An amplification medium and culture method for human adult epidermal stem cells

Abstract

The application discloses an amplification culture medium and a culture method of human adult epidermal stem cells, and relates to the technical field of stem cell in-vitro culture. The culture medium comprises the following components: fibroblast growth factor, interleukin-6, insulin-like growth factor-1, epidermal growth factor, insulin, growth hormone, hydrocortisone, Y-27632, A83-01, Forsklin, PY60, CHIR99021, linoleic acid, linolenic acid, alanine, asparagine, glutamic acid, glycine, proline, serine, arginine, N-acetyl-L-cysteine, beta-mercaptoethanol and CaCl2. The culture medium can continuously and stably culture human adult epidermal stem cells for more than 20 generations, and a more stable long-term in-vitro amplification system of epidermal stem cells is established. After continuous subculture, the epidermal stem cells can maintain a high proliferation rate and multiple differentiation potential, and have the ability to construct multilayer skin.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of stem cell in vitro culture, and more particularly to an amplification culture medium and culture method for human epidermal stem cells. BACKGROUND

[0002] Human epidermal stem cells (hEpiSCs) are a key cell population for maintaining homeostasis, repairing wounds, and regulating hair cycle in skin. Traditionally, such cells can be obtained by biopsy of the hair follicle bulge region or the basal layer, and have the potential for long-term self-renewal and directional differentiation into structures such as epidermis, hair follicle and sebaceous gland. Therefore, hEpiSCs are not only the core seed cells for regenerative medicine of large-area skin defects such as burns and chronic ulcers, but also an important tool for gene therapy, cosmeceutic screening and in vitro skin model construction.

[0003] However, hEpiSCs are prone to "stem cell exhaustion" when expanded in vitro, i.e., rapidly entering a terminal differentiation or senescence state, resulting in a decrease in clonogenicity, loss of multipotency markers (such as p63, ITGA6, KRT15), and ultimately an inability to obtain a sufficient number of functional stable cell products. This bottleneck directly limits its clinical translation and industrial application.

[0004] The prior art mainly uses a commercial epidermal keratinocyte serum-free medium (D-KSFM) and a medium based on DMEM / F12 and supplemented with B27 and 5% serum (referred to as "DMEM / F12+"). Such culture media have the following common defects: (1) dependence on animal-derived components, such as bisphenol analogue BPE, fetal bovine serum FBS, and cholesterol-rich lipid mix, which brings batch differences, exogenous contamination and ethical regulatory risks; (2) narrow growth factor concentration window, prone to "overdifferentiation" or "senescence arrest", and the purity of stem cells decreases to less than 30% after 3-4 generations of expansion; (3) the need for feeder cells, such as mouse embryonic fibroblasts 3T3-J2, dermal fibroblasts HDF or a high-oxygen environment (40% O2) to maintain clonality, which is complex in operation process, high in cost and difficult to achieve GMP-level scale-up culture; (4) lack of precise regulation of ROS stress, DNA damage repair and epigenetic homeostasis, resulting in a survival rate of <60% after cryopreservation and resuscitation, and a significant decrease in in vivo regenerative capacity.

[0005] In summary, the prior art has not yet solved the problem of large-scale expansion of human adult epidermal stem cells with adult source, no animal source, no feeder layer, low cost and high fidelity. Therefore, a new type of culture medium formula is urgently needed, which can maintain the long-term self-renewal and pluripotency of hEpiSCs under the condition of GMP level and completely clear chemical composition, and also consider the freezing stability and in vivo regeneration function, to meet the urgent needs of clinical level skin regeneration, gene therapy and in vitro skin model construction. SUMMARY

[0006] Therefore, the present application provides a kind of human adult epidermal stem cell expansion medium and culture method.

[0007] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0008] A kind of human adult epidermal stem cell expansion medium, characterized in that, include the following components:

[0009] 5-20 ng / mL of fibroblast growth factor, 5-20 ng / mL of interleukin-6, 5-20 ng / mL of insulin-like growth factor-1, 5-40 ng / mL of epidermal growth factor, 5-20 μg / mL of insulin, 5-200 ng / mL of growth hormone, 0.5-2 μg / mL of hydrocortisone, 1-10 μM of Y-27632, 5-20 μM of A83-01, 5-100 ng / mL of Forsklin, 10 μM of PY601, 1-10 μM of CHIR99021, 0.5-1 mg / mL of linoleic acid, 0.5-1 mg / mL of linolenic acid, 5-20 μg / mL of alanine, 10-25 μg / mL of asparagine, 10-25 μg / mL of glutamic acid, 7.5-30 μg / mL of glycine, 10-30 μg / mL of proline, 10-30 μg / mL of serine, 10-40 μg / mL of arginine, 0.5-2.0 mM of N-acetyl-L-cysteine, 1-100 μM of β-mercaptoethanol, 0.1-1.0 mM of CaCl2.

[0010] Preferably, the following components are included:

[0011] fibroblast growth factor 10 ng / mL, interleukin-6 10 ng / mL, insulin-like growth factor-1 10 ng / mL, epidermal growth factor 20 ng / mL, insulin 10 μg / mL, growth hormone 100 ng / mL, hydrocortisone 1 μg / mL, Y-27632 5 μM, A83-01 10 μM, Forsklin 50 ng / mL, PY60 5 μM, CHIR99021 1 μM, linoleic acid 0.5 mg / mL, linolenic acid 0.5 mg / mL, alanine 8.9 μg / mL, asparagine 13.3 μg / mL, glutamic acid 14.7 μg / mL, glycine 7.5 μg / mL, proline 11.5 μg / mL, serine 10.5 μg / mL, arginine 21 μg / mL, N-acetyl-L-cysteine 1 mM, β-mercaptoethanol 50 μM, CaCl2 0.2 mM.

[0012] Further, the expansion medium uses calcium ion-free DMEM / F12 as solvent.

[0013] A preparation method of an expansion medium of human adult epidermal stem cells, comprising the following steps:

[0014] (1) respectively dissolve fibroblast growth factor, interleukin-6, insulin-like growth factor-1, epidermal growth factor, alanine, asparagine, glutamic acid, glycine, proline, serine, arginine, N-acetyl-L-cysteine, calcium chloride in calcium ion-free DMEM / F12 to prepare a mother liquor;

[0015] Dissolve human insulin in 0.01M HCl to prepare a mother liquor;

[0016] Dissolve hydrocortisone, Y-27632, A-83-01, Forsklin, linoleic acid, and linolenic acid in DMSO to prepare a mother liquor;

[0017] (2) Add each mother liquor in step (1) to calcium ion-free DMEM / F12 according to the final concentration of the medium.

[0018] Further, the preparation method of the mother liquor in step (1) is:

[0019] Dissolve fibroblast growth factor in calcium ion-free DMEM / F12 to prepare a mother liquor of 50 ug / mL;

[0020] Dissolve interleukin-6 in calcium ion-free DMEM / F12 to prepare a mother liquor of 10 ug / mL;

[0021] Insulin-like growth factor-1 was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 100 ug / mL;

[0022] Growth hormone was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 100 ug / mL;

[0023] Epidermal growth factor was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 50 ug / mL;

[0024] Alanine was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 8.9 mg / mL;

[0025] Asparagine was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 13.3 mg / mL;

[0026] Glutamic acid was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 14.7 mg / mL;

[0027] Glycine was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 7.5 mg / mL;

[0028] Proline was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 11.5 mg / mL;

[0029] Serine was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 10.5 mg / mL;

[0030] Arginine was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 21 mg / mL;

[0031] Beta-mercaptoethanol was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 50 mM;

[0032] N-acetyl-L-cysteine was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 100 mM;

[0033] Calcium chloride was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 100 mM;

[0034] Human insulin was dissolved in 0.01 M HC1 overnight and made into a stock solution of 10 mg / mL;

[0035] Hydrocortisone was dissolved in DMSO to make a stock solution of 1 mg / mL;

[0036] Y-27632 was dissolved in DMSO to make a stock solution of 100 mM;

[0037] A-83-01 was dissolved in DMSO to make a stock solution of 10 mM;

[0038] Forskolin was dissolved in DMSO to prepare a stock solution of 1 mg / mL;

[0039] PY60 was dissolved in DMSO to prepare a stock solution of 100 mM;

[0040] CHIR99021 was dissolved in DMSO to prepare a stock solution of 10 mM;

[0041] Linoleic acid was dissolved in DMSO to prepare a stock solution of 5 mg / mL;

[0042] Linolenic acid was dissolved in DMSO to prepare a stock solution of 5 mg / mL.

[0043] A culture method of human adult epidermal stem cells, comprising the following steps:

[0044] 1) Culturing human primary epidermal stem cells in a mouse tail collagen-coated culture dish to adhere human adult epidermal stem cells;

[0045] 2) Collecting adherent human adult epidermal stem cells and culturing them with the expansion culture medium to obtain human adult epidermal stem cells.

[0046] Further, the step 2) culture process is carried out every other day with half-volume medium replacement.

[0047] A human adult epidermal stem cell, which is obtained by culturing with the expansion culture medium or the culture method.

[0048] Use of an expansion culture medium of human adult epidermal stem cells in the preparation of an expanded epidermal stem cell preparation.

[0049] Use of human adult epidermal stem cells for non-diagnostic therapeutic purposes, which is any of the following:

[0050] A: In vitro research on the proliferation, differentiation, and death regulation mechanism of epidermal stem cells;

[0051] B: Evaluation of skin cell treatment efficacy;

[0052] C: Construction of functional skin organoids;

[0053] D: Drug development, screening, and pharmacological potency evaluation for skin diseases;

[0054] E: Research on skin aging mechanisms and evaluation of cosmetic effects.

[0055] According to the above technical solution, compared with the prior art, the beneficial effects of the present application are:

[0056] The medium of the application can continuously and stably culture human adult epidermal stem cells for more than 20 generations, compared with the existing culture system in the world, a more stable epidermal stem cell long-term in vitro expansion system is established; the epidermal stem cells can maintain a high proliferation rate and multiple differentiation potential after continuous passage, and have the ability to construct multi-layer skin. BRIEF DESCRIPTION OF DRAWINGS

[0057] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0058] Figure 1 The morphology of P1 generation human adult epidermal stem cells cultured in Example 1 of the present application;

[0059] Figure 2 The morphology of P1-P3 generation human adult epidermal stem cells cultured in Example 1 and commercial medium of the present application;

[0060] Figure 3 The in vitro expansion rate of human adult epidermal stem cells cultured in Example 2 of the present application;

[0061] Figure 4 The cell morphology of human adult epidermal stem cells cultured in Example 2 of the present application expanded to P20 generation;

[0062] Figure 5 The marker gene identification of P1 and P20 generation human adult epidermal stem cells cultured in Example 3 of the present application;

[0063] Figure 6 The cell morphology of human adult epidermal stem cells of different generations cultured in Example 4 of the present application;

[0064] Figure 7 The stem cell marker identification of P1 and P10 generation human adult epidermal stem cells cultured in Example 5 of the present application;

[0065] Figure 8 The differentiation ability of P20 generation human adult epidermal stem cells cultured in Example 6 of the present application. DETAILED DESCRIPTION

[0066] Clearly, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those of ordinary skill in the art without creative efforts should belong to the scope of the present application.

[0067] Sources of experimental materials:

[0068] Fibroblast growth factor: MCE; FGF basic / bFGF protein; Human (154a.a), HY-P7331;

[0069] Interleukin-6: MCE; IL-6 protein, Human; HY-P7044;

[0070] Insulin-like growth factor-1: MCE; IGF-I / IGF-1 protein, Human (70a.a); HY-P7018;

[0071] Epidermal growth factor: MCE; EGF protein, Human; HY-P7109;

[0072] Insulin: Yisheng; Insulin human recombinant recombinant human insulin; 40112ES25;

[0073] Growth hormone: MCE; GH / Somatotropin protein, Human (CHO); HY-P7360;

[0074] Hydrocortisone: MCE; Hydrocortisone; HY-N0583;

[0075] Y-27632: MCE; Y-27632; HY-10071;

[0076] A83-01: MCE; A 83-01; HY-10432;

[0077] Forsklin: MCE; Forskolin; HY-15371;

[0078] PY60: MCE; PY-60; HY-141644;

[0079] CHIR99021: MCE; Laduviglusib; HY-10182;

[0080] Linoleic acid: MERK; a-Linoleic acid; L1376;

[0081] Linolelaidic acid: Yisheng; Linolelaidic acid Linolenic acid; 718568ES50;

[0082] 2-Mercaptoethanol: MCE; β-mercaptoethanol; HY-Y0326;

[0083] Calcium ion-free DMEM / F12: Shanghai Yuanpeibio; Calcium ion-free DMEM / F12; K421316

[0084] D-KSFM: ThermoFisher; Keratinocyte SFM (1X); 17005042;

[0085] DMEM / F12: ThermoFisher; DMEM / F-12; 11320033;

[0086] B27: ThermoFisher; B-27™ Supplement (50X); 17504044;

[0087] Serum: ThermoFisher; Fetal Bovine Serum, Heat-Inactivated, New Zealand; 10091148.

[0088] Unless otherwise specified, the reagents involved in the examples can be purchased through commercial channels, and the methods not mentioned are conventional experimental methods, which will not be described one by one here.

[0089] Example 1

[0090] A preparation method of an amplification culture medium for human adult epidermal stem cells

[0091] Step (1) Preparation of each component mother liquor in the culture medium

[0092] Fibroblast growth factor was dissolved in calcium ion-free DMEM / F12 to prepare a 50ug / mL mother liquor;

[0093] Interleukin-6 was dissolved in calcium ion-free DMEM / F12 to prepare a 10ug / mL mother liquor;

[0094] Insulin-like growth factor-1 was dissolved in calcium ion-free DMEM / F12 to prepare a 100ug / mL mother liquor;

[0095] Growth hormone was dissolved in calcium ion-free DMEM / F12 to prepare a 100ug / mL mother liquor;

[0096] Epidermal Growth Factor dissolved in calcium ion free DMEM / F12 to make a stock solution of 50 ug / mL;

[0097] Alanine dissolved in calcium ion free DMEM / F12 to make a stock solution of 8.9 mg / mL;

[0098] Asparagine dissolved in calcium ion free DMEM / F12 to make a stock solution of 13.3 mg / mL;

[0099] Glutamic acid dissolved in calcium ion free DMEM / F12 to make a stock solution of 14.7 mg / mL;

[0100] Glycine dissolved in calcium ion free DMEM / F12 to make a stock solution of 7.5 mg / mL;

[0101] Proline dissolved in calcium ion free DMEM / F12 to make a stock solution of 11.5 mg / mL;

[0102] Serine dissolved in calcium ion free DMEM / F12 to make a stock solution of 10.5 mg / mL;

[0103] Arginine dissolved in calcium ion free DMEM / F12 to make a stock solution of 21 mg / mL;

[0104] Beta-Mercaptoethanol dissolved in calcium ion free DMEM / F12 to make a stock solution of 50 mM;

[0105] N-Acetyl-L-Cysteine dissolved in calcium ion free DMEM / F12 to make a stock solution of 100 mM;

[0106] Calcium Chloride dissolved in calcium ion free DMEM / F12 to make a stock solution of 100 mM;

[0107] Human Insulin placed in 0.01 M HC1 overnight to dissolve, made into a stock solution of 10 mg / ml;

[0108] Hydrocortisone dissolved in DMSO to make a stock solution of 1 mg / mL;

[0109] Y-27632 dissolved in DMSO to make a stock solution of 100 mM;

[0110] A-83-01 dissolved in DMSO to make a stock solution of 10 mM;

[0111] Forsklin dissolved in DMSO to make a stock solution of 1 mg / mL;

[0112] PY60 dissolved in DMSO to make a stock solution of 100 mM;

[0113] CHIR99021 dissolved in DMSO to make a stock solution of 10 mM;

[0114] Linoleic acid was dissolved in DMSO to prepare a stock solution of 5 mg / mL.

[0115] Linolenic acid was dissolved in DMSO to prepare a stock solution of 5 mg / mL.

[0116] Preparation of the medium in Step (2)

[0117] The stock solutions in Step (1) were sequentially added to DMEM / F12 without calcium ions to prepare the final medium. The final medium contained the following components: fibroblast growth factor (10 ng / mL), interleukin-6 (10 ng / mL), insulin-like growth factor-1 (10 ng / mL), epidermal growth factor (20 ng / mL), insulin (10 μg / mL), growth hormone (100 ng / mL), hydrocortisone (1 μg / mL), Y-27632 (5 μM), A83-01 (10 μM), Forsklin (50 ng / mL), PY60 (5 μM), CHIR99021 (1 μM), linoleic acid (0.5 mg / mL), linolenic acid (0.5 mg / mL), alanine (8.9 μg / mL), asparagine (13.3 μg / mL), glutamic acid (14.7 μg / mL), glycine (7.5 μg / mL), proline (11.5 μg / mL), serine (10.5 μg / mL), arginine (21 μg / mL), N-acetyl-L-cysteine (1 mM), β-mercaptoethanol (75 μM), and CaCl2 (0.2 mM).

[0118] Step (3) Expansion culture of human adult epidermal stem cells

[0119] The foreskin tissue of an adult human was trimmed into small pieces of about 1 mm × 1 mm using a sterile scissors. Subsequently, the dermis and epidermis tissues were separated by digestion with 2 mg / mL Dispase II enzyme at 37°C for 2 hours. The separated epidermis tissue was collected and further digested with 0.25% trypsin at 37°C for 15 minutes. After digestion, the reaction was terminated by adding DMEM / F12 containing 20% fetal bovine serum (FBS), and the cell suspension was then filtered and centrifuged to obtain an epidermal single-cell suspension.

[0120] The obtained cells were seeded into culture dishes pre-treated with rat tail collagen and cultured using the medium in Step (2). The initial culture was performed for 48 hours to promote the adhesion of epidermal stem cells, and the culture medium was then replaced every two days. The first passage was performed on the 14th day of culture. In subsequent culture processes, the cells were passaged at a ratio of 1:5 when they covered 80% of the bottom area of the culture dish.

[0121] Figure 1The cell morphology observed at different time points when P1 human adult epidermal stem cells were cultured in the medium provided by Example 1 of the present application is shown.

[0122] As shown in Figure 1 the whole culture process, the cells continuously presented typical small spindle and round morphology and maintained good cell refractivity, which was an intuitive morphological indication that the stem cells were undifferentiated and healthy. More importantly, no morphological changes such as cell body enlargement, spreading or vacuolization, which indicated cell aging, occurred during the culture period.

[0123] To evaluate the performance of the medium provided by Example 1 of the present application, two control media were set up for parallel comparison: a commercialized epidermal keratinocyte serum-free medium (D-KSFM) and a medium based on DMEM / F12 and supplemented with B27 and 5% serum (DMEM / F12+).

[0124] As shown in Figure 2 compared with the medium of the present application, the two control media showed significant deficiency in maintaining the stemness and proliferative capacity of epidermal stem cells. Specifically, the cells cultured in the D-KSFM and DMEM / F12+ control media showed obvious signs of cell aging or differentiation at the second passage, including changes in cell morphology, appearance of antenniform protrusions at the cell edge, significant spreading and thinning of the cell body, and increase in specific surface area, etc.

[0125] Further, when the culture entered the third passage, the cells in the two control media lost the ability to adhere to the wall, a large number of cells floated, and it was difficult to form a stable adherent cell layer, making it difficult to carry out effective subculture.

[0126] The results of this control experiment clearly show that the existing commercialized medium and conventional formula containing serum supplement cannot support the long-term in vitro expansion of epidermal stem cells, thereby proving the significant advantages and creativity of the medium of the present application in maintaining cell stemness, promoting stable proliferation and prolonging the culture period.

[0127] Example 2

[0128] A method for preparing an expansion medium for human adult epidermal stem cells:

[0129] Step (1) Preparation of mother liquor of each component in the medium

[0130] Fibroblast growth factor was dissolved in calcium ion-free DMEM / F12 to prepare a mother liquor of 75 ug / mL;

[0131] Interleukin-6 was dissolved in calcium ion-free DMEM / F12 to prepare a mother liquor of 15 ug / mL;

[0132] Insulin-like growth factor-1 was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 150 ug / mL;

[0133] Growth hormone was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 150 ug / mL;

[0134] Epidermal growth factor was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 75 ug / mL;

[0135] Alanine was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 15 mg / mL;

[0136] Asparagine was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 18 mg / mL;

[0137] Glutamic acid was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 20 mg / mL;

[0138] Glycine was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 15 mg / mL;

[0139] Proline was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 20 mg / mL;

[0140] Serine was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 20 mg / mL;

[0141] Arginine was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 30 mg / mL;

[0142] Beta-mercaptoethanol was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 75 mM;

[0143] N-acetyl-L-cysteine was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 150 mM;

[0144] Calcium chloride was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 150 mM;

[0145] Human insulin was dissolved in 0.01 M HC1 overnight and made into a stock solution of 15 mg / ml;

[0146] Hydrocortisone was dissolved in DMSO to make a stock solution of 2 mg / mL;

[0147] Y-27632 was dissolved in DMSO to make a stock solution of 150 mM;

[0148] A-83-01 was dissolved in DMSO to make a stock solution of 15 mM;

[0149] Forsklin was dissolved in DMSO to prepare a stock solution of 2 mg / mL;

[0150] PY60 was dissolved in DMSO to prepare a stock solution of 150 mM;

[0151] CHIR99021 was dissolved in DMSO to prepare a stock solution of 15 mM;

[0152] Linoleic acid was dissolved in DMSO to prepare a stock solution of 10 mg / mL;

[0153] Linolenic acid was dissolved in DMSO to prepare a stock solution of 10 mg / mL.

[0154] Preparation of the medium in Step (2)

[0155] The stock solutions in Step (1) were sequentially added to the DMEM / F12 without calcium ions to prepare the final medium. The final medium contained the following components: fibroblast growth factor (15 ng / mL), interleukin-6 (15 ng / mL), insulin-like growth factor-1 (15 ng / mL), epidermal growth factor (30 ng / mL), insulin (15 μg / mL), growth hormone (150 ng / mL), hydrocortisone (1.5 μg / mL), Y-27632 (7.5 μM), A83-01 (15 μM), Forsklin (75 ng / mL), PY60 (7.5 μM), CHIR99021 (1.5 μM), linoleic acid (0.75 mg / mL), linolenic acid (0.75 mg / mL), alanine (12.5 μg / mL), asparagine (17.5 μg / mL), glutamic acid (20 μg / mL), glycine (10 μg / mL), proline (15 μg / mL), serine (15 μg / mL), arginine (30 μg / mL), N-acetyl-L-cysteine (1.5 mM), β-mercaptoethanol (75 μM), and CaCl2 (0.5 mM).

[0156] Expansion culture of human adult epidermal stem cells in Step (3)

[0157] The foreskin tissue of an adult human was trimmed into small pieces of about 1 mm × 1 mm using a sterile scissors. Subsequently, the dermis and epidermis tissues were separated by digestion with 1 mg / mL Dispase II enzyme at 37°C for 4 hours. The separated epidermis tissue was collected and further digested with 0.15% trypsin at 37°C for 30 minutes. After the digestion was completed, the reaction was terminated by adding DMEM / F12 containing 20% fetal bovine serum (FBS), and then the cell suspension was filtered and centrifuged to obtain an epidermis single-cell suspension.

[0158] The obtained cells were inoculated into culture dishes pretreated with rat tail collagen and cultured using the medium of step (2). The initial culture was performed for 48 hours to promote the adhesion of epidermal stem cells, and then the culture solution was replaced every two days. The first passage was performed at the 14th day of culture. During the subsequent culture, when the cells covered 80% of the bottom area of the culture dish, the cells were passaged at a ratio of 1:3.

[0159] Figure 3 The graph of the in vitro expansion rate of human adult epidermal stem cells cultured in Example 2 of the present application. As can be seen from the graph, the medium of the present application can maintain the stable in vitro expansion of human adult epidermal stem cells for at least 20 passages.

[0160] Figure 4 The cell morphology graph of human adult epidermal stem cells cultured in Example 2 of the present application expanded to P20. As can be seen from the graph, after 20 passages of culture, the cells still maintained the typical small spindle-shaped or round stem cell morphology and exhibited excellent birefringence, and no obvious signs of cell aging were observed.

[0161] The above results demonstrate that the medium of the present application can effectively support the in vitro long-term expansion of human adult epidermal stem cells.

[0162] Example 3

[0163] A method for preparing an expansion medium for human adult epidermal stem cells:

[0164] Step (1) Preparation of each component mother liquor in the medium

[0165] Fibroblast growth factor was dissolved in calcium ion-free DMEM / F12 to prepare a mother liquor of 10 ug / mL;

[0166] Interleukin-6 was dissolved in calcium ion-free DMEM / F12 to prepare a mother liquor of 5 ug / mL;

[0167] Insulin-like growth factor-1 was dissolved in calcium ion-free DMEM / F12 to prepare a mother liquor of 10 ug / mL;

[0168] Growth hormone was dissolved in calcium ion-free DMEM / F12 to prepare a mother liquor of 10 ug / mL;

[0169] Epidermal growth factor was dissolved in calcium ion-free DMEM / F12 to prepare a mother liquor of 10 ug / mL;

[0170] Alanine was dissolved in calcium ion-free DMEM / F12 to prepare a mother liquor of 5 mg / mL;

[0171] Asparagine was dissolved in calcium ion-free DMEM / F12 to prepare a mother liquor of 10 mg / mL;

[0172] Glycine was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 10 mg / mL.

[0173] Glycine was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 10 mg / mL.

[0174] Proline was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 10 mg / mL.

[0175] Serine was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 10 mg / mL.

[0176] Arginine was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 10 mg / mL.

[0177] β-Mercaptoethanol was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 20 mM.

[0178] N-Acetyl-L-cysteine was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 20 mM.

[0179] Calcium chloride was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 10 mM.

[0180] Human insulin was dissolved in 0.01 M HC1 overnight, and a stock solution of 5 mg / mL was prepared.

[0181] Hydrocortisone was dissolved in DMSO to make a stock solution of 0.5 mg / mL.

[0182] Y-27632 was dissolved in DMSO to make a stock solution of 10 mM.

[0183] A-83-01 was dissolved in DMSO to make a stock solution of 10 mM.

[0184] Forsklin was dissolved in DMSO to make a stock solution of 1 mg / mL.

[0185] PY60 was dissolved in DMSO to make a stock solution of 50 mM.

[0186] CHIR99021 was dissolved in DMSO to make a stock solution of 5 mM.

[0187] Linoleic acid was dissolved in DMSO to make a stock solution of 1 mg / mL.

[0188] Linolenic acid was dissolved in DMSO to make a stock solution of 1 mg / mL.

[0189] Step (2) Preparation of culture medium

[0190] The mother liquor in step (1) was added into DMEM / F12 without calcium ion to prepare the final medium. The final medium was composed of: fibroblast growth factor (5 ng / mL), interleukin-6 (5 ng / mL), insulin-like growth factor-1 (5 ng / mL), epidermal growth factor (10 ng / mL), insulin (5 μg / mL), growth hormone (10 ng / mL), hydrocortisone (1 μg / mL), Y-27632 (5 μM), A83-01 (5 μM), Forsklin (5 ng / mL), PY60 (2 μM), CHIR99021 (2 μM), linoleic acid (0.5 mg / mL), linolenic acid (0.5 mg / mL), alanine (10 μg / mL), asparagine (10 μg / mL), glutamic acid (10 μg / mL), glycine (10 μg / mL), proline (10 μg / mL), serine (10 μg / mL), arginine (10 μg / mL), N-acetyl-L-cysteine (1 mM), β-mercaptoethanol (10 μM), CaCl2 (0.2 mM).

[0191] Step (3) Expansion culture of human adult epidermal stem cells

[0192] The foreskin tissue of an adult human was trimmed into small pieces of about 1 mm x 1 mm using a sterile scissors. Then, the dermis and epidermis tissue were separated by using 2 mg / mL Dispase II enzyme for 2 hours at 37°C. The separated epidermis tissue was collected and further digested with 0.25% trypsin for 15 minutes at 37°C. After the digestion, the reaction was terminated by adding DMEM / F12 containing 20% fetal bovine serum (FBS), and then the cell suspension was filtered and centrifuged to obtain an epidermal single cell suspension.

[0193] The obtained cells were seeded into culture dishes pretreated with rat tail collagen and cultured using the medium in step (2). The initial culture was performed for 48 hours to promote the adhesion of epidermal stem cells, and then the culture medium was replaced every two days. The first passage was performed at the 14th day of culture. In the subsequent culture process, when the cells covered 80% of the bottom area of the culture dish, the cells were passaged at a ratio of 1:4.

[0194] Figure 5 The marker genes of the P1 and P20 generations of human adult epidermal stem cells cultured in Example 3 of the present application were identified. As can be seen from the figure, the human adult epidermal stem cells of the 20th generation continuously expanded in vitro still expressed a variety of epidermal stem cell genes, indicating that the epidermal stem cells expanded by the culture medium have a high differentiation potential.

[0195] Real-time fluorescent quantitative PCR verifies the expression of epidermal stem cell genes of in vitro amplified human adult epidermal stem cells. The specific identification method is as follows: the cells cultured for 1 generation and 20 generations and human skin tissue are fully lysed by Trizol, 1 / 2 chloroform is added, 4°C, 12000 rpm, centrifugation for 15 minutes, the upper water phase is taken and an equal volume of isopropanol is added to mix thoroughly, room temperature standing for 10 minutes, 4°C, 12000 rpm, centrifugation for 15 minutes, then the precipitate is washed with 75% ethanol, air-dried to a translucent state, and dissolved in water. RNA reverse transcription is performed using a reverse transcription kit according to the instructions (Vazyme, #R323). The expression level of the corresponding gene is detected by real-time fluorescent quantitative PCR. The reaction system of real-time fluorescent quantitative PCR is shown in Table 1, the reaction conditions are shown in Table 2, and the primer sequences are shown in Table 3.

[0196] Table 1. Reaction system of real-time fluorescent quantitative PCR

[0197]

[0198] Table 2. Reaction conditions of real-time fluorescent quantitative PCR

[0199]

[0200] Table 3. Primer sequences of real-time fluorescent quantitative PCR

[0201]

[0202] Example 4

[0203] A method for preparing an amplification medium for human adult epidermal stem cells:

[0204] Step (1) Preparation of each component mother liquor in the medium

[0205] Fibroblast growth factor is dissolved in calcium ion-free DMEM / F12 to prepare a 100 ug / mL mother liquor;

[0206] Interleukin-6 is dissolved in calcium ion-free DMEM / F12 to prepare a 100 ug / mL mother liquor;

[0207] Insulin-like growth factor-1 is dissolved in calcium ion-free DMEM / F12 to prepare a 100 ug / mL mother liquor;

[0208] Growth hormone is dissolved in calcium ion-free DMEM / F12 to prepare a 100 ug / mL mother liquor;

[0209] Epidermal growth factor is dissolved in calcium ion-free DMEM / F12 to prepare a 100 ug / mL mother liquor;

[0210] Alanine was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 20 mg / mL.

[0211] Asparagine was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 20 mg / mL.

[0212] Glutamic acid was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 20 mg / mL.

[0213] Glycine was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 20 mg / mL.

[0214] Proline was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 20 mg / mL.

[0215] Serine was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 20 mg / mL.

[0216] Arginine was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 20 mg / mL.

[0217] β-Mercaptoethanol was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 50 mM.

[0218] N-Acetyl-L-cysteine was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 200 mM.

[0219] Calcium chloride was dissolved in calcium ion-free DMEM / F12 to make a stock solution of 200 mM.

[0220] Human insulin was dissolved in 0.01 M HC1 overnight, and a stock solution of 20 mg / ml was prepared.

[0221] Hydrocortisone was dissolved in DMSO to make a stock solution of 5 mg / mL.

[0222] Y-27632 was dissolved in DMSO to make a stock solution of 200 mM.

[0223] A-83-01 was dissolved in DMSO to make a stock solution of 20 mM.

[0224] Forsklin was dissolved in DMSO to make a stock solution of 5 mg / mL.

[0225] PY60 was dissolved in DMSO to make a stock solution of 100 mM.

[0226] CHIR99021 was dissolved in DMSO to make a stock solution of 10 mM.

[0227] Linoleic acid was dissolved in DMSO to make a stock solution of 5 mg / mL.

[0228] Linolenic acid was dissolved in DMSO to prepare a stock solution of 5 mg / mL.

[0229] Preparation of the medium in Step (2)

[0230] The stock solutions in Step (1) were sequentially added to DMEM / F12 without calcium ions to prepare a final medium. The final medium contained the following components: fibroblast growth factor (20 ng / mL), interleukin-6 (20 ng / mL), insulin-like growth factor-1 (20 ng / mL), epidermal growth factor (40 ng / mL), insulin (20 μg / mL), growth hormone (200 ng / mL), hydrocortisone (2 μg / mL), Y-27632 (10 μM), A83-01 (20 μM), Forsklin (100 ng / mL), PY60 (5 μM), CHIR99021 (5 μM), linoleic acid (1 mg / mL), linolenic acid (1 mg / mL), alanine (20 μg / mL), asparagine (20 μg / mL), glutamic acid (20 μg / mL), glycine (20 μg / mL), proline (20 μg / mL), serine (20 μg / mL), arginine (20 μg / mL), N-acetyl-L-cysteine (2 mM), β-mercaptoethanol (100 μM), and CaCl2 (1 mM).

[0231] Expansion culture of human adult epidermal stem cells in Step (3)

[0232] The foreskin tissue of an adult human was trimmed into small pieces of about 1 mm × 1 mm using a sterile scissors. Subsequently, the dermis and epidermis tissues were separated by digestion with 2 mg / mL Dispase II enzyme at 37°C for 2 hours. The separated epidermis tissue was collected and further digested with 0.25% trypsin at 37°C for 15 minutes. After completion of the digestion, the reaction was terminated by adding DMEM / F12 containing 20% fetal bovine serum (FBS), and the cell suspension was filtered and centrifuged to obtain an epidermal single cell suspension.

[0233] The obtained cells were seeded into culture dishes pretreated with rat tail collagen and cultured using the medium in Step (2). The initial culture was performed for 48 hours to promote the adhesion of epidermal stem cells, and the culture medium was replaced every two days thereafter. The first passage was performed at 14 days after the culture. In the subsequent culture, the cells were passaged at a ratio of 1:5 when they covered 80% of the bottom area of the culture dish.

[0234] Figure 6Cell morphology of different generations of human adult epidermal stem cells cultured in the medium of Example 4. As can be seen from the figure, the medium and the culture method support the stable expansion culture of human adult epidermal stem cells in vitro for more than 20 generations, the cells still maintain the typical small spindle or round stem cell morphology, and show excellent birefringence, and no obvious signs of cell aging are observed.

[0235] Example 5

[0236] A method for preparing an expansion medium for human adult epidermal stem cells:

[0237] Step (1) Preparation of each component mother liquor in the medium

[0238] The same as in Example 2.

[0239] Step (2) Preparation of the medium

[0240] Each mother liquor in step (1) is sequentially added to DMEM / F12 without calcium ions to prepare the final medium. The components of the final medium are respectively: fibroblast growth factor (10 ng / mL), interleukin-6 (10 ng / mL), insulin-like growth factor-1 (10 ng / mL), epidermal growth factor (10 ng / mL), insulin (10 μg / mL), growth hormone (50 ng / mL), hydrocortisone (1 μg / mL), Y-27632 (5 μM), A83-01 (10 μM), Forsklin (50 ng / mL), PY60 (5 μM), CHIR99021 (1 μM), linoleic acid (1 mg / mL), linolenic acid (1 mg / mL), alanine (15 μg / mL), asparagine (25 μg / mL), glutamic acid (25 μg / mL), glycine (30 μg / mL), proline (30 μg / mL), serine (30 μg / mL), arginine (40 μg / mL), N-acetyl-L-cysteine (2 mM), β-mercaptoethanol (50 μM), CaCl2 (0.5 mM).

[0241] Step (3) Expansion culture of human adult epidermal stem cells

[0242] The same as in Example 1.

[0243] Figure 7The stem cell markers of the P1 and P10 human adult epidermal stem cells cultured in Example 5 of the present application were identified. As can be seen from the figure, the human adult epidermal stem cells amplified by the medium of the present application for 10 generations can still stably express key stemness markers, including transcription factor P63 (epidermal basal layer proliferative potential cell marker), KRT14 (basal stem cell marker), ITGA6 (CD49f) and ITGB1 (CD29), wherein ITGA6 and ITGB1 are integrin molecules, which are enriched on the membrane surface of stem cells, proving that the medium system can effectively support the human adult epidermal stem cells to maintain their undifferentiated state and multi-directional differentiation potential during long-term in vitro culture.

[0244] The specific steps of marker identification are as follows: cells that have been subcultured for 10 times in succession are selected, when the cells grow to about 80% confluence, the culture medium is discarded, and the cells are gently washed with pre-cooled PBS. Then, 4% paraformaldehyde solution is added to fix the cells at room temperature for 30 minutes. After fixation, the cells are washed with PBS again. Then, specific primary antibodies against key markers of epidermal stem cells, including transcription factor P63, KRT14, ITGA6 and ITGB1, are added. The cells are incubated with the primary antibodies at 4°C overnight. The next day, the cells are washed with PBS for 3 times to remove unbound primary antibodies. Then, fluorescently labeled secondary antibodies matching the species origin of the primary antibodies are added, and incubated at room temperature for 1 hour in the dark. After incubation, the cells are washed with PBS for 3 times again. Finally, DAPI staining solution is used to restain the cell nuclei, and incubated at room temperature for 5 minutes. After washing with PBS, the cells are observed under a fluorescence microscope and images are collected.

[0245] Example 6

[0246] A method for preparing an amplification medium for human adult epidermal stem cells:

[0247] Step (1) Preparation of mother liquor of each component in the medium

[0248] The same as in Example 1.

[0249] Step (2) Preparation of the medium

[0250] The mother liquor in step (1) was added into DMEM / F12 without calcium ions in sequence to prepare the final culture medium. The final culture medium was composed of: fibroblast growth factor (10 ng / mL), interleukin-6 (10 ng / mL), insulin-like growth factor-1 (5 ng / mL), epidermal growth factor (5 ng / mL), insulin (5 μg / mL), growth hormone (5 ng / mL), hydrocortisone (0.5 μg / mL), Y-27632 (1 μM), A83-01 (5 μM), Forsklin (5 ng / mL), PY60 (10 μM), CHIR99021 (3 μM) linoleic acid (1 mg / mL), linolenic acid (1 mg / mL), alanine (5 μg / mL), asparagine (20 μg / mL), glutamic acid (20 μg / mL), glycine (7.5 μg / mL), proline (20 μg / mL), serine (20 μg / mL), arginine (20 μg / mL), N-acetyl-L-cysteine (0.5 mM), β-mercaptoethanol (20 μM), CaCl2(0.1 mM).

[0251] Step (3) Expansion culture of human adult epidermal stem cells

[0252] The same as in Example 1.

[0253] Figure 8 The differentiation ability of P20 generation human adult epidermal stem cells cultured in Example 6 of the present application. As can be seen from the figure, after 20 generations of long-term in vitro expansion, the human adult epidermal stem cells cultured in the culture medium still have the ability to differentiate into functional epidermal cells, including terminally differentiated epidermal keratinocytes (KRT10), the outermost keratinocytes (Loricrin and Filaggrin markers keratin envelope), confirming that the excellent multilineage differentiation potential is maintained for a long time.

[0254] Identification of epidermal stem cell differentiation potential: cells passaged for 20 times were selected for induction differentiation experiment. On the 3rd day of cell subculture, the air-liquid interface contact method was used for induction and differentiation, and the differentiation induction medium was used for continuous culture for 14 days to stimulate the differentiation potential of different epidermal lineages. After induction, the cells were fixed with 4% paraformaldehyde solution at room temperature for 30 minutes. After fixation, PBS was used for washing. Then, specific antibodies KRT10, Loricrin and Filaggrin marking different terminally differentiated cells of epidermis were added respectively. After incubation of the antibodies with the cells at 4°C overnight, PBS was used for washing for 3 times; then, the corresponding fluorescently labeled secondary antibodies were added, and incubated at room temperature for 1 hour in the dark; after washing with PBS for 3 times again, DAPI staining solution was used for counterstaining the cell nuclei for 5 minutes; after final washing with PBS for 3 times, the fluorescence microscope was used for observation and image acquisition.

[0255] The foregoing description of the disclosed embodiments enables a person skilled in the art to make or use the application. Numerous modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein can be applied to other embodiments without the use of the inventive faculty. Therefore, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An expansion culture medium for human adult epidermal stem cells, characterized in that, Includes the following ingredients: Fibroblast growth factor 5-20 ng / mL, interleukin-6 5-20 ng / mL, insulin-like growth factor-15-20 ng / mL, epidermal growth factor 5-40 ng / mL, insulin 5-20 μg / mL, growth hormone 5-200 ng / mL, hydrocortisone 0.5-2 μg / mL, Y-27632 1-10 μM, A83-01 5-20 μM, Forsklin 5-100 ng / mL, PY60 1-10 μM, CHIR99021 1-10 μM, linoleic acid 0.5-1 mg / mL, linolenic acid 0.5-1 mg / mL mg / mL, alanine 5-20 μg / mL, asparagine 10-25 μg / mL, glutamic acid 10-25 μg / mL, glycine 7.5-30 μg / mL, proline 10-30 μg / mL, serine 10-30 μg / mL, arginine 10-40 μg / mL, N-acetyl-L-cysteine ​​0.5-2.0 mM, β-mercaptoethanol 1-100 μM, CaCl2 0.1-1.0 mM; The amplification medium uses calcium-free DMEM / F12 as the solvent.

2. The amplification culture medium according to claim 1, characterized in that, Includes the following ingredients: Fibroblast growth factor 10 ng / mL, interleukin-6 10 ng / mL, insulin-like growth factor-1 10 ng / mL, epidermal growth factor 20 ng / mL, insulin 10 μg / mL, growth hormone 100 ng / mL, hydrocortisone 1 μg / mL, Y-27632 5 μM, A83-01 10 μM, Forsklin 50 ng / mL, PY60 5 μM, CHIR99021 1 μM, linoleic acid 0.5 mg / mL, linolenic acid 0.5 mg / mL, alanine 8.9 μg / mL, asparagine 13.3 μg / mL, glutamic acid 14.7 μg / mL, glycine 7.5 μg / mL, proline 11.5 μg / mL, serine 10.5 μg / mL, arginine 21 μg / mL, N-acetyl-L-cysteine ​​1 mM, β-mercaptoethanol 50 μM, CaCl2 0.2 mM.

3. The method for preparing the expansion culture medium for human adult epidermal stem cells according to claim 1, characterized in that, Includes the following steps: (1) Fibroblast growth factor, interleukin-6, insulin-like growth factor-1, epidermal growth factor, alanine, asparagine, glutamic acid, glycine, proline, serine, arginine, N-acetyl-L-cysteine, and calcium chloride were dissolved in calcium-free DMEM / F12 to prepare a stock solution. Human insulin was dissolved in 0.01M HCl to prepare a stock solution; Hydrocortisone, Y-27632, A-83-01, Forsklin, linoleic acid, and linolenic acid were dissolved in DMSO to prepare a mother liquor; (2) Add each mother liquor from step (1) to calcium-free DMEM / F12 according to the final concentration of the culture medium.

4. The preparation method according to claim 3, characterized in that, The method for preparing the mother liquor in step (1) is as follows: Fibroblast growth factor was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 50ug / mL; Interleukin-6 was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 10 μg / mL; Insulin-like growth factor-1 was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 100 ug / mL; Growth hormone was prepared into a stock solution of 100 ug / mL in DMEM / F12 without calcium ions; Epidermal growth factor was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 50ug / mL; Alanine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 8.9 mg / mL; Asparagine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 13.3 mg / mL; Glutamic acid was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 14.7 mg / mL; Glycine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 7.5 mg / mL; Proline was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 11.5 mg / mL; Serine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 10.5 mg / mL; Arginine was dissolved in calcium-free DMEM / F12 to prepare a stock solution of 21 mg / mL; β-mercaptoethanol was dissolved in calcium-free DMEM / F12 to prepare a 50 mM stock solution; N-acetyl-L-cysteine ​​was dissolved in calcium-free DMEM / F12 to prepare a 100mM stock solution; Calcium chloride was dissolved in DMEM / F12, a solution free of calcium ions, to prepare a 100 mM stock solution; Human insulin was dissolved in 0.01M HCl overnight to prepare a stock solution of 10 mg / ml; Hydrocortisone was dissolved in DMSO to prepare a stock solution of 1 mg / mL; Y-27632 was dissolved in DMSO to prepare a 100mM stock solution; A-83-01 was dissolved in DMSO to prepare a 10mM stock solution; Forsklin was dissolved in DMSO to prepare a stock solution of 1 mg / mL; PY60 was dissolved in DMSO to prepare a 100 mM stock solution; CHIR99021 was dissolved in DMSO to prepare a 10 mM stock solution; Linoleic acid was dissolved in DMSO to prepare a stock solution of 5 mg / mL; Linolenic acid was dissolved in DMSO to prepare a stock solution of 5 mg / mL.

5. A method for culturing human adult epidermal stem cells, characterized in that, Includes the following steps: 1) Human primary epidermal stem cells were cultured in a culture dish coated with rat tail collagen to allow human adult epidermal stem cells to adhere to the dish. 2) Collect adherent human adult epidermal stem cells and culture them in the expansion medium described in claim 1 or 2 to obtain human adult epidermal stem cells.

6. The cultivation method according to claim 5, characterized in that, In step 2), half of the medium is replaced every other day during the culture process.

7. The use of the amplification culture medium according to claim 1 or 2 in the preparation of amplified epidermal stem cell products.

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