Human epithelial stem cell 3D-hEpSC derived from human pluripotent stem cell 3D differentiation as well as culture medium and application of human epithelial stem cell 3D-hEpSC

Abstract

The invention discloses a human epithelial stem cell 3D-hEpSC derived from human pluripotent stem cell 3D differentiation as well as a culture medium and application thereof, and belongs to the field of biological medicine, the preservation number of the human epithelial stem cell 3D-hEpSC is CGMCC NO.46003, expression markers of the human epithelial stem cell 3D-hEpSC comprise keratin 15, keratin 18, transcription factors p63 and ITGA6, and keratin 14, OCT4 and NANOG are not expressed or extremely low expressed. The new human epithelial stem cell 3D-hEpSC different from HEK and traditional hair follicle-derived hEpSC is obtained, the cell cluster (EB ball or EB for short) obtained by the method can be efficiently differentiated and is not easy to break or become poor in adherence, and the differentiation stability is greatly improved. And the differentiation strategy is rapid, stable and efficient, and can also be mixed with DPC cells for transplantation, so that nude mice successfully grow a large amount of hair, and the method has huge clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to human epithelial stem cells 3D-hEpSC derived from 3D differentiation of human pluripotent stem cells, and a culture medium and application thereof. Background Art

[0002] Hair follicle morphogenesis and regeneration rely on the close cooperation of epithelial (epithelial stem cells [EpSC]) and hair-inducing mesenchymal (dermal papilla cells [DPC]) components, also known as epithelial-mesenchymal interaction (EMI). EMI is a prerequisite for functional hair follicle formation, regeneration, and cycling and serves as the theoretical basis for tissue engineering for hair follicle regeneration.

[0003] Current strategies for regenerating hair follicles in vivo aim to mimic EMI, mainly by combining epithelial (epithelial stem cells [EpSC] or human epidermal keratinocytes (HEK)) and mesenchymal (dermal papilla cells [DPC] or skin-derived precursors (SKP)) cells to induce hair follicle regeneration.

[0004] Currently, human epidermal keratinocytes HEK, dermal hair papilla cells DPC, skin-derived precursor cells SKP and other cells can be obtained in vitro. Studies have reported that transplanting a mixture of dermal cells and epithelial cells can regenerate functional hair follicles, but mice that only receive dermal cells (such as DPC) have no visible hair growth (DPC may be able to induce hair follicle structure, but there is no visible hair growth, see Figure 8 ), indicating that although dermal cells (such as DPCs) cannot induce detectable hair growth when injected alone, when dermal cells (such as DPCs) are mixed with epithelial cells (such as HEKs), they help hair to grow out, which is of great significance for studying hair regeneration.

[0005] However, research on hair regeneration still faces many challenges. For example, there are two main sources of human epithelial cells. One is primary isolation from human skin or hair follicle tissue (such as primary isolated HEK cells), but the source is limited and difficult to proliferate; the other is differentiation using pluripotent stem cells, which can solve the problem of cell source. There are two pathways for pluripotent stem cell differentiation. One is the 2D differentiation pathway of monolayer cell attachment culture, which has the advantages of simple operation, low cost, easy observation and large-scale production; the other is the 3D differentiation pathway of first forming cell clusters (embryoid bodies or EB balls) and then adherent differentiation. Although 3D differentiation technology is relatively complex, with high turnover and great difficulty in observation and operation, it can better simulate the three-dimensional environment in the body and its functions are closer to those of in vivo cells, thus having greater research value.

[0006] However, there are still great challenges in the current 3D differentiation, including:

[0007] (a) Currently, there are many methods and culture media that can make pluripotent stem cells form cell clusters (embryoid bodies or EB spheres). For example, self-aggregation, hanging drop method and AggreWell method can form EB spheres. However, not all methods are conducive to the differentiation of hPSC into hEpSC. For example, after the formation of EB spheres, they are prone to rupture and poor adhesion during differentiation. Or after the formation of EB spheres, the early stage of adhesion and differentiation is normal, but in the later stage of differentiation, they are prone to cracking and poor adhesion.

[0008] (b) Existing 3D differentiation culture systems take too long (generally 30-50 days) and have unstable differentiation efficiency (continuous subculture and purification are required to obtain human epithelial cells), which limits their clinical application. Summary of the invention

[0009] Purpose of the Invention

[0010] In order to overcome the above-mentioned shortcomings, the purpose of the present invention is to provide a human epithelial stem cell 3D-hEpSC derived from 3D differentiation of human pluripotent stem cells, and a culture medium and application thereof. In the process of studying the 3D differentiation of human pluripotent stem cells (hPSC) to obtain HEK cells, the present invention unexpectedly obtained a new human epithelial stem cell 3D-hEpSC that is different from HEK and traditional hair follicle-derived hEpSC. The differentiated human epithelial stem cell 3D-hEpSC highly expresses epithelial stem cell markers cytokeratin 5 (KRT15), keratin 18 (K18), p63 and ITGA6 (integrin subunit α6) at the mRNA level, but does not express or lowly expresses HEK-specific marker keratin 14 (K14) (different from HEK) and hPSC-related markers OCT4 and NANOG (high safety, no potential tumorigenicity), and also extremely low expression of CD200 (different from traditional hair follicle-derived hEpSC), and the relative expression of ITGA6 is also much lower than that of traditional hair follicle-derived hEpSC. The present invention can rapidly, stably and efficiently differentiate human epithelial stem cells 3D-hEpSC through human pluripotent stem cells (hPSC), and the differentiation time is greatly shortened (differentiation time only takes 14 to 20 days). The cell clusters (EB balls) obtained by the present invention can be efficiently differentiated, not easy to break or adhere to the wall, and the differentiation stability is greatly improved. In addition, the human epithelial stem cells 3D-hEpSC of the present invention are obtained from human pluripotent stem cells (hPSC), which are easy to obtain. When the human epithelial stem cells 3D-hEpSC obtained by the present invention are mixed with DPC cells for transplantation, they can successfully make nude mice grow a lot of hair, and have great clinical application value.

[0011] Solution

[0012] To achieve the purpose of the present invention, the technical solution adopted by the present invention is as follows:

[0013] In the first aspect, the present invention provides a human epithelial stem cell 3D-hEpSC derived from 3D differentiation of human pluripotent stem cells, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 20, 2024, with the deposit number CGMCC NO.46003.

[0014] Furthermore, compared with the H9 cell line in hPSC cells (human pluripotent stem cells, abbreviated as hPSC), human epithelial stem cells 3D-hEpSC highly expressed markers cytokeratin 15, keratin 18, transcription factors p63 and ITGA6, did not express or very low expression of NANOG, OCT4, and very low expression of CD200;

[0015] Human epithelial stem cells 3D-hEpSCs do not express or express very low levels of keratin 14 compared to primary HEK cells.

[0016] Furthermore, the expression of keratin 18 at the mRNA level in human epithelial stem cells 3D-hEpSC was more than 8 times that of the H9 cell line.

[0017] Furthermore, the expression level of ITGA6 at the mRNA level in human epithelial stem cells 3D-hEpSC is 5 to 8 times, optionally 5 to 6 times, that of the H9 cell line.

[0018] Furthermore, the expression of keratin 15 at the mRNA level in human epithelial stem cells 3D-hEpSC was more than 40 times that of the H9 cell line.

[0019] Furthermore, the expression of transcription factor p63 at the mRNA level in human epithelial stem cells 3D-hEpSC is more than 1000 or 1200 times that of the H9 cell line.

[0020] Furthermore, the expression levels of NANOG and OCT4 in human epithelial stem cells 3D-hEpSC at the mRNA level were close to 0 relative to those in the H9 cell line.

[0021] Furthermore, the expression level of CD200 in human epithelial stem cells 3D-hEpSC at the mRNA level was 1 / 5 or lower than that of the H9 cell line.

[0022] In a second aspect, the present invention provides a differentiation medium for differentiating to obtain the human epithelial stem cells 3D-hEpSCs described in the first aspect, comprising differentiation medium 1 or differentiation medium 2, or a combination of differentiation medium 1 and differentiation medium 2;

[0023] Wherein, differentiation medium 1 comprises: DMEM medium, and contains: 20% serum replacement, 1×NEAA, 1×GlutaMAX, 0.055mM β-mercaptoethanol, 1×penicillin-streptomycin double antibody solution, 1-10μM retinoic acid, 10-50μM bone morphogenetic protein 4 and 10μM ROCK inhibitor;

[0024] Differentiation medium 2 includes: DKSFM medium, and contains 1-10 μM retinoic acid, 10-50 μM bone morphogenetic protein 4 (BMP4) and 10 μM ROCK inhibitor, and optionally the ROCK inhibitor is Y27632.

[0025] Further, differentiation medium 1 includes DMEM medium and contains: 20% serum replacement, 1×NEAA, 1×GlutaMAX, 0.055mM β-mercaptoethanol, 1×penicillin-streptomycin double antibody solution, 1μM retinoic acid (RA), 25μM bone morphogenetic protein 4 (BMP4) and 10μM ROCK inhibitor, optionally the ROCK inhibitor is Y27632.

[0026] Further optionally, the differentiation medium 2 comprises: DKSFM medium, and contains 1 μM retinoic acid (RA), 25 μM bone morphogenetic protein 4 (BMP4), and 10 μM Y27632.

[0027] Further optionally, in the differentiation medium 1, the serum replacement is KnockOut-Serum Replacement.

[0028] Further optionally, in the differentiation medium 1, the DMEM medium is KnockOut DMEM.

[0029] Further optionally, in the differentiation medium 1, 1×NEAA is obtained by diluting 100×MEM non-essential amino acid solution into DMEM medium.

[0030] In a third aspect, a differentiation method for obtaining the human epithelial stem cells 3D-hEpSCs described in the first aspect is provided, comprising the following steps:

[0031] 1) Expand and culture hPSC cells into clones and disperse the cells into single cells;

[0032] 2) 3D culture the single cell of step 1) to form a cell cluster using EB formation medium; optionally, the EB formation medium includes: DMEM medium, and contains: 20% serum replacement, 1xNEAA, 1xGlutaMAX, 0.055mMβ-mercaptoethanol, 1xpenicillin-streptomycin double antibody solution, 1-10μM retinoic acid and 10-50μM bone morphogenetic protein 4 and 100μM ROCK inhibitor; optionally, the ROCK inhibitor is Y27632;

[0033] 3) Differentiating the cell cluster obtained in step 2); using the differentiation medium described in the second aspect for differentiation culture, wherein first using differentiation medium 1 for adaptive differentiation for 1 to 2 days, then using differentiation medium 2 for differentiation culture for 3 to 5 days, and then transferring to D-KSFM for culture for 4 to 6 days to obtain differentiated human epithelial stem cells 3D-hEpSC.

[0034] Furthermore, in step 3), the differentiation medium 2 is used and the culture medium is replaced once every 1 to 2 days during the differentiation culture process;

[0035] And / or, during the D-KSFM culture process, the culture medium is replaced every 20 to 28 hours, and the replacement time is optionally 24 hours.

[0036] Furthermore, in step 1), the culture plate is coated with Vitronectin.

[0037] Furthermore, in step 1), the hPSC culture medium is used for expanded culture.

[0038] Furthermore, in step 1), when the cells are passaged until the clone edges are clear, the nuclear-cytoplasmic ratio is high, the morphology is uniform, the cells in the clone are in close contact, and the confluence is about 60%, the clones are dispersed and resuspended into a cell suspension for cell cluster culture.

[0039] Furthermore, in step 2), the cell cluster formation method includes at least one of a self-aggregation method, a hanging drop method, and an AggreWell method.

[0040] Furthermore, in step 2), the cell culture dish is not moved during the cell aggregation process.

[0041] Further, in step 2), after the cell clusters are formed, they are transferred to a low-adsorption culture dish; optionally, after the cell clusters are formed, they are cultured for 1 to 2 days using EB formation medium; optionally, after continued culture, the cell clusters are inoculated into a collagen IV-coated well plate, and the EB formation medium and 2% FBS described in step 2) are added and cultured overnight for differentiation.

[0042] In a fourth aspect, the present invention provides a cell composition for hair regeneration, comprising human dermal papilla cells (DPCs) and the human epithelial stem cells 3D-hEpSCs described in the first aspect or the human epithelial stem cells 3D-hEpSCs obtained by the differentiation method described in the second aspect;

[0043] Optionally, the number ratio of DPC cells to human epithelial stem cells is 1:(1-3), optionally 1:(2-3).

[0044] In a fifth aspect, the present invention provides a use of the human epithelial stem cells 3D-hEpSC described in the first aspect, the human epithelial stem cells 3D-hEpSC obtained by the differentiation method described in the fourth aspect, or the cell composition described in the fifth aspect in the preparation of hair regeneration products.

[0045] On the other hand, the present invention provides a method for hair regeneration using the human epithelial stem cell 3D-hEpSC described in the first aspect, the human epithelial stem cell 3D-hEpSC obtained by the differentiation method described in the third aspect, or the cell composition described in the fourth aspect, wherein a preventive or therapeutically effective amount of the cell composition containing human epithelial stem cells 3D-hEpSC and DPC cells is administered to a subject in need.

[0046] Beneficial Effects

[0047] In the process of studying the 3D differentiation of human pluripotent stem cells (hPSC) to obtain HEK cells, the present invention unexpectedly obtained a new human epithelial stem cell 3D-hEpSC that is different from HEK and traditional hair follicle-derived hEpSC. The differentiated human epithelial stem cell 3D-hEpSC highly expresses epithelial stem cell markers cytokeratin 5 (KRT15), keratin 18 (K18), p63 and ITGA6 at the mRNA level, but does not express or low-expresses HEK-specific marker keratin 14 (K14) (different from HEK) and hPSC-related markers OCT4 and NANOG (high safety, no potential tumorigenicity), and also extremely low-expresses CD200 (different from traditional hair follicle-derived hEpSC), and the relative expression of ITGA6 is also much lower than that of traditional hair follicle-derived hEpSC. The present invention can rapidly, stably and efficiently differentiate human epithelial stem cells 3D-hEpSC through human pluripotent stem cells (hPSC), and the differentiation time is greatly shortened (differentiation time only takes 14 to 20 days). The cell clusters (EB balls) obtained by the present invention can be efficiently differentiated, not easy to break or adhere to the wall, and the differentiation stability is greatly improved. In addition, the human epithelial stem cells 3D-hEpSC of the present invention are obtained from human pluripotent stem cells (hPSC), which are easy to obtain. When the human epithelial stem cells 3D-hEpSC obtained by the present invention are mixed with DPC cells for transplantation, they can successfully make nude mice grow a lot of hair, and have great clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0049] Figure 1 This is a 3D cell differentiation flow chart of Example 1 of the present invention and typical cell morphologies at each stage.

[0050] Figure 2 These are cell morphology diagrams of Experimental Example 1 of the present invention using different differentiation mediums on different days, wherein 1 μM RA+50 ng / ml BMP4 refers to cells cultured using the differentiation medium of Example 2; 10 μM RA+10 ng / ml BMP4 refers to cells cultured using the differentiation medium of Example 3.

[0051] Figure 3 The cell morphology of cell clusters (EBs) formed by different EB formation mediums in Test Example 2 of the present invention at different days in the sphere formation or differentiation process, wherein EB formation medium 1 represents EB formation medium 1 of Comparative Example 1, EB formation medium 2 represents EB formation medium 2 of Comparative Example 2, and EB formation medium 3 represents EB formation medium 3 of Example 1.

[0052] Figure 4 These are cell morphology diagrams of cell clusters (EBs) obtained by self-aggregation, hanging drop method, and AggreWell method during the differentiation process of different days in Test Example 2 of the present invention.

[0053] Figure 5 It is the relative gene expression level of the markers (OCT4, NANOG, CD200) of the human epithelial stem cell 3D-hEpSC at the mRNA expression level in Test Example 3 of the present invention (relative to the expression levels of OCT4, NANOG, CD200 in hPSC (H9 cell line) respectively).

[0054] Figure 6 The relative gene expression levels of markers KRT15, ​​K18, p63 and ITGA6 in the differentiation process of Example 1 in Test Example 3 of the present invention (relative to the expression levels of KRT15, ​​K18, p63 and ITGA6 in the H9 cell line, respectively);

[0055] Figure 7It is the relative expression level of the gene of K14, a marker of human epithelial stem cells 3D-hEpSC and primary HEK cells in Test Example 3 of the present invention (the expression level of primary HEK cells relative to human epithelial stem cells 3D-hEpSC).

[0056] Figure 8 This is the result of identification of the germinal function of co-transplantation of epithelial stem cells and DP cells in Test Example 4 of the present invention. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0058] In addition, in order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In some embodiments, raw materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail, so as to highlight the main purpose of the present invention.

[0059] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising”, etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0060] The human epithelial stem cells 3D-hEpSC derived from the 3D differentiation of human pluripotent stem cells of the present invention are obtained by 3D induced differentiation of hPSC cells, with a preservation date of June 20, 2024; the preservation unit is the General Microbiology Center of China Microbiological Culture Collection Administration; the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China; the preservation number is CGMCC NO.46003.

[0061] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0062] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources, for example:

[0063] hPSC cells (including H9 cell line), H9 cell line was purchased from Zhongsheng Suyuan Biotechnology Co., Ltd.

[0064] Substrate 1: Vitronectin (purchased from Gibco, A14700).

[0065] Matrix 2: Type IV collagen (purchased from Sigma, C5533).

[0066] Digestive enzymes: ReLeSR (purchased from stemcell, 5872), Accutase (purchased from Gibco, A1110501), TrypLE enzyme (purchased from Gibco, 12604021).

[0067] Retinoic acid (RA for short, purchased from sigma, R2625), BMP-4 (purchased from mce, HY-P7007), and Y27632 (purchased from MCE, HY-10071).

[0068] hPSC medium: mTeSR medium.

[0069] EB formation medium 1: E8 + 20 μM Y27632.

[0070] EB formation medium 2: E8 / PVA+20μM Y27632. Note: E8 / PVA preparation method: Add PolyvinylAlcohol (PVA) (Sigma-Aldrich, P8136, average molecular weight 30,000-70,000) powder at 4 mg / ml into E8, stir at room temperature for 1 hour, filter and sterilize, and store at 4°C.

[0071] EB formation medium 3: KnockOut DMEM (Gibco, 10829018) + 20% KnockOut-Serum Replacement (Gibco, 10828028, alias: KnockOut TM Serum replacement, KOSR) + 1×NEAA (MEM non-essential amino acid solution (100×) purchased from Gibco, 11140050) + 1×GlutaMAX (GlutaMAX TM Additive (100×) purchased from Gibco, 35050061) + 0.055 mM β-mercaptoethanol (β-mercaptoethanol (Solarbio, M8211)) + 1×Penicillin / Streptomycin (Penicillin-Streptomycin (100×) (Gibco, 15140122)) + 100 μM Y27632.

[0072] EB formation medium 3 without Y27632: The difference from EB formation medium 3 is that it does not contain Y27632, specifically: KnockOut DMEM (Gibco, 10829018) + 20% KnockOut-Serum Replacement (Gibco, 10828028, alias: KnockOut TM Serum replacement, KOSR) + 1×NEAA (MEM non-essential amino acid solution (100×) purchased from Gibco, 11140050) + 1×GlutaMAX (GlutaMAX TM Additives (100×) purchased from Gibco, 35050061) + 0.055 mM β-mercaptoethanol (β-mercaptoethanol (Solarbio, M8211)) + 1×Penicillin / Streptomycin (100×) (Gibco, 15140122)).

[0073] Differentiation medium 1: EB formation medium 3 without Y27632 + 1-10 μM retinoic acid (RA) + 10-50 μM BMP4 + 10 μM Y27632, specifically comprising: KnockOut DMEM + 20% KnockOut-Serum Replacement (KnockOut TM Serum replacement, KOSR) + 1×NEAA + 1xGlutaMAX + 0.055mMβ-mercaptoethanol (β-mercaptoethanol) + 1Penicillin / Streptomycin (Penicillin-Streptomycin (100X) (Gibco, 15140122)) + 1~10μM retinoic acid (RA) + 10~50μM BMP4 + 10μM Y27632.

[0074] Differentiation medium 2: DKSFM medium + 1-10 μM retinoic acid (RA) + 10-50 μM BMP4 + 10 μM Y27632.

[0075] Expansion medium: DKSFM medium (full name: Defined Keratinocyte serum-free medium, abbreviated as DKSFM, purchased from Gibco, 10744019).

[0076] Consumables: AggreWell 400 24-well plates (Stemcell, 34411), low attachment 6-well plates.

[0077] In the following embodiments or test examples, the PBS used is a calcium- and magnesium-free PBS buffer.

[0078] In the following embodiments or test examples, the solution was changed every other day and the operation was gentle.

[0079] In the following embodiments or test examples, the method for coating the culture plate with Vitronectin is as follows: 60ul Vitronectin frozen at -80°C is taken, DMEM / F12 is added to 6ml, 1ml / well is added to a 6-well plate pre-cooled at 4°C, and shaken until the bottom of the plate is completely covered, and the plate is coated in a 37°C incubator for at least 1h.

[0080] Example 1

[0081] 1) Culture and expansion of hPSC (H9 cell line):

[0082] Coat the culture plate with Vitronectin 1 hour in advance. When the cell confluence is about 80%, wash once with PBS, add ReLeSR enzyme, aspirate within 1 minute, and then incubate at room temperature for 5-8 minutes. Add 1mL / well hPSC culture medium (mTeSR) and beat vigorously until the clone block is about 200μm. Centrifuge at 150g for 5 minutes, and subculture the cells and hPSC culture medium (mTeSR) at a ratio of 1:24. When the cells are subcultured to the state of "clear clone edge, high nuclear-cytoplasmic ratio, uniform morphology, and close contact between cells in the clone", and the confluence is about 60%, differentiation can be carried out.

[0083] 2) Replace the cell clones with 60% confluence in 1) with fresh culture medium (hPSC culture medium) 1-2 hours in advance, then digest with Accutase at 37°C for 5 minutes, add an equal volume of PBS, gently beat into single cells, centrifuge at 150g for 5 minutes, resuspend and count. This day is defined as Day 0.

[0084] 3) Formation of cell clusters (EBs):

[0085] (1) The above cell suspensions were respectively formed into cell clusters (EBs) according to the following methods (a) to (c):

[0086] (a) Self-aggregation method: Take the corresponding volume of cell suspension and add it to EB formation medium 3. The final cell concentration is adjusted to 10,000-35,000 cells / ml (1,000-3,500 cells / well / 96-well plate) for differentiation culture. Resuspend and mix again and aliquot 100 ul / well into a low-attachment U-bottom 96-well plate. Centrifuge at room temperature at 110 g for 6 min. 37°C, 5% CO 2 The cells were cultured for 2 days without moving, and EBs of uniform size were formed on Day 2.

[0087] (b) Hanging drop method: Add the cell suspension to EB formation medium 3, adjust the cell concentration to 50,000-150,000 cells / mL, then add 20ul / drop to the lid of the culture dish drop by drop (note that a certain distance is maintained between the droplets), and finally close the bottom of the dish and carefully flip it over to form 20ul droplets (cell number is 1000-3000 cells / 20ul / drop) hanging on the lid of the culture dish at 37°C, 5% CO 2 After 2 days of culture without any movement, EBs of uniform size were formed on Day 2.

[0088] (c) AggreWell method: Before use, rinse each well of AggreWell-400 with KnockOut-DMEM medium, add 0.5 mL of EB formation medium 3 to each well, and remove bubbles. Add 1.5 mL of cell suspension to make each well contain (2.4-12)×10 5 The cells were transferred to each well and centrifuged at 100 g for 3 min at 37 °C and 5% CO 2 The cells were cultured for 2 days without moving. On the second day (Day 2), EBs of uniform size were formed, with about 200 to 1000 cells forming one EB.

[0089] (2) After Day 2, EBs were transferred to centrifuge tubes using a 1 mL pipette, washed once with PBS, and transferred to low attachment 2-well / 6-well plates (containing EB formation medium 3) for continued culture overnight.

[0090] (3) Day 3, transfer the EBs from step 2) to a 15 ml centrifuge tube, wash 3 times with PBS, inoculate into a 6-well plate coated with collagen IV, add EB formation medium 3 + 2% FBS, and culture overnight without moving.

[0091] 4) Differentiation stage

[0092] i) Day 4, differentiation culture, the culture medium was replaced with differentiation medium 1 (EB formation medium 3 without Y27632 + 1 μM RA + 25 ng / mL BMP4 + 10 μM Y27632, i.e. KnockOut DMEM + 20% KnockOut-Serum Replacement (KnockOut TM Serum replacement, KOSR) + 1xNEAA + 1xGlutaMAX + 0.055mMβ-mercaptoethanol (β-mercaptoethanol) + 1x Penicillin / Streptomycin (Penicillin-Streptomycin (100X) (Gibco, 15140122)) + 1μM retinoic acid (RA) + 25μM BMP4 + 10μM Y27632.

[0093] ii) On Day 6 and Day 8, the culture medium was replaced with differentiation medium 2 (DKSFM medium 3 + 1 μM RA + 25 ng / mL BMP4 + 10 μM Y27632, i.e., DKSFM medium + 1 μM retinoic acid (RA) + 25 μM BMP4 + 10 μM Y27632, during which dead cells were washed with PBS.

[0094] iii) Starting from Day 10, the medium was replaced with D-KSFM, and then the medium was changed every other day until Day 15.

[0095] 5) Subculture and expansion stage

[0096] a) Around Day 15, when the cell population becomes round and bright, it can be passaged.

[0097] b) After washing with PBS, add TrypLE enzyme, incubate at 37°C for 10 min, and centrifuge at 200 g for 5 min.

[0098] c) Resuspend and count the cells in D-KSFM and inoculate them into collagen IV-coated culture plates at a ratio of 1:3. The culture medium is D-KSFM + 10 μM Y27. At this time, some cells can be retained for subsequent marker expression detection.

[0099] d) Change the medium to D-KSFM every other day. When the cell confluence is about 80%, it can be subcultured again. At this time, the cells can be used for nude mouse germination experiments.

[0100] The cells differentiated in this example were named epithelial stem cells 3D-H9-EpSC-P1.

[0101] The differentiation flow chart of this embodiment is as follows Figure 1 .

[0102] Example 2

[0103] The difference from Example 1 is that the differentiation medium 1 in step 3) of the differentiation stage is replaced with: EB formation medium 3 without Y27632 + 1 μM RA + 50 ng / ml BMP4 + 10 μM Y27632, and the differentiation medium 2 in step 3) of the differentiation stage is replaced with: DKSFM medium + 1 μM RA + 50 ng / ml BMP4 + 10 μM Y27632 (referred to as 1 μM RA + 50 ng / ml BMP4 group)

[0104] Example 3

[0105] The difference from Example 1 is that in step 3), the differentiation medium 1 in the differentiation stage is replaced by: EB formation medium 3 without Y27632 + 10 μM RA + 10 ng / ml BMP4 + 10 μM Y27632, and the differentiation medium 2 in step 3) is replaced by: DKSFM medium + 10 μM RA + 10 ng / ml BMP4 + 10 μM Y27632 (referred to as 10 μM RA + 10 ng / ml BMP4 group).

[0106] Comparative Example 1

[0107] The difference from Example 1 is that the EB formation medium 3 (and the EB formation medium 3 without Y27632) used in steps 3) and 4) is replaced with EB formation medium 1 (E8+20 μM Y27632), referred to as EB formation medium 1 group.

[0108] Comparative Example 2

[0109] The difference from Example 1 is that the EB formation medium 3 (and EB formation medium 3 without Y27632) used in steps 3) and 4) is replaced with EB formation medium 2 (E8 / PVA+20 μM Y27632), referred to as EB formation medium group 2.

[0110] Test Example 1: Effect of small molecule concentration in culture medium

[0111] The cells obtained in the differentiation stage of step 3) of Example 2 and 3 were examined under a microscope, and the results were as follows: Figure 2 The results show that Examples 2 and 3 can also obtain consistent typical cell morphology results at different times during the differentiation stage, and the differentiation medium of the present invention has good differentiation performance.

[0112] Test Example 2: Cell morphology detection

[0113] The cells obtained at different stages of step 3) and 4) of Example 1, Comparative Example 1, and Comparative Example 2 were observed and photographed under a 4× microscope. Some of the results are shown in the figure. Figure 3 .

[0114] Figure 3The results showed that the cell morphology of EB spheres obtained by different EB formation mediums to differentiate into hEpSCs was quite different. For example, both Comparative Example 1 (EB formation medium group 1) and Comparative Example 2 (EB formation medium group 2) could successfully form EB spheres, but Comparative Example 1 (EB formation medium group 1) was easily broken and not attached to the wall after sphere formation on Day 4, while Comparative Example 2 (EB formation medium group 2) was attached to the wall and differentiated normally in the early stage of differentiation, but in the later stage of differentiation, cracking and poor adhesion began to occur on Day 8; similar situations also occurred when other EB formation methods (hanging drop method or AggreWell method) were used. Example 1 (EB formation medium group 3) had good cell adhesion efficiency on Day 2, Day 8, and Day 12, and could differentiate into consistent typical cell morphology.

[0115] Combination Figure 4 EB formation medium 3 can differentiate hPSC into hEpSC regardless of whether it is self-aggregation, hanging drop method or AggreWell method. The cell adhesion efficiency is good on Day 2, Day 8 or Day 15, and the cells can differentiate into consistent typical cell morphology.

[0116] Test Example 3: Marker Detection

[0117] The cells obtained in step c) of step 5) of the passage and amplification stage of Example 1, Comparative Example 1, and Comparative Example 2 were respectively extracted with an RNA extraction kit (TIANGEN, DP420) for total RNA, and the total RNA was synthesized with a cDNA synthesis kit (TransGen, AE311-02). Then, marker expression was detected using SYBR Green premix (Thermo, A25742) and the following primers. The primer sequences are as follows:

[0118] Table 1. Primer sequences

[0119]

[0120] Test results such as Figure 5 , 6 , as shown in 7.

[0121] Figure 5 The results showed that the relative expression levels of OCT4 and NANOG at the mRNA level of the human epithelial stem cells 3D-hEpSC obtained in Example 1 relative to hPSC were close to 0, indicating that human hPSC had completely transformed into epithelial stem cells 3D-hEpSC, and the risk of tumorigenesis was extremely low.

[0122] Figure 5It is also shown that the relative expression level of CD200 mRNA level of the human epithelial stem cells 3D-hEpSC obtained in Example 1 relative to that of hPSC is also very low, less than 1 / 5 of that of hPSC.

[0123] Figure 6 The results showed that the human epithelial stem cell 3D-hEpSC obtained in Example 1 had significantly increased expression levels of the markers KRT15, ​​ITGA6, K18 and p63 at the mRNA level relative to hPSC. The expression level of K18 at the mRNA level was increased by more than 6 times relative to hPSC, the expression level of ITGA6 at the mRNA level was increased by about 6 times relative to hPSC, the expression level of KRT15 at the mRNA level was increased by more than 40 times, and the expression level of p63 at the mRNA level was increased by more than 1000 times.

[0124] Combination Figure 5 , Figure 6 At the mRNA level, the human epithelial stem cells 3D-hEpSC obtained in Example 1 have an expression level of CD200 that is approximately 1 / 5 of that of hPSC (the expression level of CD200 of traditional hair follicle-derived hEpSC is equivalent to that of hPSC), and an expression level of ITGA6 that is approximately 6 times higher than that of hPSC (the expression level of ITGA6 of traditional hair follicle-derived hEpSC is approximately 20 times that of hPSC). The expression levels of CD200 and ITGA6 relative to hEpSC are much lower than those of traditional hair follicle-derived hEpSC cells relative to hPSC, indicating that the human epithelial stem cells 3D-hEpSC obtained in the present invention are also different from traditional hair follicle-derived hEpSC cells, and a new human epithelial stem cell 3D-EpSC is obtained.

[0125] This is because the traditional hair follicle-derived hEpSC cells express markers including KRT15, ​​K18, P63, CD200 and ITGA6, and the expression level of CD200 of the traditional hair follicle-derived hEpSC is comparable to that of hPSC, and the expression level of ITGA6 of the traditional hair follicle-derived hEpSC is about 20 times that of hPSC. For reference, see Yang, Ruifeng et al. "Generation of offolliculogenic human epithelial stem cells from induced pluripotent stem cells." Nature communications vol. 5 (2014): 3071. doi: 10.1038 / ncomms4071.

[0126] Figure 7The results showed that the human epithelial stem cells 3D-hEpSC differentiated by the present invention also did not express or extremely low expressed keratin 14 (K14), a marker specifically associated with HEK, while HEK cells could highly express K14 (the expression level of keratin 14 in HEK cells at the mRNA level was more than 2000 times that of human epithelial stem cells 3D-hEpSC, that is, the human epithelial stem cells 3D-hEpSC differentiated by the present invention did not express or extremely low expressed keratin 14), that is, unlike traditional HEK cells, the primary HEK cells used in the test were purchased from Meisen cells (CTCC-103-HUM).

[0127] The marker detection results show that the differentiated human epithelial stem cells 3D-hEpSC of the present invention express epithelial stem cell markers cytokeratin 15 (KRT15), keratin 18 (K18), p63 and ITGA6 at the mRNA level, but do not express or extremely low expression of the HEK-specific marker keratin 14 (K14) (different from HEK) and hPSC-specific markers OCT4 and NANOG (high safety, no potential tumorigenicity), and do not express or extremely low expression of CD200 (and the relative expression level of ITGA6 is also lower than that of traditional hair follicle-derived hEpSC, that is, human epithelial stem cells 3D-hEpSC are also different from traditional hair follicle-derived hEpSC).

[0128] Test Example 4: Identification of hair growth function in nude mice

[0129] Nine 5-week-old nude mice (Nu / Nu-403 from Vital River) were purchased, weighing about 18-20 g. After acclimating to culture for 3-5 days, they were divided into three groups, with 3 mice in each group:

[0130] 1) The first group (3D-hEpSC+DPC): 12×10 4 Differentiated human epithelial stem cells 3D-hEpSC with 6×10 4 DPC cells (purchased from ScienCell, 2400) were resuspended and mixed to make 180 μL cell suspension. Six points were injected intradermally in the hairless area on the back of each nude mouse, with 10 μL of cell suspension at each point. After transplantation, the mice were observed and photographed every day, and the observation was continued for about 4 weeks to determine the hair growth and penetration.

[0131] 2) The second group (HEK+DPC): 12×10 4 Primary HEK cells (purchased from Meisen Cell, CTCC-103-HUM) were cultured with 6×10 4DPC cells (purchased from ScienCell, 2400) were resuspended in physiological saline to make 180 μL cell suspension. Six points were injected intradermally in the hairless area on the back of each nude mouse, with 10 μL of cell suspension at each point. After transplantation, the mice were observed and photographed every day, and the observation was continued for about 4 weeks to determine the hair growth and outgrowth.

[0132] 3) The third group (DPC only): 6×10 4 DPC cells (purchased from ScienCell, 2400) were resuspended in physiological saline to make 180 μL cell suspension. Six points were injected intradermally in the hairless area on the back of each nude mouse, with 10 μL of cell suspension at each point. After transplantation, the mice were observed and photographed every day, and the observation was continued for about 4 weeks to determine the hair growth and outgrowth.

[0133] The results are as follows Figure 8 The results showed that the injection of 3D-hEpSC and DPC cells (Group 1) of the present invention could induce a large amount of hair growth in nude mice, which was comparable to the hair growth effect of the combination of primary HEK and DPC cells (Group 2) (generally speaking, HEK will lose its hair growth performance when mixed with DPC cells at the P3 generation with the increase of the number of passages, that is, the hair growth effect of primary HEK is the best). However, nude mice that only received DPC cells (Group 3) had no visible hair growth, even when the number of DPC cells was increased to 18×10 4 DPC cells were injected, and no visible hair growth occurred. There was no visible hair growth when human epithelial stem cells 3D-hEpSC were injected alone, which was similar to the situation with traditional hair follicle-derived hEpSC (no visible hair growth when traditional hair follicle-derived hEpSC were injected alone). This indicates that when DPC cells or human epithelial stem cells 3D-hEpSC were injected alone, it was not possible to induce visible hair growth in nude mice. When DPC cells were mixed with the human epithelial stem cells 3D-hEpSC induced by the present invention and transplanted, it was unexpectedly found that a large amount of hair growth could be induced ( Figure 8first row); and the hair growth effect induced by the second group of primary HEK and DPC is equivalent, but primary HEK or traditional EpSC need to obtain hair follicle tissue to obtain, and the source is relatively limited. The human epithelial stem cell 3D-hEpSC of the present invention is derived from human pluripotent stem cells (hPSC) by 3D induction differentiation. The cell source is easy to obtain, and the differentiation cycle is short (only 14 to 20 days), which is easy to obtain in large quantities, that is, the required EpSC cells can be obtained infinitely through hPSC differentiation and subculture methods; it overcomes the problem of limited sources caused by the need to obtain hair follicle tissue for primary HEK or traditional EpSC, and also solves the problem of long time period for inducing differentiation of human pluripotent stem cells (hPSC) into epithelial cells (such as HEK). The human epithelial stem cell 3D-hEpSC of the present invention can be continuously obtained through induced differentiation, and the material collection and portability are far superior to those of primary HEK.

[0134] In summary, the differentiation culture system of the present invention has clear components and does not contain serum, which avoids the influence of serum batch on differentiation stability and makes differentiation more stable. The differentiated human epithelial stem cells 3D-hEpSC of the present invention express epithelial stem cell markers cytokeratin 15 (KRT15), keratin 18 (K18), p63 and ITGA6 at the mRNA level, but do not express or extremely low express the marker keratin 14 (K14) specifically related to HEK (different from HEK), and do not express or extremely low express the markers OCT4 and NANOG related to hPSC (high safety, no potential tumorigenicity), and also extremely low express CD200 (different from traditional hair follicle-derived hEpSC), and the relative expression of ITGA6 is also much lower than that of traditional hair follicle-derived hEpSC.

[0135] The induction differentiation time of the present invention is greatly shortened, and human epithelial stem cells 3D-hEpSC with good hair growth effect can be obtained in only 14 to 20 days. It can not only achieve the effect of hair growth function similar to that of primary HEK, but also solve the problem of limited source of primary HEK or traditional EpSC due to the need to obtain hair follicle tissue: when mixed with DPC cells for transplantation, it can successfully make nude mice grow a large amount of hair, which has great clinical application value.

[0136] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A human epithelial stem cell 3D-hEpSC derived from 3D differentiation of human pluripotent stem cells was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 20, 2024, with the deposit number CGMCC NO.46003.

2. The human epithelial stem cell 3D-hEpSC according to claim 1, characterized in that Compared with the H9 cell line in hPSC cells, human epithelial stem cells 3D-hEpSC highly expressed markers of cytokeratin 15, keratin 18, transcription factors p63 and ITGA6, did not express or very low expression of NANOG and OCT4, and very low expression of CD200; Human epithelial stem cells 3D-hEpSC do not express or very low expression of keratin 14 relative to primary HEK cells; Alternatively, human epithelial stem cells 3D-hEpSC expressed more than 8 times more keratin 18 at the mRNA level than the H9 cell line; Optionally, the expression of ITGA6 at the mRNA level in human epithelial stem cells 3D-hEpSC is 5 to 8 times, optionally 5 to 6 times, higher than that in the H9 cell line; Alternatively, human epithelial stem cells 3D-hEpSC expressed keratin 15 at the mRNA level more than 40 times that of the H9 cell line; Optionally, the expression of transcription factor p63 at the mRNA level in human epithelial stem cells 3D-hEpSC is more than 1000 or 1200 times that of the H9 cell line; Optionally, the expression of NANOG and OCT4 in human epithelial stem cells 3D-hEpSC at the mRNA level relative to that of the H9 cell line is close to 0; Optionally, the expression level of CD200 in the human epithelial stem cells 3D-hEpSC at the mRNA level is 1 / 5 or lower than that of the H9 cell line.

3. A differentiation medium for obtaining the human epithelial stem cells 3D-hEpSC according to claim 1 or 2 by 3D differentiation, characterized in that: It includes differentiation medium 1 or differentiation medium 2, or a combination of differentiation medium 1 and differentiation medium 2; Wherein, differentiation medium 1 comprises: DMEM medium, and contains: 20% serum replacement, 1×NEAA, 1×GlutaMAX, 0.055mM β-mercaptoethanol, 1×penicillin-streptomycin double antibody solution, 1-10μM retinoic acid, 10-50μM bone morphogenetic protein 4 and 10μM ROCK inhibitor, and optionally the ROCK inhibitor is Y27632; Differentiation medium 2 comprises: DKSFM medium, and contains 1-10 μM retinoic acid, 10-50 μM bone morphogenetic protein 4 and 10 μM ROCK inhibitor, and optionally the ROCK inhibitor is Y27632.

4. The differentiation medium according to claim 3, characterized in that Differentiation medium 1 includes: DMEM medium, and contains: 20% serum replacement, 1×NEAA, 1×GlutaMAX, 0.055mM β-mercaptoethanol, 1× penicillin-streptomycin double antibody solution, 1μM retinoic acid, 25μM bone morphogenetic protein 4 and 10μM ROCK inhibitor; And / or, differentiation medium 2 comprises: DKSFM medium, and contains 1 μM retinoic acid, 25 μM bone morphogenetic protein 4, and 10 μM Y27632.

5. The differentiation medium according to claim 3 or 4, characterized in that In Differentiation Medium 1, the serum replacement was KnockOut-Serum Replacement; and / or, in the differentiation medium 1, the DMEM medium is KnockOut DMEM; And / or, in the differentiation medium 1, 1×NEAA is obtained by diluting 100×MEM non-essential amino acid solution into DMEM medium.

6. A method for differentiating the human epithelial stem cells 3D-hEpSCs according to claim 1 or 2, characterized in that: The steps include: 1) Expand and culture hPSC cells into clones and disperse the cells into single cells; 2) The single cells in step 1) are subjected to 3D culture and cell clusters are formed using EB formation medium; Optionally, the EB formation medium comprises: DMEM medium, and contains: 20% serum replacement, 1×NEAA, 1×GlutaMAX, 0.055mM β-mercaptoethanol, 1×penicillin-streptomycin double antibody solution, 1-10μM retinoic acid, 10-50μM bone morphogenetic protein 4 and 100μM ROCK inhibitor; optionally, the ROCK inhibitor is Y27632; 3) Differentiating the cell cluster obtained in step 2); using the differentiation medium described in any one of claims 3 to 5 for differentiation culture, wherein first using differentiation medium 1 for adaptive differentiation for 1 to 2 days, then using differentiation medium 2 for differentiation culture for 3 to 5 days, and then transferring to D-KSFM for culture for 4 to 6 days to obtain differentiated human epithelial stem cells 3D-hEpSC.

7. The differentiation method according to claim 6, characterized in that: In step 3), differentiation medium 2 is used and the medium is replaced every 1 to 2 days during the differentiation culture process; And / or, during the D-KSFM culture process, the culture medium is replaced every 20 to 28 hours, and the replacement time is optionally 24 hours.

8. The differentiation method according to claim 6 or 7, characterized in that: In step 1), the culture plate is coated with Vitronectin; And / or, in step 1), using hPSC culture medium for expanded culture; And / or, in step 1), when the cells are passaged until the clone edges are clear, the nuclear-cytoplasmic ratio is high, the morphology is uniform, the cells in the clone are in close contact, and the confluence is about 60%, the clones are dispersed and resuspended into a cell suspension for cell cluster culture; And / or, in step 2), the cell cluster formation method includes at least one of a self-aggregation method, a hanging drop method, and an AggreWell method; and / or, in step 2), the cell culture dish is not moved during the cell clustering process; And / or, in step 2), after the cell clusters are formed, they are transferred to a low-adsorption culture dish; optionally, after the cell clusters are formed, they are cultured for 1 to 2 days using EB formation medium; optionally, after continued culture, the cell clusters are inoculated into a collagen IV-coated well plate, and the EB formation medium and 2% FBS in step 2) are added and cultured overnight for differentiation.

9. A cell composition for hair regeneration, characterized in that: Comprising human dermal papilla cells and the human epithelial stem cells 3D-hEpSC according to claim 1 or 2 or the human epithelial stem cells 3D-hEpSC obtained by the differentiation method according to any one of claims 6 to 8; Optionally, the number ratio of DPC cells to human epithelial stem cells 3D-hEpSC is 1:(1-3), optionally 1:(2-3).

10. Use of the human epithelial stem cell 3D-hEpSC according to claim 1 or 2, the human epithelial stem cell 3D-hEpSC obtained by the differentiation method according to any one of claims 6 to 8, or the cell composition according to claim 9 in preparing a hair regeneration product.

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