Induced differentiation method of sebaceous gland cells

Abstract

The invention discloses an induced differentiation method of sebaceous gland cells, which comprises the following specific steps: adding epidermal stem cells into an induction culture medium, and carrying out induced culture for 5-7 days to obtain the sebaceous gland cells, wherein the adopted induction culture medium is not added with insulin and specifically comprises the following components: 50% of a keratinocyte culture medium, 50% of a sebaceous gland cell culture medium, 1% of antibiotics, 20-40 ng / mL of EGF, 10-40 ng / mL of KGF and 5-20 ng / mL of rosiglitazone. Growth factors EGF and KGF and rosiglitazone are simultaneously added into the induction culture medium, insulin is completely not added, normal division and proliferation of cells are guaranteed, and sebaceous gland cells which are good in growth state and high in differentiation degree are obtained in a short culture period; compared with SZ95, the expression quantities of various sebaceous gland cell markers are similar.

Description

Technical Field

[0001] This invention relates to the field of cell differentiation technology, and in particular to a method for inducing the differentiation of sebaceous gland cells. Background Technology

[0002] The skin is the largest organ in the human body, and sebaceous glands (SGs) are important skin appendages. Their main functions include regulating lipid production on the skin surface, secreting sebum to moisturize the skin, and forming a lipid film with hair and sweat to protect the skin. Therefore, sebaceous glands play a crucial role in maintaining skin homeostasis. Abnormal sebaceous gland function can cause various skin problems, such as acne, seborrheic dermatitis, and abnormal sebum secretion. To elucidate the mechanisms by which these skin problems are caused by sebaceous gland abnormalities and to develop appropriate skincare products, it is necessary to utilize sebaceous gland cells cultured in vitro.

[0003] Currently, human sebaceous gland cell lines available for experimental use mainly come from the following sources: First, sebaceous gland cells isolated from human tissues and cultured in vitro. While primary human sebaceous gland cells cultured in vitro retain their main characteristics, their growth cycle is short, only 3-6 generations can be cultured in vitro, and the cell growth state is uneven, which makes it impossible to conduct long-term large-scale cell experiments. Second, immortalized human sebaceous gland cell lines obtained through gene editing. For example, transfecting human sebaceous gland cells with the giant T antigen DNA of SV40 yields the immortalized human sebaceous gland cell line SZ95. This cell line retains the main characteristics of normal sebaceous gland cells, such as differentiation, sebum synthesis and accumulation as cell volume increases, and the ability to specifically express sebaceous gland cell marker proteins. Third, sebaceous gland cells obtained through directed induction of skin stem cell differentiation. Sebaceous gland cells obtained through stem cell differentiation ensure that the growth state of each batch of cells is uniform and stable, and the cells can normally synthesize and excrete lipids and express sebaceous gland cell marker proteins. Among them, epidermal stem cells are more suitable for the differentiation of sebaceous gland cells due to their availability and proliferation potential in culture.

[0004] Sebaceous gland cell differentiation can be divided into four stages: epidermal stem cells, sebaceous gland progenitor cells, mature glandular cells, and fully mature cells. Different differentiation stages express different differentiation markers. Among them, MUC1, BLIMP1, and K7 are usually markers of mature sebaceous gland cells, while LRIG1 and MYC are markers of stem cell characteristics. The expression levels of MUC1, BLIMP1, and K7 are usually increased in mature sebaceous gland cells, while the expression levels of LRIG1 and MYC are decreased.

[0005] The differentiation, proliferation, and sebum synthesis and secretion of sebaceous gland cells are regulated by multiple factors. Current research has found that the PI-3K / Akt pathway, the PPAR pathway, and certain receptor-ligand pathways are related to sebaceous gland cell differentiation and sebum synthesis. Among these, IGF-1 or insulin can promote sebaceous gland cell proliferation and stimulate sebum synthesis by activating the PI-3K / Akt pathway. In current techniques, when designing culture media to induce stem cell differentiation into sebaceous gland cells, insulin is typically added to activate the PI-3K / Akt pathway and promote lipid synthesis in sebaceous gland cells. For example, patent documents CN11730522A and CN118185857A both describe the use of insulin-containing culture media to obtain sebaceous gland cells. However, the addition of insulin usually faces the following problems: (1) Because insulin is easily degraded in the culture system, insulin concentrations of tens of times the physiological concentration are usually added in actual in vitro culture operations, and the actual effective concentration of insulin in the culture medium is difficult to control. (2) When the effective concentration of insulin in the culture system is too high, it will activate cell proliferation through IGF-1R, thereby causing undifferentiated epidermal stem cells to rapidly divide and spread. The high concentration of cells will affect the differentiation of sebaceous gland cells. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a method for inducing the differentiation of sebaceous gland cells.

[0007] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides an induction culture medium for differentiating epidermal stem cells (ESCs) into sebaceous gland cells, wherein the induction culture medium does not contain insulin and specifically comprises the following components: 50% keratinocyte culture medium, 50% sebaceous gland cell culture medium, 1% antibiotic, 20-40 ng / mL EGF, 10-40 ng / mL KGF, and 5-20 ng / mL rosiglitazone; The keratinocyte culture medium comprises the following components: EPLife medium, 1% HKGS, and 1% antibiotics; The sebaceous gland cell culture medium comprises the following components: Sebomed medium, 10% FBS, 10 ng / mL LEGF, 3 ng / mL KGF, and 10 -9 M cholera toxin, 1% antibiotic, 0.4 μg / mL hydrocortisone; The 1% HKGS comprises the following components: 0.2% BPE, 0.01 μg / mL recombinant human insulin-like growth factor, 0.18 μg / mL hydrocortisone, 5 μg / mL bovine transferrin, and 0.2 ng / mL EGF.

[0008] As a preferred embodiment, the induction culture medium specifically comprises the following components: 50% keratinocyte culture medium, 50% sebaceous gland cell culture medium, 1% antibiotic, 20-40 ng / mL EGF, 20-40 ng / mL KGF, and 20 ng / mL rosiglitazone.

[0009] As a preferred embodiment, the induction culture medium specifically comprises the following components: 50% keratinocyte culture medium, 50% sebaceous gland cell culture medium, 1% antibiotic, 30 ng / mL EGF, 30 ng / mL KGF, and 20 ng / mL rosiglitazone.

[0010] Secondly, the present invention provides a method for inducing differentiation of sebaceous gland cells, comprising the following steps: After adjusting the density of epidermal stem cells, they are seeded into well plates. The next day, the epidermal stem cells are replaced with the induction culture medium described in any one of claims 1-3 and induced for 5-7 days to obtain sebaceous gland cells.

[0011] As a preferred embodiment, the epidermal stem cells are adjusted for cell density using a keratinocyte culture medium; the keratinocyte culture medium comprises the following components: EPLife medium, 1% HKGS, and 1% antibiotics; The 1% HKGS comprises the following components: 0.2% BPE, 0.01 μg / mL recombinant human insulin-like growth factor, 0.18 μg / mL hydrocortisone, 5 μg / mL bovine transferrin, and 0.2 ng / mL EGF.

[0012] As a preferred embodiment, the seeded cell density is 15,000-25,000 / well.

[0013] As a preferred embodiment, the induction culture is carried out at 37°C and a volume concentration of 5% CO2.

[0014] As a preferred embodiment, the induction culture time is 6 days.

[0015] As a preferred embodiment, the method for obtaining the epidermal stem cells includes the following steps: A1. After cleaning the isolated foreskin tissue, remove the subcutaneous tissue and blood vessels, cut the skin tissue into pieces, and digest it with thermophilic protease to obtain the epidermis; A2. After the epidermis is allowed to stand, trypsin is added for digestion, centrifugation is performed, and the cell suspension is prepared and then transferred to a pre-coated T175 culture flask and allowed to stand. After the culture flask with the cell suspension attached is washed, it is added to keratinocyte culture medium for culture. A3. When the epidermal stem cells reach a confluence of 80%-90%, perform passage and expansion culture.

[0016] As a preferred embodiment, the amplification culture time is 20-28 h; more preferably, the amplification culture time is 24 h.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention obtains sebaceous gland cells with good growth status and high degree of differentiation in a shorter culture period by simultaneously adding growth factors EGF and KGF and rosiglitazone to the induction culture medium without adding insulin, while ensuring normal cell division and proliferation. The expression levels of various sebaceous gland cell markers are similar to those of SZ95 after induction.

[0018] 2. This invention further optimizes the content of EGF, KGF and rosiglitazone in the induction culture medium, resulting in sebaceous gland cells with the best growth status and differentiation degree after induction, which are closest to the expression levels of various sebaceous gland cell markers of SZ95.

[0019] 3. The induction culture medium used in this invention does not contain added insulin, thereby reducing culture costs and waste liquid pollution. Attached Figure Description

[0020] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 Microscopic (100x) images of sebaceous gland cells obtained by the induction differentiation method in Examples 2-11; Figure 2 Microscopic (100x) images of sebaceous gland cells obtained by the induction differentiation methods of Comparative Examples 1, 2, 6, and 7; Figure 3 The expression levels of MUC1 mRNA in sebaceous gland cells obtained by the induction differentiation methods of Examples 2-11 and Comparative Examples 1-2 and 6 are shown. Figure 4 The KRT7 mRNA expression levels of sebaceous gland cells obtained by the induction differentiation methods of Examples 2-11 and Comparative Examples 1-2 and 6 are shown. Figure 5 The expression levels of LRIG1 mRNA in sebaceous gland cells obtained by the induction differentiation methods of Examples 2-11 and Comparative Examples 1-2 and 6 are shown. Figure 6 The MYC mRNA expression levels of sebaceous gland cells obtained by the induction differentiation methods of Examples 2-11 and Comparative Examples 1-2 and 6 are shown. Detailed Implementation

[0021] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0022] All raw materials and reagents used in the experiments in this invention are provided by our company and / or purchased from the market.

[0023] Example 1 This embodiment provides a method for obtaining human epidermal stem cells (ESCs), the specific steps of which are as follows: 1. Preparation and culture of human epidermal stem cells (ESCs) Discarded foreskin tissue was obtained from Jinshan Hospital in Shanghai after surgery. The tissue was washed with 10 mL of 75% ethanol for 1 min, followed by 2-3 washes with 10 mL of PBS to remove subcutaneous tissue and blood vessels. Blood was gently scraped from the inner surface of the skin. The skin tissue was then cut into strips approximately 2 mm wide and less than 3 cm long in a culture dish and placed in 14 mL of 0.5 mg / mL thermophilic protease (purchased from Yuanye, S10236) solution. Digestion was carried out overnight at 4 ℃. The next day, under sterile conditions, the digested dermis and epidermis were separated using forceps. T175 culture flasks were pre-coated with 10 mL of 20 ug / mL Col IV (purchased from Sigma, C5533) at 37 ℃ for 1 h, washed twice with PBS, and then used for further processing. The isolated epidermis was placed in 10 mL PBS and allowed to stand for 5 min. The epidermis was then placed in 0.05% trypsin (purchased from Gibco, 25300062), pipetted and aspirated for 2 min, then incubated in a 37 ℃ water bath for 2 min. This process was repeated, with the digestion time not exceeding 16 min. The amount of trypsin used was generally 10–20 mL. 20 mL of complete culture medium (purchased from Yuanpei Biotechnology, L110KJ) was added to terminate the enzyme reaction. The cells were passed through a cell sieve (70 μm pore size) and centrifuged at 1000 rpm for 10 min. The culture medium was discarded, and another 20 mL of complete culture medium was added and mixed thoroughly. The mixture was then transferred to a pre-coated T175 culture flask and allowed to stand for 10 min. The unattached cell suspension was then aspirated, and the cells were washed twice with PBS. Finally, 25 mL of complete culture medium was added for further culture.

[0024] 2. Expansion culture of ESC cells After separation and culture in step 1, ESC cells can be expanded when their confluence reaches 80%-90%. The specific steps are as follows: discard the old culture medium, wash twice with PBS, digest with 0.05% trypsin (purchased from Gibco, 25300062) for 5 min, add complete culture medium containing 10% serum (purchased from Yuanpei Biotechnology, L110KJ) to stop digestion, pipette the cells from the culture flask to make a single-cell suspension, and evenly seed the cells into a new culture plate for expansion culture for 24 h to obtain ESC cells for use.

[0025] Example 2 This embodiment provides a method for inducing differentiation of sebaceous gland cells (ESC-1), the specific steps of which are as follows: 1. The culture medium for inducing the differentiation of human epidermal stem cells into sebaceous gland cells consists of the following components: Keratinocyte culture medium: EPLife medium (Gibco) + 1% HKGS (Gibco) + 1% antibiotic; prepared by adding the appropriate percentage of HKGS and antibiotic to EPLife medium.

[0026] The final concentrations of the added components in the above keratinocyte culture medium after the addition of 1% HKGS were: bovine pituitary extract (BPE): 0.2% v / v; recombinant human insulin-like growth factor: 0.01 μg / mL; hydrocortisone: 0.18 μg / mL; bovine transferrin: 5 μg / mL; human epidermal growth factor (EGF): 0.2 ng / mL. Sebaceous gland cell culture medium: Sebomed (Merk F8205) medium + 10% FBS (Gibco) + 10 ng / mL EGF + 3 ng / mL KGF + 10 -9 M cholera toxin + 1% antibiotic + 0.4 μg / mL hydrocortisone; prepared by adding the appropriate percentage or final concentration of FBS, EGF, KGF, cholera toxin, antibiotic and hydrocortisone to Sebomed medium.

[0027] Induction medium: 50% keratinocyte culture medium + 50% sebaceous gland cell culture medium + 1% antibiotic + 20 ng / mL EGF + 5 μM rosiglitazone + 10 ng / mL KGF; prepared by adding the appropriate percentage or final concentration of antibiotics EGF, rosiglitazone and KGF to a mixed culture medium (mixed volume ratio of 50%:50%) formed by keratinocyte culture medium and sebaceous gland cell culture medium.

[0028] The above culture media contained the following: HKGS (purchased from Gibco); FBS (purchased from Gibco); DMEM (purchased from Shanghai Yuanpei Biotechnology); EGF (purchased from Gibco, PHG0311L); KGF (purchased from Gibco, 100-19-10μg); cholera toxin (purchased from WHELAB, G0508); hydrocortisone (purchased from MCE); rosiglitazone (purchased from MCE); and the antibiotic was a penicillin-streptomycin-amphotericidal B solution, purchased from Beyotime, catalog number C0224.

[0029] 2. The ESC cells obtained in Example 1 were adjusted to the desired cell density using the keratinocyte culture medium prepared in Step 1, and seeded at a density of 20,000 cells / well in 24-well plates. The next day, the ESC cells were transferred to the induction medium prepared in Step 1. The induction medium was changed every two days, and the cells were cultured continuously at 37 °C in a 5% CO2 incubator for 6 days. The gene expression levels of sebaceous gland cell markers were detected after induction. Total RNA was extracted from the cells using the Novizan RNA Extraction Kit, and the extracted total RNA was synthesized into cDNA using the Takara Reverse Transcription Kit. Intracellular sebaceous gland cell markers, including cytokeratin (KRT7), type I transmembrane mucin (MUC-1), leucine-rich repeats and immunoglobulin-like domain 1 (LRIG1), and myeloma oncogene (MYC), were detected by RT-qPCR.

[0030] Example 3 This embodiment provides a method for inducing differentiation of sebaceous gland cells (ESC-2). The specific steps are basically the same as those in Embodiment 2, except that the induction culture medium used in this embodiment consists of: 50% keratinocyte culture medium + 50% sebaceous gland cell culture medium + 1% antibiotic + 20 ng / mL EGF + 10 μM rosiglitazone + 10 ng / mL KGF.

[0031] Example 4 This embodiment provides a method for inducing differentiation of sebaceous gland cells (ESC-3). The specific steps are basically the same as those in Embodiment 2, except that the induction culture medium used in this embodiment consists of: 50% keratinocyte culture medium + 50% sebaceous gland cell culture medium + 1% antibiotic + 20 ng / mL EGF + 20 μM rosiglitazone + 10 ng / mL KGF.

[0032] Example 5 This embodiment provides a method for inducing differentiation of sebaceous gland cells (ESC-4). The specific steps are basically the same as those in Embodiment 2, except that the induction culture medium used in this embodiment consists of: 50% keratinocyte culture medium + 50% sebaceous gland cell culture medium + 1% antibiotic + 20 ng / mL EGF + 5 μM rosiglitazone + 20 ng / mL KGF.

[0033] Example 6 This embodiment provides a method for inducing differentiation of sebaceous gland cells (ESC-5). The specific steps are basically the same as those in Embodiment 2, except that the induction culture medium used in this embodiment consists of: 50% keratinocyte culture medium + 50% sebaceous gland cell culture medium + 1% antibiotic + 20 ng / mL EGF + 10 μM rosiglitazone + 20 ng / mL KGF.

[0034] Example 7 This embodiment provides a method for inducing differentiation of sebaceous gland cells (ESC-6). The specific steps are basically the same as those in Embodiment 2, except that the induction culture medium used in this embodiment consists of: 50% keratinocyte culture medium + 50% sebaceous gland cell culture medium + 1% antibiotic + 20 ng / mL EGF + 20 μM rosiglitazone + 20 ng / mL KGF.

[0035] Example 8 This embodiment provides a method for inducing differentiation of sebaceous gland cells (ESC-7). The specific steps are basically the same as those in Embodiment 2, except that the induction culture medium used in this embodiment consists of: 50% keratinocyte culture medium + 50% sebaceous gland cell culture medium + 1% antibiotic + 30 ng / mL EGF + 20 μM rosiglitazone + 20 ng / mL KGF.

[0036] Example 9 This embodiment provides a method for inducing differentiation of sebaceous gland cells (ESC-8). The specific steps are basically the same as those in Embodiment 2, except that the induction culture medium used in this embodiment consists of: 50% keratinocyte culture medium + 50% sebaceous gland cell culture medium + 1% antibiotic + 40 ng / mL EGF + 20 μM rosiglitazone + 20 ng / mL KGF.

[0037] Example 10 This embodiment provides a method for inducing differentiation of sebaceous gland cells (ESC-9). The specific steps are basically the same as those in Embodiment 2, except that the induction culture medium used in this embodiment consists of: 50% keratinocyte culture medium + 50% sebaceous gland cell culture medium + 1% antibiotic + 30 ng / mL EGF + 20 μM rosiglitazone + 30 ng / mL KGF.

[0038] Example 11 This embodiment provides a method for inducing differentiation of sebaceous gland cells (ESC-10). The specific steps are basically the same as those in Embodiment 2, except that the induction culture medium used in this embodiment consists of: 50% keratinocyte culture medium + 50% sebaceous gland cell culture medium + 1% antibiotic + 30 ng / mL EGF + 20 μM rosiglitazone + 40 ng / mL KGF.

[0039] The composition of the induction culture medium used in the induction differentiation methods of sebaceous gland cells in Examples 2-11 is summarized in Table 1.

[0040] Table 1 Comparative Example 1 This comparative example provides a method for inducing the differentiation of sebaceous gland cells (ESC-11). The specific steps are basically the same as in Example 2, except that the induction medium used in this comparative example consists of EPLife complete medium + 10 -6 M dexamethasone + 10 μg / ml insulin + 20 μM rosiglitazone + 1 uM XAV939.

[0041] Comparative Example 2 This comparative example provides a method for inducing differentiation of sebaceous gland cells (ESC-12). The specific steps are basically the same as those in Example 2, except that the induction culture medium used in this comparative example consists of: DMEM / F12 (1:1) + 2.5% FBS + 10 ng / ml insulin + 3 ng / ml EGF + 45.2 ng / ml hydrocortisone + 1 μM isoproterenol + 24 μg / ml adenine + 2 mM Glu.

[0042] Comparative Example 3 The induction differentiation method of sebaceous gland cells (ESC-13) in this comparative example is basically the same as that in Example 2, except that the induction culture medium used in this comparative example consists of 50% keratinocyte culture medium + 50% sebaceous gland cell culture medium + 1% antibiotic + 5 μM rosiglitazone.

[0043] Comparative Example 4 The induction differentiation method of sebaceous gland cells (ESC-14) in this comparative example is basically the same as that in Example 2, except that the induction culture medium used in this comparative example consists of 50% keratinocyte culture medium + 50% sebaceous gland cell culture medium + 1% antibiotic + 20 ng / mL EGF + 5 μM rosiglitazone.

[0044] Comparative Example 5 The induction differentiation method of sebaceous gland cells (ESC-15) in this comparative example is basically the same as that in Example 2, except that the induction culture medium used in this comparative example consists of 50% keratinocyte culture medium + 50% sebaceous gland cell culture medium + 1% antibiotic + 5 μM rosiglitazone + 10 ng / mL KGF.

[0045] Comparative Example 6 The induction differentiation method of sebaceous gland cells (ESC-16) in this comparative example is basically the same as that in Example 2, except that the induction culture medium used in this comparative example consists of: 100% keratinocyte culture medium + 1% antibiotic + 20 ng / mL EGF + 5 μM rosiglitazone + 10 ng / mL KGF.

[0046] Comparative Example 7 The induction differentiation method of sebaceous gland cells (ESC-17) in this comparative example is basically the same as that in Example 2, except that the induction culture medium used in this comparative example consists of: 100% sebaceous gland cell culture medium + 1% antibiotic + 20 ng / mL EGF + 5 μM rosiglitazone + 10 ng / mL KGF.

[0047] Microscopic images of the sebaceous gland cells and SZ95 obtained by the induction differentiation methods in Examples 2-11 above are shown below. Figure 1 As shown, photographs of sebaceous gland cells obtained by the induction differentiation methods in Comparative Examples 1-2 are as follows. Figure 2 As shown. Figure 1 The photographs show cells obtained by the induced differentiation method in Examples 2-11, with significantly enlarged nuclei, polygonal cell morphology, a tendency for cell clustering, and smaller intercellular spaces; while Figure 2 The photos show that the cells induced by Comparative Examples 1, 2, 6, and 7 have no change in nuclear volume and are spindle-shaped. The cells induced by Comparative Examples 1, 2, and 7 cover the container and do not show any tendency to grow in clusters. In contrast, the cells induced by Comparative Example 6 have obvious intercellular gaps and do not show any tendency to grow in clusters.

[0048] The qPCR detection results of sebaceous gland cells obtained by the induction differentiation methods of Examples 2-11 and Comparative Examples 1-2 and 6 are as follows: Figures 3 to 6 As shown. Figure 3 MUC1 mRNA expression level, Figure 4 This refers to the expression level of KRT7 (i.e., K7) mRNA. Figure 5 This refers to the LRIG1 mRNA expression level. Figure 6 The values ​​represent MYC mRNA expression levels. In each figure, SZ95 represents the publicly reported human immortalized sebaceous gland cell line.

[0049] The results above show that the expression levels of key genes MUC1 and KRT7 in ESCs significantly increased after induction with the induction medium of this invention, and this increase was dose-dependent on the concentrations of rosiglitazone and KGF. The key to the significant increase in MUC1 expression was the addition of KGF to the induction medium; simply increasing the concentration of rosiglitazone did not induce an increase in MUC1 gene expression. The expression of epidermal stem cell markers LRIG1 and MYC was downregulated after ESC induction, indicating that epidermal stem cells are differentiating into sebaceous gland cells. In particular, the expression levels of various sebaceous gland cell markers were most similar to those of SZ95 in ESC-7, ESC-8, and ESC-9 obtained using the methods in Examples 8-10. Further comparison of the expression levels of various sebaceous gland cell markers at ESC-6, ESC-7, and ESC-8 showed that the expression levels of various sebaceous gland cell markers at ESC-7 were most similar to those at SZ95. Comparison of the expression levels of various sebaceous gland cell markers at ESC-7, ESC-9, and ESC-10 showed that the expression levels of various sebaceous gland cell markers at ESC-9 were most similar to those at SZ95. This indicates that adding 30 ng / mL EGF + 30 ng / mL KGF + 20 μM EGF-rosiglitazone is the optimal combination.

[0050] ESCs induced with the induction media of Comparative Examples 1 and 2 also showed increased expression of key genes MUC1 and KRT7, and downregulated expression of epidermal stem cell markers LRIG1 and MYC, but the effect was significantly worse than that of the induction media of this invention.

[0051] After induction with the induction medium of Comparative Examples 3-4, ESCs, without the addition of KGF, did not differentiate into sebaceous gland cells. Instead, they grew into keratinocytes, similar to Comparative Example 1, and the expression levels of key genes MUC1 and KRT7 were lower than in Example 2. After induction with the induction medium of Comparative Example 5, without the addition of EGF, the induced cells showed a tendency to differentiate into sebaceous gland cells, but the intercellular spaces were obvious and the expression levels of key genes MUC1 and KRT7 were also lower.

[0052] This invention has many specific applications, and the above description is only a preferred embodiment. It should be noted that the above embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention. For those skilled in the art, several improvements can be made without departing from the principle of this invention, and these improvements should also be considered within the scope of protection of this invention.

Claims

1. A culture medium for inducing the differentiation of epidermal stem cells into sebaceous gland cells, characterized in that, The induction culture medium does not contain insulin and specifically includes the following components: 50% keratinocyte culture medium, 50% sebaceous gland cell culture medium, 1% antibiotic, 20-40 ng / mL EGF, 10-40 ng / mL KGF, and 5-20 ng / mL rosiglitazone; The keratinocyte culture medium comprises the following components: EPLife medium, 1% HKGS, and 1% antibiotics; The sebaceous gland cell culture medium comprises the following components: Sebomed medium, 10% FBS, 10 ng / mL EGF, 3 ng / mL KGF, and 10 -9 M cholera toxin, 1% antibiotic, 0.4 μg / mL hydrocortisone; The 1% HKGS comprises the following components: 0.2% BPE, 0.01 μg / mL recombinant human insulin-like growth factor, 0.18 μg / mL hydrocortisone, 5 μg / mL bovine transferrin, and 0.2 ng / mL EGF.

2. The induction culture medium for differentiating epidermal stem cells into sebaceous gland cells according to claim 1, characterized in that, The induction culture medium specifically includes the following components: 50% keratinocyte culture medium, 50% sebaceous gland cell culture medium, 1% antibiotic, 20-40 ng / mL EGF, 20-40 ng / mL KGF, and 20 ng / mL rosiglitazone.

3. The induction culture medium for differentiating epidermal stem cells into sebaceous gland cells according to claim 2, characterized in that, The induction culture medium specifically comprises the following components: 50% keratinocyte culture medium, 50% sebaceous gland cell culture medium, 1% antibiotic, 30 ng / mL EGF, 30 ng / mL KGF, and 20 ng / mL rosiglitazone.

4. A method for inducing differentiation of sebaceous gland cells, characterized in that, Includes the following steps: After adjusting the cell density of epidermal stem cells, they are seeded into well plates. The next day, the epidermal stem cells are added to the induction culture medium described in any one of claims 1-3 and induced for 5-7 days to obtain sebaceous gland cells.

5. The method for inducing differentiation of sebaceous gland cells according to claim 4, characterized in that, The epidermal stem cells were adjusted for cell density using a keratinocyte culture medium; the keratinocyte culture medium contained the following components: EPLife medium, 1% HKGS, and 1% antibiotics; The 1% HKGS comprises the following components: 0.2% BPE, 0.01 μg / mL recombinant human insulin-like growth factor, 0.18 μg / mL hydrocortisone, 5 μg / mL bovine transferrin, and 0.2 ng / mL EGF.

6. The method for inducing differentiation of sebaceous gland cells according to claim 4, characterized in that, The cell density for seeding is 15,000-25,000 per well.

7. The method for inducing differentiation of sebaceous gland cells according to claim 4, characterized in that, The induction culture was conducted at 37°C and a volume concentration of 5% CO2.

8. The method for inducing differentiation of sebaceous gland cells according to claim 4, characterized in that, The induction culture time is 6 days.

9. The method for inducing differentiation of sebaceous gland cells according to claim 4, characterized in that, The method for obtaining the epidermal stem cells includes the following steps: A1. After cleaning the isolated foreskin tissue, remove the subcutaneous tissue and blood vessels, cut the skin tissue into pieces, and digest it with thermophilic protease to obtain the epidermis; A2. After the epidermis is allowed to stand, trypsin is added for digestion, centrifugation is performed, and the cell suspension is prepared and then transferred to a pre-coated T175 culture flask and allowed to stand. After the culture flask with the cell suspension attached is washed, it is added to complete culture medium for culture. A3. When the epidermal stem cells reach a confluence of 80%-90%, proceed with expansion culture.

10. The method for inducing differentiation of sebaceous gland cells according to claim 9, characterized in that, The amplification culture time is 20–28 h.

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