Method for inducing directional differentiation of human induced pluripotent stem cells into mesenchymal stem cells in stages and application of method

Abstract

The invention discloses a method for inducing directional differentiation of human induced pluripotent stem cells into mesenchymal stem cells in stages and application of the method, and develops a mesenchymal induction system which has definite components, is reasonably added with induced differentiation factors and has a reasonable concentration and is subjected to induced differentiation in stages. The induced pluripotent stem cells are regulated to efficiently and directionally induce and differentiate into the mesoderm cells, and then the mesoderm cells are directionally induced to induce and convert into the MSCs, so that the differentiation period of inducing and differentiating the human induced pluripotent stem cells into the mesenchymal stem cells can be shortened to 2-3 weeks, the differentiation rate is high, and the batch consistency can be ensured.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell induction and differentiation, and particularly relates to a method for inducing human induced pluripotent stem cells to differentiate into mesenchymal stem cells in stages and application thereof. BACKGROUND

[0002] Mesenchymal stem cells (MSCs) are a kind of adult stem cells with multi-directional differentiation potential, which widely exist in bone marrow, fat, umbilical cord and other tissues. Because MSCs can differentiate into osteoblasts, chondrocytes, adipocytes and other cell types, and have immune regulation, anti-inflammatory and tissue repair functions, MSCs show great application potential in the fields of regenerative medicine, cell therapy and tissue engineering. For example, MSCs have been used for the treatment of bone defect repair, graft-versus-host disease (GVHD), diabetes, rheumatoid arthritis and neurodegenerative diseases. However, traditional adult-derived MSCs face many limitations in practical application, including donor damage, limited cell number, low in vitro expansion capacity, significant cell heterogeneity and donor age dependence. In addition, the extraction process of adult MSCs is invasive, and its function is easily affected by donor aging, which is difficult to meet the clinical scale demand.

[0003] To solve the above problems, researchers turn their attention to human induced pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells. hPSCs have unlimited proliferation capacity, and can obtain functionally uniform MSCs (hPSCs-MSCs) through directional differentiation. Because it can be derived from the body, there is no ethical controversy and unlimited proliferation capacity, it is considered as an ideal alternative source. However, the existing differentiation method still has the following key problems:

[0004] (1) The traditional embryoid body method (EB method) needs to be suspended in culture to form embryoid bodies, and then induced to differentiate adherently, which is complicated and relies on spontaneous differentiation, with low efficiency (usually less than 30%). In addition, the cell signal inside EB is complex, which is easy to produce non-target cell types.

[0005] (2) The monolayer induction method simplifies the process, but it mostly relies on serum-containing medium, and the undefined composition leads to batch difference and risk of heterologous contamination.

[0006] (3) The existing differentiation system lacks directionality, and most of them do not regulate the differentiation of mesoderm progenitor cells to MSCs in stages, and the timing and concentration of functional compounds (such as BMP4, TGF-β1, etc.) are not optimized, which cannot effectively simulate the in vivo development microenvironment.

[0007] (4) Commercial serum-free medium (such as MSC-SFM) avoids animal-derived components, but the effect of supporting cell survival and differentiation is significantly lower than that of the serum-containing system, and the mechanism of action of key inducing factors is unknown. Therefore, it is necessary to develop a more efficient method with a higher differentiation rate and a higher orientation to induce human induced pluripotent stem cells to differentiate into mesenchymal stem cells. SUMMARY

[0008] Therefore, based on the above background, the present application provides a method for inducing human induced pluripotent stem cells to differentiate into mesenchymal stem cells in stages and its application. The present application develops a mesenchymal induction system with clear components and reasonable addition of inducing differentiation factors and their concentrations, which can induce differentiation in stages. By first regulating the efficient directional induction of induced pluripotent stem cells into mesoderm cells, and then inducing the directional induction of mesoderm cells into MSCs, the differentiation cycle of human induced pluripotent stem cells into mesenchymal stem cells can be shortened to 2-3 weeks, the differentiation rate is high, and batch consistency can be ensured.

[0009] The technical scheme provided by the present application is:

[0010] An induction culture system for inducing human induced pluripotent stem cells to differentiate into mesenchymal stem cells, the induction culture system comprising three kinds of culture media, mesoderm induction medium A, mesoderm induction medium B and mesenchymal induction medium;

[0011] The mesoderm induction medium A is composed of the following components:

[0012] RPMI 1640 basic medium

[0013]

[0014] The mesoderm induction medium B is composed of the following components:

[0015] RPMI 1640 basic medium

[0016] Vitamin C phosphate 50-500 μg / mL

[0017] Recombinant human serum albumin 100-1000 μg / mL

[0018] LY294002 1-20 μmol / L.

[0019] The mesenchymal induction medium is prepared by mixing E8 and MSC-SFM at a volume ratio of 1:1, and contains 5 μmol / L Y-27632.

[0020] Based on the same inventive concept, the present application further provides an application of the induction culture system for inducing human induced pluripotent stem cells to differentiate into mesenchymal stem cells.

[0021] Based on the same inventive concept, the present application further provides a method for inducing human induced pluripotent stem cells to differentiate into mesenchymal stem cells in stages based on the induction culture system, which comprises the following steps:

[0022] ①The human induced pluripotent stem cells are cultured in the mesoderm induction medium A for 2 days, and the fresh mesoderm induction medium A is replaced every 24 hours;

[0023] ②The mesoderm induction medium A is replaced by the mesoderm induction medium B, and the cells are continuously cultured for 3 days; after the fresh mesoderm induction medium B is replaced every 24 hours, the human induced pluripotent stem cells can be induced to differentiate into mesoderm cells.

[0024] ③The cells induced and differentiated in step ② are washed, dissociated into single cells by digestion, and then added into the mesenchymal induction medium for culture; the fresh mesenchymal induction medium is replaced every 24 hours, and when the cell confluence reaches 100%, the cells are continuously subcultured for 1 generation or more.

[0025] Further, in step ①, the human induced pluripotent stem cells with a confluence of 70%-85% are added into the mesoderm induction medium A for culture.

[0026] Further, in step ③, the cells dissociated by digestion are inoculated onto the culture plate at a cell density of 200,000 cells·mL -1 for culture.

[0027] Further, in step ③, the cells dissociated by digestion are inoculated onto the gelatin-coated culture plate for culture.

[0028] Specifically, the operation of coating the culture plate with gelatin is as follows: 0.1 g of gelatin powder is added into 100 mL of DPBS liquid, the gelatin powder is heated to completely dissolve at a temperature of 60℃ by using a water bath, and then filtered by using a 0.22 μm filter membrane while hot to obtain a 0.1% gelatin solution. Meanwhile, 1 mL of the gelatin solution is added into each hole of a 6-hole plate while hot, and the plate is incubated in an incubator overnight.

[0029] Further, in step ③, the cells are dissociated by digestion using a trypsin-EDTA digestion solution.

[0030] Based on the same inventive concept, the present application further provides the mesenchymal stem cells prepared by the method for inducing human induced pluripotent stem cells to differentiate into mesenchymal stem cells in stages.

[0031] Based on the same inventive concept, the application also provides the use of mesenchymal stem cells prepared by the method for preparing mesenchymal stem cells from human induced pluripotent stem cells by directional differentiation in stages in the preparation of products applicable to bone tissue engineering or immunotherapy.

[0032] The application achieves the following beneficial effects:

[0033] 1. The application shortens the differentiation period of human induced pluripotent stem cells to 2-3 weeks by adding specific functional compounds in stages, optimizing the culture conditions, and developing a serum-free induction culture system, and the differentiation rate can reach more than 90%, and batch consistency can be ensured.

[0034] 2. The application optimizes the composition of the induction culture system, which can reduce the risk of heterologous contamination, avoid ethical restrictions and immunogenicity problems.

[0035] 3. The mesenchymal stem cells prepared by the application have high expression of CD73 and CD44, and have the potential for osteogenic differentiation, adipogenic differentiation and the like.

[0036] 4. The application optimizes the cell density (such as 200,000 cells / mL) and the passage strategy to obtain a high-purity CD73+ and CD44+ cell population, which meets the functional requirements of clinical applications.

[0037] 5. The application establishes a monolayer culture system, which can simplify the operation process and reduce production costs, and provides a stable cell source for regenerative medicine.

[0038] Drawings of the specification

[0039] Figure Figure 1 Figure

[0040] Figure Figure 2 Figure Figure

[0041] Figure Figure 3 Figure

[0042] Figure Figure 4Figure 6 shows the expression of MSCs related genes (a: CD73; b: CD105; c: CD90; d: CD29; e: CD44) in hNF-C1 hiPSCs after mesoderm induction for 2 days and mesenchymal differentiation for 5 days and passaging twice, according to an embodiment of the present application.

[0043] Figure 7 shows the positive expression rate of CD73 in hNF-C1 hiPSCs after mesoderm induction for 2 days and mesenchymal differentiation for 5 days and passaging twice, according to an embodiment of the present application. Figure 5 Figure 8 shows the positive expression rate of CD44 in hNF-C1 hiPSCs after mesoderm induction for 2 days and mesenchymal differentiation for 5 days and passaging twice, according to an embodiment of the present application.

[0044] Figure 6 Figure 9 shows the cell morphology of hNF-C1 hiPSCs during mesoderm induction for 5 days and mesenchymal differentiation and passaging, according to an embodiment of the present application.

[0045] Figure 10 shows the cell activity of hNF-C1 hiPSCs after mesoderm induction for 5 days, according to an embodiment of the present application. Figure 7 Figure 11 shows the expression of mesoderm marker genes (a: MSX1; b: MEOX1; c: MIXL1; d: T), endoderm marker genes (a: AFP; b: GATA4), neural crest marker genes (c: FOXD3) and ectoderm related genes (d: SOX1) in hNF-C1 hiPSCs after mesoderm induction for 5 days, according to an embodiment of the present application.

[0046] Figure 8 Figure 12 shows the expression of MSCs related genes (a: CD73; b: CD105; c: CD90; d: CD29; e: CD44) in hNF-C1 hiPSCs after mesoderm induction for 5 days and mesenchymal differentiation and passaging twice, according to an embodiment of the present application.

[0047] Figure 13 shows the positive expression rate of CD73 in hNF-C1 hiPSCs after mesoderm induction for 5 days and mesenchymal differentiation and passaging twice, according to an embodiment of the present application. Figure 9 Figure 14 shows the positive expression rate of CD44 in hNF-C1 hiPSCs after mesoderm induction for 5 days and mesenchymal differentiation and passaging twice, according to an embodiment of the present application.

[0048] Figure 10 Figure 15 shows the cell morphology of hNF-C1 hiPSCs during mesoderm induction for 5 days and mesenchymal differentiation and passaging, according to an embodiment of the present application.

[0049] Figure 16 shows the cell activity of hNF-C1 hiPSCs after mesoderm induction for 5 days, according to an embodiment of the present application. Figure 11 ​​​The positive expression rate of CD73 in the cell sample when CD73 is detected by flow cytometry in the mesoderm differentiation P1 after the mesoderm differentiation of the hNF-C1 hiPSCs induced by different functional compounds for 5 days.

[0050] Figure 8 shows the cell survival rate after the mesoderm induction of the hNF-C1 hiPSCs for 5 days, and then seeding to the gelatin surface for 12 hours using different cell digestion methods. Figure 12 Figure 9 shows the cell morphology change on the second day after the mesoderm induction of the hNF-C1 hiPSCs for 5 days, and then seeding to the gelatin surface with different densities.

[0051] Figure 13 Figure 10 shows the cell morphology change after the mesenchymal induction of the cells seeded to the gelatin with different densities after the mesoderm induction of the hNF-C1 hiPSCs for 5 days.

[0052] Figure 11 shows the expression of MSCs marker genes (a: CD73; b: CD44; c: CD29; d: CD90) detected by RT-PCR after the mesenchymal induction of the cells seeded to the gelatin surface with different densities after the mesoderm induction of the hNF-C1 hiPSCs for 5 days. Figure 14 Figure 12 shows the cell morphology change after the mesenchymal induction of the cells seeded to the gelatin surface with single cell after the mesoderm induction of the hNF-C1 hiPSCs for 5 days.

[0053] Figure 15 Figure 13 shows the expression of MSCs related genes detected by RT-PCR after the mesenchymal induction of the cells seeded to the gelatin surface with single cell after the mesoderm induction of the hNF-C1 hiPSCs for 5 days. a is MSCs marker genes; b is negative expression genes.

[0054] Figure 14 shows the cell morphology change after the mesenchymal induction of the cells seeded to the gelatin surface with single cell after the mesoderm induction of the hNF-C1 hiPSCs for 5 days. Figure 16 Figure 15 shows the expression of MSCs related genes detected by RT-PCR after the mesenchymal induction of the cells seeded to the gelatin surface with single cell after the mesoderm induction of the hNF-C1 hiPSCs for 5 days. a is MSCs marker genes; b is negative expression genes.

[0055] Figure 17 Figure 16 shows the cell morphology change after the mesenchymal induction of the cells seeded to the gelatin surface with single cell after the mesoderm induction of the hNF-C1 hiPSCs for 5 days.

[0056] Figure 17 shows the expression of MSCs related genes detected by RT-PCR after the mesenchymal induction of the cells seeded to the gelatin surface with single cell after the mesoderm induction of the hNF-C1 hiPSCs for 5 days. a is MSCs marker genes; b is negative expression genes. Figure 18 Figure 18 shows the cell morphology change after the mesenchymal induction of the cells seeded to the gelatin surface with single cell after the mesoderm induction of the hNF-C1 hiPSCs for 5 days.

[0057] Figure 19 ​​​​After mesoderm induction of hNF-C1 hiPSCs for 5 days of the embodiment of the present application, the cells were seeded on a gelatin surface after digestion for mesenchymal induction, and the expression of CD73, CD44 and CD34 was detected by flow cytometry, and the expression of UC-MSCs was also detected.

[0058] Figure 6 shows the staining results of hiPSCs-MSCs and hBMSCs after osteogenic induction for 14 days, and adipogenic and chondrogenic induction for 21 days. Figure 20 Figure 6 shows the staining results of hiPSCs-MSCs and hBMSCs after osteogenic induction for 14 days, and adipogenic and chondrogenic induction for 21 days.

[0059] Figure 6 shows the staining results of hiPSCs-MSCs and hBMSCs after osteogenic induction for 14 days, and adipogenic and chondrogenic induction for 21 days. Figure 21 Figure 6 shows the staining results of hiPSCs-MSCs and hBMSCs after osteogenic induction for 14 days, and adipogenic and chondrogenic induction for 21 days.

[0060] Figure 6 shows the staining results of hiPSCs-MSCs and hBMSCs after osteogenic induction for 14 days, and adipogenic and chondrogenic induction for 21 days. Figure 22 Figure 6 shows the staining results of hiPSCs-MSCs and hBMSCs after osteogenic induction for 14 days, and adipogenic and chondrogenic induction for 21 days. DETAILED DESCRIPTION

[0061] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme of the embodiments of the present application will be described below in detail, obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0062] The present application provides an induction culture system for directional differentiation of human induced pluripotent stem cells into mesenchymal stem cells, which is serum-free and has clear components, and includes three kinds of culture media, i.e., mesoderm induction medium A, mesoderm induction medium B and mesenchymal induction medium.

[0063] The mesoderm induction medium A is composed of the following components:

[0064] Basic medium RPMI 1640

[0065]

[0066] The mesoderm induction medium B is composed of the following components:

[0067] Basic medium RPMI 1640

[0068] Vitamin C phosphate 50-500 μg / mL

[0069] Recombinant human serum albumin 100-1000 μg / mL

[0070] LY294002 1-20 μmol / L.

[0071] The mesenchymal induction medium is prepared by E8 and MSC-SFM at a volume ratio of 1:1, and contains 5 μmol / L Y-27632.

[0072] The following examples were used:

[0073] MSC-SFM is a serum-free mesenchymal stem cell culture medium (Sbiotech, CA1004500);

[0074] E8 medium is II Human pluripotent stem cell medium (Sbiotech, CA1014500);

[0075] RPMI 1640 (Gibco, C22400500BT)

[0076] The MSC medium used in the following examples is a traditional serum-containing mesenchymal stem cell induction medium, i.e., α-MEM (Biosharp, BL306A) containing 15% FBS (Vistech, SE100-A), 1% L-glutaMax (Gibco, ), 1% non-essential amino acids (Solarbio, N1250), and 1% penicillin / streptomycin double-antibiotic solution (Solarbio, P1400).

[0077] A method for inducing human induced pluripotent stem cells to differentiate into mesenchymal stem cells in stages based on the above induction culture system, characterized in that it comprises the following steps:

[0078] ①The human induced pluripotent stem cells are cultured in the mesoderm induction medium A for 2 days, and the fresh mesoderm induction medium A is replaced every 24 hours;

[0079] ②The mesoderm induction medium A is replaced with the mesoderm induction medium B, and the cells are further cultured for 3 days, and after the fresh mesoderm induction medium B is replaced every 24 hours, the human induced pluripotent stem cells can be differentiated into mesoderm cells, and the differentiation efficiency is more than 90%.

[0080] ③ The cells induced and differentiated in step ② are washed, dissociated into single cells, and then added into mesenchymal induction medium for culture. Fresh mesenchymal induction medium is replaced every 24 hours. When the cell confluence reaches 100%, the cells are subcultured for one generation or more. The proportion of P1 generation mesenchymal stem cells is 30-70%, and the proportion of P2 and P3 generation mesenchymal stem cells can reach 90% or more.

[0081] Example 1: Stage-by-stage directional induction of human induced pluripotent stem cells into mesoderm cells

[0082] (1) Cell culture

[0083] The hNF-C1 hiPSCs cell line was provided by Guangzhou Institutes of Biomedicine and Health.

[0084] First, the human pluripotent stem cell line was seeded into Matrigel (BD Biosciences, Canada)-coated culture plates at a cell density of 15,000 cm -2 The cells were cultured in an incubator at 37°C, 100% humidity, and 5% CO2. Matrigel was diluted with pre-cooled sterile DPBS at a ratio of 1:230 (operated on ice), and 1 ml per well was added to a 6-well plate at 37°C. The cells were replaced daily, and subcultured at a ratio of 1:3-6 every 3-4 days by 0.5 mM EDTA 37°C digestion for 4-5 min. In this embodiment, the cells were subcultured at a ratio of 1:3.

[0085] The operation of coating the culture plate with Matrigel (BD Biosciences, Canada) is as follows: First, Matigel is diluted with pre-cooled sterile DPBS at a ratio of 1:230 (operated on ice), then 1 mL per well is added to a 6-well plate, shaken to flatten, and placed in a 37°C incubator for incubation for more than 1 hour, preferably overnight.

[0086] (2) Directional induction of hiPSCs into mesoderm cells

[0087] When the cell confluence of hNF-C1 hiPSCs cultured on the Matrigel surface reaches 80%, the E8 medium is replaced with mesoderm induction medium to induce hNF-C1 hiPSCs to differentiate into mesoderm. The mesoderm induction and differentiation is divided into two stages, D0-D2 stage (i.e., the first two days of induction and differentiation) and D3-D5 stage (i.e., the third day after induction and differentiation), using an in vitro culture system.

[0088] In the mesoderm induction of D0-D2, when the cell fusion degree of the hNF-C1 hiPSCs reaches 80%, the cells are washed once with DPBS, and the mesoderm induction medium A (i.e., C01 group) is replaced,

[0089] The mesoderm induction medium A is RPMI 1640 as the basic medium, which contains 213 μg·mL -1 Vitamin C phosphate sodium, 500 μg·mL -1 Recombinant albumin, and 6 μmol·L -1 CHIR99021, 10 μmol·L -1 SB431542.

[0090] Then, five control groups corresponding to the mesoderm induction medium A are additionally prepared, and the five control groups are compared with the mesoderm induction medium A, in which 5 μmol·L -1 CYC, 10 μmol·L - 1 LY294002, 20 ng·mL -1 bFGF, 10 ng·mL -1 BMP4, and 40 ng·mL -1 RA replaces SB431542, and the cell culture operation of the control group is the same as that of the mesoderm induction medium A culture, and the medium is replaced every day.

[0091] During the experiment, the phase contrast microscope and the CCD camera are used to record the cell morphology change, and the RNA sample and the flow sample are collected for detection, and the primer sequence is shown in Table 2.

[0092] The subsequent second stage and the mesenchymal differentiation stage of the control group in this stage are the same as the specific operation of the present application.

[0093] In the mesoderm induction of D3-D5 (i.e., continue to induce for 3 days after the first stage);

[0094] In this stage, the mesoderm induction medium B is used for culture, and the mesoderm induction medium B (i.e., C03 group) is RPMI 1640 as the basic medium, which contains 213 μg·mL -1 Vitamin C phosphate sodium, and 500 μg·mL -1 Recombinant albumin, and 10 μmol·L -1 LY294002;

[0095] Then, five control groups corresponding to the mesoderm induction medium B are additionally prepared, and the five control groups are compared with the mesoderm induction medium B, in which 5 μmol·L -1 CYC, 20 ng·mL-1 bFGF, 10 ng·mL -1 BMP4, 10 μmol·L -1 SB431542 and 40 ng·mL -1 RA instead of LY294002, the cells in the control group were cultured in the mesoderm induction medium B, and the medium was changed every day.

[0096] The cells in the control group in this stage were cultured by using the mesoderm induction medium A in the first stage.

[0097] During the experiment, phase contrast microscope and CCD camera were used to record the changes in cell morphology, and RNA samples and flow samples were collected for detection.

[0098] Cell activity detection

[0099] After completing the mesoderm induction of D0-D2 and D3-D5, i.e. on the 2nd and 5th day of induction, the cells were washed once with DPBS. CCK-8 diluent was prepared: CCK-8 reagent was diluted with F12 / DMEM basic medium at a ratio of 1:20, and mixed well for use. 500 μL of CCK8 diluent was added to each well of the cells cultured in a 12-well plate, and the same light-avoiding operation was performed. Then it was incubated in a 37°C incubator for 2 hours until the CCK-8 reaction solution turned orange yellow. Shake gently and mix well, and take 100 μL of reaction solution per well and place it in a 96-well plate. Each well of the 12-well plate can be taken to 4 96-well plates, which is a group. Then use Bio-Rad full-wavelength enzyme marker to measure the absorbance value at 450 nm.

[0100] (3) Mesenchymal differentiation of hiPSCs-derived mesodermal progenitor cells

[0101] After completing the mesoderm induction in the two stages of D0-D2 and D3-D5, the cells were washed once with DPBS, and the differentiated cells were dissociated into single cells by using 0.25% trypsin-EDTA for 2-4 min, and counted by using a cell counting plate. The cells were seeded on a 0.1% gelatin-coated culture plate at a cell density of 200,000 cells·mL -1 , and mesenchymal induction medium was used for mesenchymal induction, and 5 μmol·L -1 Y-27632 was added to the medium, which was recorded as P1. After 7 days, the cells were continued to be digested by using 0.25% trypsin-EDTA, and subcultured at a cell density of 20,000·mL -1 , and the mesenchymal induction medium was continued to be used for mesenchymal induction, which was recorded as P2. During the mesenchymal induction, phase contrast microscope and CCD camera were used to record the changes in cell morphology, and P1, P2 generation RNA samples and flow samples were collected for detection, and the results are shown in the following table. Figure 2To the annex Figure 12 as shown.

[0102] To the annex Figure 2 The cell morphology during the process of mesoderm induction after 2 days of differentiation of hNF-C1 hiPSCs induced by different functional compounds is shown. As can be seen from the figure, after 2 days of mesoderm induction, the cells proliferate obviously and arrange very densely. The cells of the control group (control group without induction and differentiation) arrange obviously in single layer, and the cells of the other groups arrange densely in circles. Among them, obvious collagen formation can be seen in the C01 group. After digestion and subculture for mesoderm induction, the cell morphology has changed obviously. At P1, under the microscope, the cells of C01 and each experimental group adhere well, arrange densely, arrange obviously in single layer, the cell volume increases, the cell nucleus is obvious, the cell morphology is not uniform, and obvious collagen deposition can be seen. The cell volume of the control group and the SB431542 group is larger than that of the other groups, and the cell body edge is clear. The cell morphology of the control group is relatively consistent. It is worth noting that the cell morphology of the SB431542 group arranges obviously in single layer, and the cell arrangement in some areas shows a certain directionality. At P2, the cell morphology of the SB431542 group has changed to uniform long spindle shape, and the cell arrangement has directionality. The cell morphology of the control group and the other experimental groups still shows the heterogeneity of the morphology, and the cell arrangement is chaotic. The RA group shows different cell states at P2, the cell volume increases, the cell morphology is irregular, the cell body edge is blurred, and shows "tearing shape", and obvious cell apoptosis and slow cell proliferation can be seen.

[0103] Figure 3 The results show the cell activity of hNF-C1 hiPSCs after 2 days of mesoderm induction by different functional compounds. As can be seen, the number of cells in the control group is the least, followed by the LY294002 group and the SB431542 group. The cell activity of the BMP4 group and the bFGF group is the highest. This is consistent with the results presented in the cell morphology diagram.

[0104] Figure 4The different functional compounds in mesoderm induction role after 2 days, reseeded to gelatin surface by cell digestion, mesenchymal induction medium was used to continue mesenchymal induction differentiation, accompanied by 2 passages. The expression of mesenchymal stem cell marker genes (CD73, CD105, CD90, CD29) and surface markers (CD73, CD44) was detected by RT-PCR technology. With the extension of cell induction time, the gene expression level of CD73 was significantly increased, and the gene expression level of SB431542 group was significantly higher than that of other groups (p<0.01) at P1. For the expression of CD105, the gene expression level of SB431542 group was significantly increased with the extension of induction time. While the gene expression level of C01 group, CYC group, LY294002 group and BMP4 group showed a downward trend at P2. The gene expression of CD90 is as follows: at P1, the gene expression of LY294002 group was significantly higher than that of the other groups (p<0.05), and the control group was lowly expressed CD90 during the whole induction. At P2, except for the control group, SB431542 group and RA group, the other groups were high in CD90 gene expression. The expression of CD29 gene was similar to that of CD105. The gene expression level of SB431542 group was significantly increased with the extension of induction time, and was significantly higher than that of the other groups. The gene expression level of CD44 showed a different trend. At P1, CYC group was relatively high in CD44 gene expression, and at P2, the gene expression level showed a significant downward trend. At P2, the control group was high in CD44 expression, followed by the RA group. We speculate that the mesodermal progenitor cells inhibit the EMT process during mesenchymal induction, which further affects the gene level of CD44.

[0105] Figures 5-6 The different functional compounds in mesoderm induction role after 2 days, continue to perform mesenchymal induction and pass 2 times, and the positive expression rate of CD73 and CD44 at P2 was detected by flow cytometry (FCM). The analysis results showed that the cells treated with SB431542 expressed the highest level of CD73 and CD44 marker proteins (CD73 was 16.5%, and CD44 was 68.6%). Therefore, these results show that it is suggested to add SB431542 to accelerate the induction of mesenchymal stem cell progenitor cells.

[0106] Figure 7The cell morphology after mesoderm induction for 5 days and after 2 times of continuous passage is shown. After mesoderm induction for 5 days, the arrangement is very dense, and small area gaps between cells can be seen in C03 group, CYC group, LY294002 group, and BMP4 group, and there are no adherent cells, which may be caused by apoptosis. After mesoderm induction for 5 days, mesenchymal induction is performed, and the cell morphology has changed obviously. At P1 generation, the cell morphology of the rest groups is mostly long spindle-shaped except for the control group and the RA group. With the extension of the induction time, at P2 generation, the cell morphology is more uniform except for the control group and the RA group, which is similar to the morphology of fibroblast cells, and is arranged in a fingerprint pattern.

[0107] Figure 8 The results show the cell activity after mesoderm induction for 5 days. The cell number of the SB431542 group is the most, followed by the RA group, the SB431542+control group, and the bFGF group. The cell activity of the rest groups has little difference, and there is no obvious difference in the cell number relative to the control group.

[0108] Figure 9 The expression of three germ layer marker genes and neural crest marker genes after 5 days of mesoderm induction by different functional compounds is shown. Comprehensive analysis shows that SB431542 has obvious advantages in promoting mesoderm induction, and D0-D2 addition and D3-D5 continuous addition have certain effects on promoting mesoderm differentiation. LY294002 continues to play a role in inhibiting the differentiation of ectoderm during mesoderm induction.

[0109] Figure 10 The expression of mesenchymal stem cell marker genes (CD73, CD105, CD90, CD29) and surface markers (CD73, CD44) after 5 days of mesoderm induction by different functional compounds is shown. The results show that the addition of functional compounds during the D3-D5 stage of mesoderm induction can significantly promote the differentiation of mesodermal progenitor cells into mesenchymal-like cells, especially bFGF, LY294002, and BMP4 compounds.

[0110] Figures 11-12 The CD73 and CD44 positive expression rates at P2 generation by flow cytometry (FCM) are shown. The results show that bFGF can effectively promote the induction efficiency of hiPSCs during the initial mesenchymal differentiation of mesoderm, but LY294002 has a stronger effect on promoting the maturation of mesenchymal-like cells.

[0111] Digestion method and initial density for mesoderm cell induction of Example 2

[0112] hiPSCs were subcultured by using mesoderm induction medium A, mesoderm induction medium B and mesenchymal induction medium in stages as in Example 1.

[0113] Mesoderm induction and differentiation of hiPSCs

[0114] When the confluence of hiPSCs reached 80%, the E8 medium was replaced with mesoderm induction medium to initiate mesoderm differentiation. Mesoderm induction and differentiation was divided into two stages, D0-D2 stage, using in vitro culture system. Mesoderm induction medium A was RPMI 1640 base medium containing 213 μg / mL sodium phosphate ascorbate, 500 μg / mL recombinant human serum albumin, 6 μmol / L CHIR99021 and 10 μmol / L SB431542. Fresh mesoderm medium was replaced every 24 hours, and the culture was continued for 2 days.

[0115] Then D3-D5, mesoderm induction medium B was RPMI 1640 base medium containing 213 μg / mL sodium phosphate ascorbate, 500 μg / mL recombinant human serum albumin and 10 μmol / L SB431542. -1 LY294002, fresh mesoderm induction medium was replaced after the cells were washed with DPBS every 24 hours.

[0116] After 5 days of mesoderm induction, the cells were washed once with DPBS, and the cells were digested and dissociated using 0.25% trypsin-EDTA. The 0.25% trypsin-EDTA described herein is 0.25% trypsin-0.02% EDTA, which is prepared using DPBS without calcium and magnesium (- / -).

[0117] A control group was also set up, and the cells were digested and dissociated into single cells using commonly used cell digestion solutions 0.5 mmol / L EDTA, 2 mmol / L EDTA, 2 mg / mL type I collagenase and 2 mg / mL type II collagenase, respectively.

[0118] All the cell digestion solutions were prepared using DPBS without calcium and magnesium (- / -).

[0119] After digestion of each group, the cells were counted using a cell counting plate, and 200,000 cells / mL of each group were inoculated into a 6-well plate. -1 The cells were inoculated on the surface of a culture plate coated with 0.1% gelatin, and this was recorded as P1 generation. After adhering for 12 hours, the survival rates of different digestion methods were compared using the CCK-8 method and crystal violet staining, and the cell morphology and growth after cell adhesion were observed.

[0120] Starting density

[0121] Mesoderm progenitor cells were counted after 0.25% trypsin-EDTA digestion, and cells were seeded at a density of 200,000 cells / mL (high-density group) on 0.1% gelatin-coated culture plates, and induced to differentiate by adding 5 μmol·L -1 MSC mesenchymal induction medium Y-27632 induced differentiation.

[0122] A control group was set up, and mesoderm progenitor cells were counted after 0.25% trypsin-EDTA digestion, and cells were seeded at a density of 50,000 cells / mL (low-density group) and 100,000 cells / mL (medium-density group) on 0.1% gelatin-coated culture plates, and induced to differentiate as in the high-density group.

[0123] Cell morphology was recorded with a phase contrast microscope and a CCD camera during the induction process, and fresh medium was replaced the next day, and then every 2 days. After the P1 generation of cells was fully grown, they were digested with 0.25% trypsin-EDTA and subcultured at a density of 20,000 cells / mL to the P3 generation, and cell morphology was continuously observed during this period.

[0124] RNA extraction and reverse transcription

[0125] P1-P3 generation cells were collected, washed with DPBS, and then lysed with 500 μL Trizol, and RNA was extracted by the chloroform-isopropanol precipitation method. The RNA concentration and purity (260 / 280 and 260 / 230 ratios) were detected with a NanoDrop 2000, and the integrity of the RNA was analyzed by agarose gel electrophoresis. cDNA was synthesized by the PrimeScript RT Reagent Kit for RT-PCR.

[0126] Fluorescent quantitative PCR

[0127] A 20 μL reaction system (containing SYBR Green Master Mix, upstream and downstream primers, cDNA, etc.) was configured, and the program was pre-denatured at 95°C for 10 min, 95°C for 15 s, and 57.6°C for 45 s for 40 cycles, and the data were analyzed by the 2 -ΔΔCt method.

[0128] Flow cytometry detection

[0129] When the cell confluence reached 90%, the cells were digested with 0.25% trypsin-EDTA, fixed and washed, and then incubated with CD73, CD44 (FITC-labeled, 1:50 and 1:100 dilution) and CD34 (FITC-labeled, 1:100 dilution) antibodies in the dark, and the expression of surface markers was detected by flow cytometry (BD LSRFortessa), and the positive rate of CD73 and CD44 and the negative rate of CD34 were analyzed by Flowjo software.

[0130] Figure 13 The results show that the cell survival rate is the highest using 0.5 mmol / L EDTA, followed by 0.25% trypsin-EDTA. However, considering that mesenchymal induction digestion is most suitable for single cells, 0.25% trypsin-EDTA is selected for cell digestion after comprehensive analysis. Although the cell survival rate is the highest using 0.5 mmol / L EDTA, the cells cannot be completely digested into single cells after digestion, which is not conducive to the subsequent mesenchymal differentiation. In order to ensure high survival rate and high differentiation efficiency, the present application preferably uses 0.25% trypsin-EDTA for cell digestion and dissociation.

[0131] Figure 14 The cell density and cell morphology were observed 48 hours after inoculation. Under the microscope, it can be seen that the cells adhere well, and the cell morphology changes significantly compared with the control group, and the cell volume becomes larger. The cells inoculated at low and medium density are distributed in single cells, and the cell body is small and irregular, and the cell body edge is not clear. The cells inoculated at high density are arranged in a single layer, and the cell body edge is not clear, and the cells contact each other.

[0132] Figure 15 The cell morphology changes of mesenchymal induction cells inoculated at different densities into gelatin after mesoderm induction of hNF-C1 hiPSCs for 5 days are shown. Under the microscope, the cell volume is obviously larger, the nucleus is not obvious, the cell body edge is not clear, and the cell morphology is not uniform, and part of the cell morphology is short spindle-shaped. The cells in the low and medium density groups are arranged randomly, and the cells in the high density group may have a certain directionality.

[0133] Figure 16 The expression of MSC marker genes after mesenchymal induction of hNF-C1 hiPSCs mesoderm induction for 5 days and inoculation into gelatin at different densities is shown by RT-PCR technology. Figure 4 It can be seen that the initial differentiation density has a significant effect on mesenchymal induction and differentiation. The expression of MSC marker genes in the high density group (200,000 cells / mL) is the highest.

[0134] Figure 17The morphology of the cells after mesenchymal induction in gelatin at different densities and continuous passage for 3 times is shown. It can be seen that the cell morphology changes obviously with the increase of the passage number and the extension of the induction time. The P1 generation cells are arranged in monolayer, and the morphology is irregular, and part of the cells are short spindle-shaped. The P2 generation cell morphology is long spindle-shaped, like fibroblast morphology, and the cells are arranged regularly, and the cells arranged in vortex are visible. The P3 generation cell morphology has no obvious difference with the adult mesenchymal stem cells, and vortex-shaped colonies are formed, and arranged in polarity. The expression of mesenchymal stem cell markers can reach more than 93% after three generations, which is consistent with the expression level of the extracted human umbilical cord mesenchymal stem cell markers. The expression of mesenchymal stem cell markers after continuous passage is not much different from that of P3 generation. It can be seen that the mesenchymal stem cells prepared by the present application are very close to the level of mesenchymal stem cells directly extracted from adult human body.

[0135] Figure 18 The expression of MSCs related marker genes CD73, CD44, CD90, CD105 and CD34, CD14, CD19 in the induction process is detected by RT-PCR technology. The results show that the positive expression level of CD73, CD90 and CD105 genes increases steadily with the increase of the passage number, and the gene expression level of P3 generation is obviously higher than that of P1 generation (p<0.05), which indicates that more and more mesenchymal-like cells appear with the extension of the induction time and continuous passage.

[0136] Figure 19 The positive expression rate of MSCs related marker genes CD73, CD44 and CD34 is detected by flow cytometry. The results are basically consistent with the RT-PCR detection results. With continuous passage for three times, the positive expression amount of CD73 and CD44 gradually increases, and CD34 is negatively expressed in the whole induction process.

[0137] Example 3: Effect of culture medium on induction and differentiation of hiPSC into mesenchymal stem cells The passage culture of hiPSC is the same as that in Example 1.

[0138] hiPSC-MSCs culture

[0139] Once the confluence of hiPSCs reached 80%, the E8 medium was replaced with mesodermal induction medium to initiate mesodermal differentiation. An in vitro culture system was used, and mesodermal differentiation was induced in two stages, D0-D2. Mesodermal induction medium A was RPMI 1640 basal medium containing 213 μg / mL sodium vitamin C phosphate, 500 μg / mL recombinant human serum albumin, 6 μmol / L CHIR99021, and 10 μmol / L SB431542. Fresh mesodermal medium was added every 24 hours, and the culture was continued for 2 days.

[0140] From days 3 to 5, the mesotherapy induction medium B was RPMI 1640 basal medium containing 213 μg / mL sodium vitamin C phosphate, 500 μg / mL recombinant human serum albumin, and 10 μmol / L LY294002. Cells were washed with DPBS every 24 hours and then replaced with fresh mesotherapy induction medium. The induction was carried out at 200,000 mL / min. -1 Inoculate onto the surface of a culture plate coated with 0.1% gelatin.

[0141] Use with 5 μmol·L -1 Y-27632 mesenchymal stem cell induction medium (E8+SFM(V)) E8 :V MSC-SFM Cells were induced to differentiate into mesenchymal cells using a 1:1 ratio, resulting in generation P1. After the P1 generation cells reached confluence, they were digested with 0.25% trypsin-EDTA and passaged at a density of 20,000 cells / mL to generation P3. Cell morphology was continuously observed during this period, and RNA samples and flow cytometry samples from generations P1 and P2 were collected for analysis.

[0142] Control groups were set up separately, and each group was treated with 5 μmol·L⁻¹ solution. -1 Y-27632's E8 medium, MSC-SFM, E8+B27 (i.e., E8 medium containing 2% B27), SFM+B27 (i.e., MSC-SFM medium containing 2% B27), and MSC medium replaced with 5 μmol·L⁻¹ -1 The mesenchymal stem cell induction medium of Y-27632 was used to induce mesenchymal differentiation of cells.

[0143] Figure 20 The results showed that during the mesenchymal differentiation stage, the E8+SFM group was the most effective in promoting mesenchymal differentiation, and the composition was clearly defined, avoiding the use of serum and reducing the problem of heterologous contamination.

[0144] Example 4: Identification of hiPSC-MSCs

[0145] Cell proliferation curve

[0146] hiPSCs are induced to differentiate into mesenchymal-like cells, i.e. hiPSCs-MSCs, by using the induction system of the present application.

[0147] hiPSCs are subcultured as in Example 1.

[0148] hiPSC-MSCs culture

[0149] When the confluency of hiPSCs reaches 80%, the E8 medium is replaced with mesoderm induction medium to initiate mesoderm differentiation. The mesoderm induction differentiation is divided into two stages, D0-D2 stage, using in vitro culture system. The mesoderm induction medium A is RPMI 1640 base medium containing 213 μg / mL sodium phosphate ascorbate, 500 μg / mL recombinant human serum albumin, 6 μmol / L CHIR99021 and 10 μmol / L SB431542. Fresh mesoderm medium is replaced every 24 hours, and the culture is maintained for 2 days.

[0150] Then D3-D5, the mesoderm induction medium B is RPMI 1640 base medium containing 213 μg / mL sodium phosphate ascorbate, 500 μg / mL recombinant human serum albumin and 10 μmol / L LY294002. Fresh mesoderm induction medium is replaced every 24 hours after the cells are washed with DPBS.

[0151] The cells are dissociated by digestion with 0.25% trypsin-EDTA, and are seeded at a density of 200,000 cells / mL in a 6-well plate. -1 The cells are seeded on the surface of a culture plate coated with 0.1% gelatin, and are induced to differentiate into mesenchymal cells using mesenchymal induction medium containing 5 μmol / L Y-27632. -1 The mesenchymal induction medium containing Y-27632 is used to induce the cells to differentiate into mesenchymal cells, and the P1 generation is obtained. After the P1 generation cells are fully grown, they are digested with 0.25% trypsin-EDTA, and are cultured at a density of 20,000 cells / mL until the cells are fully grown, and the P2 generation is obtained. The culture is subcultured to the P5 generation. RNA samples and flow cytometry samples of the P1 to P5 generations are collected for detection.

[0152] hiPSCs-MSCs of the P5 generation are selected to draw cell proliferation curves using a CCK-8 kit. hiPSCs-MSCs obtained by induction are seeded on the surface of gelatin at a cell density of 10,000 cells / mL, and human bone marrow mesenchymal stem cells (hBMSCs) of the P5 generation are selected as controls. The cells are cultured using mesenchymal induction medium, and fresh medium is replaced every 48 hours. CCK-8 diluent is used to incubate the cells in the dark for 2 hours at 1, 3 and 7 days of culture, respectively, and then the absorbance value at 450 nm is measured by a Bio-Rad full-wavelength enzyme marker. -1

[0153] ​Osteogenic induction of hiPSCs-MSCs

[0154] hiPSCs-MSCs cultured to P5 were selected for multipotent induction differentiation. In osteogenic induction, when the confluence of hiPSCs-MSCs reached 80%, the culture medium was replaced with osteogenic induction medium (DMEM / F12 basal medium containing 15% FBS, 1% non-essential amino acids, 1% L-glutaMax, 0.1 mmol·L -1 β-mercaptoethanol, 1% penicillin / streptomycin double-antibiotic solution, 50 μg·mL -1 vitamin C, 10 mmol·L -1 sodium glycerophosphate and 10 -8 mol·L -1 dexamethasone) for 14 days, and fresh osteogenic induction medium was replaced every two days. During this period, the cell morphology in osteogenic induction was recorded using phase contrast microscopy and a CCD camera.

[0155] Alizarin red staining

[0156] An alizarin red solution with a mass fraction of 2% and a pH of 4.2 was prepared based on a 0.01 mol·L -1 Tris buffer solution and saturated NaOH solution. After mixing, it was filtered through a medium-speed filter paper. The cell samples in the 24-well plate were washed with DPBS solution and then fixed with 4% paraformaldehyde solution at room temperature for 30 min. They were washed with DPBS 3 times, 500 μL of the alizarin red solution was added to each well, and they were observed under a microscope. After about 10 min of reaction (observing while staining to avoid non-specific staining), they were repeatedly washed with pure water until the solution was clear. Then, the plate was photographed using a camera and an inverted microscope containing a CCD, and the alizarin red staining of the cells was photographed (the plate should not be too dry when taking pictures, and an appropriate amount of water should be added).

[0157] Adipogenic induction of hiPSCs-MSCs

[0158] hiPSCs-MSCs cultured to P5 were selected for multipotent induction differentiation. In adipogenic induction, when the confluence of hiPSCs-MSCs reached 80%, the culture medium was replaced with adipogenic induction medium (high-sugar DMEM basal medium containing 10% FBS, 1% penicillin / streptomycin double-antibiotic solution, 10 μg·mL -1 insulin, 200 μM indomethacin, 500 μM 3-isobutyl-1-xanthine and 1 μM dexamethasone) for 21 days, and fresh adipogenic induction medium was replaced every three days. During this period, the cell morphology in osteogenic induction was recorded using phase contrast microscopy and a CCD camera.

[0159] Oil red O staining

[0160] The cell samples in the 24-well plate were washed with DPBS solution and then fixed with 4% paraformaldehyde solution at room temperature for 30 min. After washing with DPBS for 3 times, 500 μL of oil red O staining solution was added to each well, and the staining was performed at room temperature for 30 min, followed by gentle washing with PBS for 2-3 times. 500 μL of DPBS was added to each well, and the plate photographing and cell staining photographing were performed by using an inverted microscope with CCD.

[0161] hiPSCs-MSCs were selected at P5 for multi-potential induction and differentiation. In the chondrogenic induction, when the confluence of the hiPSCs-MSCs reached 80%, the culture medium was replaced with chondrogenic induction medium (high glucose DMEM basal medium containing 1% penicillin / streptomycin double-antibiotic solution, 50 μg·mL -1 ascorbic acid-2-phosphate, 40 μg·mL -1 L-proline, 100 μg·mL -1 sodium pyruvate, 1x insulin-transferrin-selenium, 10 ng mL -1 TGF-β3 and 0.1 μM dexamethasone) for chondrogenic induction for 21 days, and the fresh chondrogenic induction medium was replaced every three days. The cell morphology in the chondrogenic induction was recorded by using a phase contrast microscope and a CCD camera.

[0162] Alcian blue staining

[0163] The cell samples in the 24-well plate were washed with DPBS solution and then fixed with 4% paraformaldehyde solution at room temperature for 30 min. After washing with DPBS for 3 times, 500 μL of oil red O staining solution was added to each well, and the staining was performed at room temperature for 30 min, followed by gentle washing with PBS for 2-3 times. 500 μL of DPBS was added to each well, and the plate photographing and cell staining photographing were performed by using an inverted microscope with CCD.

[0164] Figure 22 The staining results of hiPSCs-MSCs and hBMSCs after osteogenic, adipogenic and chondrogenic induction for 21 days are shown. As can be seen from the figures, the hiPSCs-MSCs have similar in vitro proliferation ability and multi-directional differentiation potential as the adult MSCs.

[0165] The above description of the present application and its embodiments is not restrictive, and the embodiments shown in the examples are only one of the embodiments of the present application, and the actual structure is not limited thereto. In general, if a person skilled in the art is inspired thereby, without departing from the purpose of the present application, without creative design, similar structure and embodiments of the technical solutions can be designed, which shall belong to the protection scope of the present application.

Claims

1. An induction culture system for differentiating human induced pluripotent stem cells into mesenchymal stem cells, characterized by, The induction culture system comprises three kinds of culture media, mesoderm induction medium A, mesoderm induction medium B and mesenchymal induction medium. The mesoderm induction medium A is composed of the following components: The mesoderm induction medium B is composed of the following components: The mesenchymal induction medium is prepared by mixing E8 and MSC-SFM at a volume ratio of 1:1 and containing 5 μmol / L Y-27632.

2. The use of the induction culture system for directing the differentiation of human induced pluripotent stem cells into mesenchymal stem cells in the induction of human induced pluripotent stem cells into mesenchymal stem cells.

3. A method for the stage-wise induction of the directed differentiation of human induced pluripotent stem cells into mesenchymal stem cells based on the induction culture system according to claim 1, characterized in that, It comprises the following steps: ① Culturing human induced pluripotent stem cells in the mesoderm induction medium A of claim 1 for 2 days, and replacing the fresh mesoderm induction medium A every 24 hours; ② Replacing the mesoderm induction medium A with the mesoderm induction medium B of claim 1 and continuing to culture the cells for 3 days, and replacing the fresh mesoderm induction medium B every 24 hours, then the human induced pluripotent stem cells can be differentiated into mesoderm cells; ③ Washing and digesting the cells induced and differentiated in step ② into single cells, then adding the mesenchymal induction medium for culture, replacing the fresh mesenchymal induction medium every 24 hours, and continuing to subculture the cells for 1 generation or more when the confluence of the cells reaches 100%.

4. The method of claim 3, wherein the method is for inducing differentiation of human induced pluripotent stem cells into mesoderm cells. In step ①, the human induced pluripotent stem cells with a confluence of 70%-85% are added into the mesoderm induction medium A for culture.

5. The method for directing differentiation of human induced pluripotent stem cells into mesenchymal stem cells in stages according to claim 3, characterized in that, In step 3, the cells after dissociation by digestion were seeded onto the culture plate at a cell density of 200,000 cells mL -1 for culture.

6. The method for differentiating human induced pluripotent stem cells into mesenchymal stem cells in stages according to claim 3, characterized in that, In step ③, the digested and dissociated cells are inoculated into gelatin-coated culture plates for culture.

7. The method for directing differentiation of human induced pluripotent stem cells into mesenchymal stem cells in stages according to claim 6, characterized in that, In step ③, the cells are digested and dissociated by using trypsin-EDTA digestion solution.

8. The mesenchymal stem cells prepared by the method for directing the differentiation of human induced pluripotent stem cells into mesenchymal stem cells in stages according to any one of claims 3 to 7.

9. The use of the mesenchymal stem cells prepared by the method for directing the differentiation of human induced pluripotent stem cells into mesenchymal stem cells in stages according to any one of claims 3 to 7 in the preparation of bone tissue engineering or immunotherapy products.

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