCs.

[0329] Example 6: Expression intensity of CD44 and CD90 in MSCs

[0330] As important functional groups of MSCs, MSC-associated surface proteins play a crucial role in distinguishing and defining different MSC populations. CD44, a hyaluronic acid receptor, is involved in cell-cell interactions, cell adhesion, and migration. It is considered a key surface biomarker for MSCs and is used as a positive marker for MSCs, even though it is not included in the MSC expression profile of ISCT. CD90, known as Thy-1, mediates cell-cell adhesion, cell-matrix interactions, immune responses, stem cell proliferation, and differentiation.

[0331] These proteins, and their functions in cell adhesion and cell-matrix interaction, also affect the cell's migration ability.

[0332] To investigate the global, intracellular, and subcellular expression of CD44 and CD90 in mecocells and compare them with ADMSCs and BMMSCs, immunofluorescence staining and imaging were performed under the same conditions, followed by quantitative analysis of global (whole cell) and local (membrane vs. cytoplasm) signal intensities.

[0333] method

[0334] 10,000 mecoscrons (n=4 donors, 3 biological experiments per donor, 2 technical replicates per experiment), ADMSCs (n=4, 3 biological experiments, 2 technical replicates), and BMMSCs (n=4, 3 biological experiments, 2 technical replicates) were seeded on 8-well slides and attached under hypoxic conditions (5% O2, 5% CO2, 37°C) for 24 hours. MSCs were fixed with 4% PFA, blocked with 10% normal goat serum, and incubated overnight at 4°C with primary antibodies against CD44 (mouse mIgG2b anti-human CD44 antibody, 1:200 dilution) and CD90 (mouse mIgG1 anti-human CD90 antibody, 1:3 dilution), respectively. After washing, Alexa594-labeled secondary antibody (goat anti-mouse IgG, 1:400 dilution) was incubated with DAPI (4',6-diamino-2-phenylindole, 1:500 dilution) at room temperature for 30 minutes. After rinsing, use Fluoromount TM Water-based mounting medium is used for mounting.

[0335] Imaging was performed using a Keyence BZ-9000 fluorescence microscope. The exposure time for CD44 was 1 / 20 of a second, and for CD90, it was 1 / 10 of a second. Twenty images were randomly taken from each well. Fluorescence intensity was quantitatively analyzed using ImageJ Version 53a software. In MSC cells, to quantify the different intracellular expression patterns of CD44 and CD90, a "cell membrane / cytoplasm index" was established to describe the ratio of fluorescence signal intensity at the cell membrane edge to that in the cytoplasm.

[0336] Statistical analysis

[0337] CD44 and CD90 signal intensities, as well as the cell membrane / cytoplasm index, were statistically evaluated using analysis of variance (ANOVA) or the nonparametric Kruskal-Wallis test. The normality and uniformity of the data differences were assessed using the Shapiro-Wilk normality test and Bartlett's test for equal variances. Bonferroni's post-test and Dunn's post-test were used for pairwise comparisons. A p-value <0.05 was considered statistically significant.

[0338] result

[0339] Figure 10 The signal intensities of immunofluorescence staining for CD44 and CD90 under the same conditions are shown. A representative image of CD44 expression is shown below. Figure 10 As shown in (A), different intracellular expression patterns were found in MSCORS, ADMSCs and BMMSCs.

[0340] The signal strength of CD44 and CD90 (not shown) is higher in BMMSCs than in MECORS and ADMSCs. The signal strength in the outer membrane is higher than that in the cytoplasm of MSCORs, while in ADMSCs and BMMSCs, the signal strength in the membrane and cytoplasm is comparable. Figure 10 Quantitative data in (B) showed that BMMSCs exhibited the highest expression levels (intensity) of CD44 and CD90 per single cell compared with MSCORS and ADMSCs (p<0.001); no significant differences were found between MSCORS and ADMSCs.

[0341] like Figure 10 As shown in (A), MSCORS showed that CD44 signaling along the cell membrane edge was stronger than that in the intracellular cytoplasm. Figure 10 Quantitative analysis of the cell membrane / cytoplasm index in (B) also supports this conclusion, with the index for CD44 being greater than 1. Furthermore, the cell membrane / cytoplasm indices of CD44 and CD90 in MSCORS were significantly higher than those in ADMSCs (p<0.001).

Claims

1. A method for generating mesenchymal stem cells, comprising the following steps: (i) Excise the hair follicle bulb from which the hair was plucked; (ii) The remaining portion of the hair follicle is incubated with collagenase to obtain a hair follicle containing stem cells and partially degraded extracellular matrix; (iii) The hair follicles obtained in step (ii) are cultured on a Transwell permeable membrane using a first culture medium to allow stem cells to migrate from the hair follicles under conditions that induce stem cell proliferation without inducing their differentiation; and (iv) Using a second culture medium, the stem cells obtained in step (iii) are further cultured on a liquid-impermeable solid matrix, which induces stem cell proliferation without inducing their differentiation; in, The composition of the first culture medium is as follows: 88% low glucose DMEM, 10% human serum, 1% ITS Premix, 10 ng / ml fibroblast growth factor, 20 ng / ml recombinant human epidermal growth factor, 2 mM L-glutamine, and penicillin / streptomycin at a volume concentration provided by 1% Sigma-Aldrich. The composition of the second culture medium is as follows: 89% low glucose DMEM, 10% fetal bovine serum, 10 ng / ml fibroblast growth factor, 20 ng / ml recombinant human epidermal growth factor, 2 mM L-glutamine, 10 ng / ml IL-6, and penicillin / streptomycin at a volume concentration provided by 1% Sigma-Aldrich. The culture was carried out under hypoxic conditions, which refer to an oxygen concentration of less than 20%. The culture in step (iii) lasts for 14 to 25 days. Cell layers fuse within 3 weeks of culture. Cells are harvested from the permeate membrane to obtain a cell suspension. The harvesting method using multi-step trypsin digestion is as follows: Aspirate the culture medium from the upper and lower chambers on both sides of the permeate membrane and add pre-warmed PBS for rinsing; gently rinse the cell layer and hair follicles, repeating the rinsing step 3 times; add 0.5 ml of 0.04% / 0.03% trypsin / EDTA to the membrane and incubate for 6–8 minutes, observing under a microscope; when many cells shrink into spherical shapes, gently tap the 6-well plate and gently rinse the cell layer with a pipette; collect the supernatant and aspirate it into a 15 ml centrifuge tube containing 0.5 ml of FBS for neutralization; add another 0.5 ml of trypsin / EDTA to the Transwell and repeat the trypsin digestion process 2–3 times until all cells on the permeate membrane detach; collect all supernatant along with the cells into the same 15 ml conical tube and add 0.5 ml of FBS again for neutralization. The culture in step (iv) lasts for 21 to 35 days.

2. The method of claim 1, wherein the hair removal is the removal of human hair.

3. The method of claim 1, wherein the hair removal is the removal of human hair in the growth phase.

4. The method of any one of claims 1-3, wherein multiple plucked hairs are used to isolate mesenchymal stem cells.

5. The method of claim 4, wherein at least 30 plucked hairs are used to isolate mesenchymal stem cells.

6. The method of claim 4, wherein at least 50% of the hair follicles are capable of generating proliferating mesenchymal stem cells.

7. The method of any one of claims 1-3, wherein step (iv) comprises culturing the isolated stem cells until the number of proliferating stem cells reaches at least 6 × 10⁻⁶. 6 .

8. The method of any one of claims 1-3, further comprising step (v), differentiating the proliferating stem cells obtained from step (iv) to obtain other cells.

9. The method of claim 8, wherein the differentiated cells include chondrocytes, osteoblasts, adipocytes, endothelial cells, and smooth muscle cells.

Images