A culture medium, method and use for inducing embryonic stem cells to differentiate into ectoderm chondrogenic progenitor cells

Abstract

The present application relates to the field of stem cell technology, and in particular to a culture medium, method and use for inducing embryonic stem cells to differentiate into ectodermal chondrocytes. The present invention can efficiently differentiate ectodermal chondrocytes by step-by-step induction in different culture media, and the obtained ectodermal chondrocytes have good stability, proliferation ability and potential for spontaneous differentiation into chondrocytes; compared with traditional mesoderm-derived chondrocytes, the ectodermal chondrocytes differentiated by the present invention are closer to the temporomandibular joint condylar chondrocytes in the entire expression spectrum, and have extremely high research value for cartilage originating from the ectoderm.

Description

Technical Field

[0001] The present application relates to the field of stem cell technology, and in particular to a culture medium, method and use for inducing directed differentiation of embryonic stem cells into ectoderm chondrogenic progenitor cells. Background Art

[0002] Cartilage is an avascular tissue. Once it is damaged or destroyed, it is extremely difficult to repair. At present, with the transformation of basic research into clinical medicine, stem cell therapy has become a hot topic of research at home and abroad. Mesenchymal stem cells (MSCs) have become the most commonly used seed cells due to their abundant sources and convenient collection. However, due to uncontrollable factors such as their limited proliferation ability, multiple differentiations and lack of load-bearing capacity of differentiated cells, the application of BMSCs in clinical repair of articular cartilage is limited. Embryonic stem cells (ESCs) are cells with multidirectional differentiation potential, and inducing them to differentiate into chondrocytes has also attracted the attention of many research groups. Using ESCs to achieve directed differentiation into cartilage is expected to become an effective solution for repairing damaged cartilage tissue.

[0003] At present, a considerable amount of data has been accumulated on the experimental research on the directed differentiation of ESCs into cartilage. Most basic and clinical research focuses on differentiating ESCs into chondrocytes via the mesoderm pathway. Although these technical methods have good application prospects in the repair of other joint cartilages, they are not applicable to the field of temporomandibular joint (TMJ). The reason is that during embryonic development, the TMJ is significantly different from other large joints in the body. In terms of germ layer origin, almost all articular cartilage comes from the mesoderm, while the TMJ condylar cartilage comes from the neural ectoderm. This leads to significant differences between the TMJ condylar cartilage and other articular cartilages in terms of biological composition, tissue structure, body metabolism, and growth pattern. Therefore, the mainstream ESCs-to-chondrogenic induction method is not suitable for the repair of TMJ cartilage damage.

[0004] With the in-depth development of basic research, evidence of regulating stem cell fate through spatiotemporal control of small molecules is gradually increasing. Using a step-by-step induction method to simulate the developmental pathway of TMJ condylar cartilage, ESCs can be differentiated into a type of chondrogenic stem cell via the neuroectoderm pathway, which is expected to provide new ideas and cell sources for the repair of TMJ condylar cartilage damage. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide a method and use of inducing embryonic stem cells to differentiate into ectoderm chondrogenic progenitor cells, so as to solve the problems in the prior art.

[0006] To achieve the above-mentioned purpose and other related purposes, the first aspect of the present application provides a culture medium for inducing embryonic stem cells to differentiate into ectodermal chondrogenic progenitor cells in vitro, the culture medium comprising a neural crest induction medium and / or a CSSEDF culture medium, the components of the neural crest induction medium comprising a basal culture medium, BMP4 and a TGF-β receptor inhibitor; the components of the CSSEDF culture medium comprising a basal culture medium, a GSK-3 inhibitor, a TGF-β receptor inhibitor, a smoothened agonist, EGF, FGF2 and a BMP receptor inhibitor.

[0007] The second aspect of the present application provides the use of the culture medium for inducing embryonic stem cells to differentiate into ectoderm chondrogenic progenitor cells in vitro.

[0008] The third aspect of the present application provides a method for inducing embryonic stem cells to differentiate into ectodermal chondrogenic progenitor cells in vitro, comprising the following steps:

[0009] 1) Resuscitating and expanding embryonic stem cells to obtain cells for subculture;

[0010] 2) Inoculating the subcultured cells in step 1) into the culture medium for culture to obtain ectoderm chondrogenic progenitor cells.

[0011] A fourth aspect of the present application provides an ectodermal chondrocyte progenitor cell, wherein the ectodermal chondrocyte progenitor cell is prepared by the method described.

[0012] The fifth aspect of the present application provides the use of the ectodermal chondrogenic cells in the preparation of a product for repairing cartilage defects.

[0013] Compared with the prior art, the beneficial effects of this application are:

[0014] 1. It can efficiently differentiate into ectodermal chondrocytes, and the ectodermal chondrocytes obtained have good stability, proliferation ability and potential to spontaneously differentiate into chondrocytes.

[0015] 2. Compared with traditional mesoderm-derived chondrocytes, the ectoderm chondrocytes differentiated by the present invention are closer to the temporomandibular joint condylar chondrocytes in terms of the entire expression spectrum, and have extremely high research value for cartilage originating from the ectoderm. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1a It is a schematic diagram of the step-by-step induction of embryonic stem cells to differentiate into ectoderm chondrogenic progenitor cells in the present invention.

[0017] Figure 1b PCA diagram of transcriptome data of different cell lines at each time point.

[0018] FIG. 2 is a diagram for verifying the differentiation pathway of mesoectodermal chondrogenic cells of the present invention. Figure 2a Bulk RNA-seq cluster analysis of ectoderm marker expression during induction. Figure 2b Bulk RNA-seq cluster analysis of neural crest marker expression during induction. Figure 2c GO enrichment analysis was performed to identify differentially expressed genes in the osteo-cartilage system during cell induction. Figure 2d Bulk RNA-seq cluster analysis of cartilage marker expression during induction.

[0019] Figure 3 This is a graph for detecting the self-renewal ability of mesodermal chondrogenic progenitor cells of the present invention. Figure 3 a is a diagram of single-cell clone formation of ectoderm chondrogenic progenitor cells. Figure 3 b is the cell immunofluorescence staining of SIX1, an ectoderm marker of ectoderm chondrogenic progenitor cells. Figure 3 c is the cell immunofluorescence staining of NESTIN, an ectoderm marker of ectoderm chondrogenic progenitor cells. Figure 3 d is the cell immunofluorescence staining of ETS1, an ectoderm marker of ectoderm chondrogenic progenitor cells. Figure 3 e is the cell immunofluorescence staining of SOX9, a cartilage marker of ectodermal chondrogenic progenitor cells. Figure 3 f is the cell immunofluorescence staining of RUNX2, a cartilage marker of ectodermal chondrogenic progenitor cells. Figure 3 g is the cell immunofluorescence staining of SOX5, a cartilage marker of ectodermal chondrogenic progenitor cells. Figure 3 h is the cell immunofluorescence staining of TWIST1, a cartilage marker of ectodermal chondrogenic progenitor cells. Figure 3 i is an immunofluorescence staining image of the cartilage marker CD29 of ectodermal chondrogenic cells. Figure 3 j is the cell immunofluorescence staining of FOXC1, a maxillofacial development marker of ectodermal chondrogenic progenitor cells. Figure 3 k is the cell immunofluorescence staining image of FOXC2, a maxillofacial development marker of ectodermal chondrogenic progenitor cells. Figure 3 l is the cell immunofluorescence staining of MSX1, a maxillofacial development marker of ectodermal chondrogenic progenitor cells. Figure 3 m is the flow cytometric analysis of Ki67 and cartilage-related marker genes SOX5, SOX9, TWIST1, and CD29 in ectoderm chondrogenic progenitor cells of different generations. Figure 3 n is the expression of cartilage-related markers SOX9, COL1A1, SOX5, COL2A1, OCN, RUNX2, and ACAN in ectodermal chondrogenic progenitor cells of different generations.

[0020] Figure 4 This is a graph showing the effect of the spontaneous differentiation potential of ectodermal chondrogenic cells of the present invention. Figure 4 a is a general picture of ectodermal chondrogenic progenitor cells after spontaneous differentiation and culture. Figure 4 b is the HE staining image of ectoderm chondrogenic progenitor cells after spontaneous differentiation culture. Figure 4 c is the trypan blue staining image after spontaneous differentiation culture of ectoderm chondrogenic progenitor cells. Figure 4 d is the Alcian blue staining image of ectoderm chondrogenic progenitor cells after spontaneous differentiation culture. Figure 4 e is the image of Safranin O fast green staining after spontaneous differentiation of ectodermal chondrogenic progenitor cells. Figure 4 f is the Collagen II immunohistochemical staining after spontaneous differentiation of ectoderm chondrogenic progenitor cells. Figure 4 g is the Collagen X immunohistochemical staining of ectoderm chondrogenic progenitor cells after spontaneous differentiation culture. Figure 4 h is the immunohistochemical staining of Aggrecan after spontaneous differentiation of ectodermal chondrogenic progenitor cells. Figure 4 i is the immunohistochemical staining of RUNX2 after spontaneous differentiation of ectodermal chondrogenic progenitor cells. Figure 4 j is the immunohistochemical staining of Collagen I after spontaneous differentiation of ectoderm chondrogenic progenitor cells. Figure 4 k is the immunohistochemical staining of Lubricin after spontaneous differentiation of ectoderm chondrogenic progenitor cells.

[0021] FIG5 is a single cell transcriptome expression diagram of mesoectodermal chondrocytes, classical mesodermal chondrocytes and human ectodermal condylar chondrocytes of the present invention. Figure 5a Single-cell transcriptome expression profile of human ectodermal condylar chondrocytes. Figure 5b Single-cell transcriptome expression profile of ectodermal chondrogenic progenitor cells. Figure 5c This is a functional clustering analysis of single-cell transcriptomes of ectodermal chondrocytes, classical mesodermal chondrocytes and human ectodermal condylar chondrocytes.

[0022] Figure 6 This is a diagram showing the effect of the mesodermal chondrogenic progenitor cells of the present invention repairing cartilage defects in vivo. Figure 6 a is the knee cartilage defect and repair model. Figure 6 b is the GFP green fluorescence of ectoderm chondrogenic cells repairing cartilage defects in vivo observed directly under a stereoscope. Figure 6 c is the GFP green fluorescence of the control group (single sponge scaffold) repairing cartilage defects in vivo observed directly under a stereoscope. Figure 6 d is a picture of GFP green fluorescence in cartilage tissue sections after ectoderm chondrogenic cells repaired cartilage defects in vivo, observed under a fluorescence microscope. Figure 6e is the GFP green fluorescence of cartilage tissue sections observed under fluorescence microscopy after the control group (single sponge scaffold) repaired the cartilage defect in vivo. Figure 6 f is the HE staining image of cartilage tissue section after ectoderm chondrogenic cells repaired cartilage defects in vivo. Figure 6 g is the Collagen I immunohistochemical staining of cartilage tissue section after ectoderm chondrogenic cells repaired cartilage defects in vivo. Figure 6 h is the CollagenII immunohistochemical staining of cartilage tissue section after ectoderm chondrogenic cells repaired cartilage defects in vivo. Figure 6 i is a picture of cartilage tissue section stained with safranin O fast green after ectodermal chondrogenic cells repaired cartilage defects in vivo. Figure 6 j is the Collagen X immunohistochemical staining of cartilage tissue sections after ectoderm chondrogenic cells repaired cartilage defects in vivo. Figure 6 k is the Lubricin immunohistochemical staining of cartilage tissue sections after ectoderm chondrogenic cells repaired cartilage defects in vivo. DETAILED DESCRIPTION

[0023] In order to make the invention purpose, technical scheme and beneficial effect of the present application clearer, the present application is further described below in conjunction with the examples. It should be understood that the examples are only used to explain the present application and are not used to limit the scope of the application. The test methods used in the following examples are conventional methods unless otherwise specified, and those familiar with the technology can easily understand other advantages and effects of the present application from the contents disclosed in this description.

[0024] After extensive exploration and research, the inventors of the present application have discovered a method and use for inducing embryonic stem cells to differentiate into ectodermal chondrocytes. Through the step-by-step induction method in different culture media, ectodermal chondrocytes can be efficiently differentiated and obtained, and the obtained ectodermal chondrocytes have good stability, proliferation ability and the potential to spontaneously differentiate into chondrocytes. Most importantly, compared with traditional mesoderm-derived chondrocytes, the ectodermal chondrocytes differentiated by the present invention are closer to the temporomandibular joint condylar chondrocytes in the entire expression spectrum. On this basis, the present application was completed.

[0025] The present application provides a culture medium for inducing embryonic stem cells to differentiate into ectodermal chondrogenic progenitor cells in vitro, characterized in that the culture medium comprises a neural crest induction medium and / or a CSSEDF medium, the components of the neural crest induction medium comprising a basal culture medium, BMP4 and a TGF-β receptor inhibitor; the components of the CSSEDF culture medium comprising a basal culture medium, a GSK-3 inhibitor, a TGF-β receptor inhibitor, a smoothened agonist, EGF, FGF2 and a BMP receptor inhibitor.

[0026] In the medium for inducing embryonic stem cells to differentiate into ectodermal chondrogenic progenitor cells in vitro provided in the present application, when the medium includes a neural crest induction medium and a CSSEDF medium, it means that two culture media are used respectively during the culture process. The neural crest induction medium is used to induce neural crest cells, and the CSSEDF medium is used to promote the self-renewal of chondrogenic stem cells.

[0027] In the medium for inducing embryonic stem cells to differentiate into ectoderm chondrogenic progenitor cells in vitro provided by the present application, the concentration of BMP4 in the neural crest induction medium is 0.1-100 ng / ml based on the total volume of the basal medium. Specifically, the concentration of BMP4 can be 0.1-10 ng / ml, 10-20 ng / ml, or 20-100 ng / ml. In a preferred embodiment, the concentration of BMP4 is 20 ng / ml. The BMP4 refers to the addition of bone morphogenetic protein 4.

[0028] In the medium for inducing embryonic stem cells to differentiate into ectodermal chondrogenic progenitor cells in vitro provided by the present application, the amount of the TGF-β receptor inhibitor added in the neural crest induction medium is 0.01 to 100 μM based on the total volume of the basal medium. Specifically, the amount of the TGF-β receptor inhibitor added can be 0.01 to 2 μM, 2 to 20 μM, or 20 to 100 μM, etc. In a preferred embodiment, the concentration of the TGF-β receptor inhibitor is 2 μM. The TGF-β is a transforming growth factor. The TGF-β receptor inhibitor is selected from 616452, LY2109761, Pirfenidone, Repsox (E-616452), SB431542, A77-01, Tranilast, Galunisertib (LY2157299), A8301, GW788388, ITD-1, SD208, SB525334, LY364947, ASP3029, D4476 and SB505124. In a preferred embodiment, the TGF-β receptor inhibitor is selected from SB431542.

[0029] In the culture medium for inducing embryonic stem cells to differentiate into ectoderm chondrogenic progenitor cells in vitro provided by the present application, in the CSSEDF culture medium, the amount of the GSK-3 inhibitor added is 0.1 to 50 μM based on the total volume of the basal culture medium. Specifically, the amount of the GSK-3 inhibitor added can be 0.1 to 1 μM, 1 to 10 μM, or 10 to 50 μM, etc. In a preferred embodiment, the concentration of the GSK-3 inhibitor is 1 μM. The GSK3 refers to a serine protein kinase. The GSK-3 inhibitor is selected from a combination of one or more of CHIR99021, BIO and LY2090314. In a preferred embodiment, the GSK-3 inhibitor is selected from CHIR99021.

[0030] In the culture medium for inducing embryonic stem cells to differentiate into ectoderm chondrogenic progenitor cells in vitro provided by the present application, in the CSSEDF culture medium, the amount of the TGF-β receptor inhibitor added is 0.01 to 100 μM based on the total volume of the basal culture medium. Specifically, the amount of the TGF-β receptor inhibitor added can be 0.01 to 2 μM, 2 to 10 μM, or 10 to 100 μM, etc. In a preferred embodiment, the concentration of the TGF-β receptor inhibitor is 2 μM. The TGF-β receptor inhibitor is selected from 616452, LY2109761, Pirfenidone, Repsox (E-616452), SB431542, A77-01, Tranilast, Galunisertib (LY2157299), A8301, GW788388, ITD-1, SD208, SB525334, LY364947, ASP3029, D4476 and SB505124. In a preferred embodiment, the TGF-β receptor inhibitor is selected from SB431542.

[0031] In the culture medium for inducing embryonic stem cells to differentiate into ectoderm chondrogenic progenitor cells in vitro provided by the present application, in the CSSEDF culture medium, based on the total volume of the basal culture medium, the amount of the smoothened agonist added is 0.01-20 μM. Specifically, the amount of the smoothened agonist added can be 0.01-0.2 μM, 0.2-5 μM, 5-10 μM, or 10-20 μM, etc. In a preferred embodiment, the concentration of the smoothened agonist is 0.2 μM. The smoothened agonist is selected from a combination of one or more of purmorphamine, SAG and GSA 10. In a preferred embodiment, the smoothened agonist is selected from SAG.

[0032] In the culture medium for inducing embryonic stem cells to differentiate into ectodermal chondrogenic progenitor cells in vitro provided by the present application, the concentration of EGF in the CSSEDF culture medium is 0.1-500 ng / ml based on the total volume of the basal culture medium. Specifically, the concentration of EGF can be 0.1-10 ng / ml, 10-20 ng / ml, or 20-500 ng / ml, etc. In a preferred embodiment, the concentration of EGF is 20 ng / ml. The EGF refers to epidermal growth factor.

[0033] In the culture medium for inducing embryonic stem cells to differentiate into ectoderm chondrogenic progenitor cells in vitro provided by the present application, the concentration of FGF2 in the CSSEDF culture medium is 0.1-500ng / mlng / ml based on the total volume of the basal culture medium. Specifically, the concentration of FGF2 can be 0.1-15ng / ml, 15-20ng / ml, or 20-500ng / ml, etc. In a preferred embodiment, the concentration of FGF2 is 20ng / ml. The FGF2 refers to basic fibroblast growth factor.

[0034] In the culture medium for inducing embryonic stem cells to differentiate into ectodermal chondrogenic progenitor cells in vitro provided by the present application, in the CSSEDF culture medium, the amount of the BMP receptor inhibitor added is 0.01 to 50 μM, based on the total volume of the basal culture medium. Specifically, the amount of the BMP receptor inhibitor added can be 0.01 to 1 μM, 1 to 10 μM, or 10 to 50 μM, etc. In a preferred embodiment, the concentration of the BMP receptor inhibitor is 1 μM. The BMP refers to bone morphogenetic protein. The BMP receptor inhibitor is selected from a combination of one or more of DMH1, K02288, LDN-193189, and Noggin. In a preferred embodiment, the BMP receptor inhibitor is selected from DMH1.

[0035] In the culture medium for inducing embryonic stem cells to differentiate into ectoderm chondrogenic progenitor cells in vitro provided by the present application, the basal culture medium is selected from one or more of MEM, DEM, DEM / F12, RPMI1640 or F12. Preferably, the basal culture medium is DEM / F12 containing N2 and B27. Preferably, based on the volume of the basal culture medium, the concentration of N2 is 0.1-10% (v / v), specifically, the concentration of N2 is 0.1-0.5%, 0.5-1%, or 1-10%, etc. Preferably, based on the volume of the basal culture medium, the concentration of B27 is 0.1-10% (v / v). Specifically, the concentration of B27 is 0.1-0.5%, 0.5-1%, or 1-10%, etc. In a preferred embodiment, the concentration of N2 is 0.5% (v / v) and the concentration of B27 is 0.5% (v / v). Both the N2 and the B27 refer to a serum-free cell culture supplement.

[0036] The present application also provides the use of the culture medium for inducing embryonic stem cells to differentiate into ectoderm chondrogenic progenitor cells in vitro. A person skilled in the art can select the culture medium for inducing differentiation of embryonic stem cells according to actual needs.

[0037] The present application also provides a method for inducing embryonic stem cells to differentiate into ectoderm chondrogenic progenitor cells in vitro, comprising the following steps:

[0038] 1) Resuscitating and expanding embryonic stem cells to obtain cells for subculture;

[0039] 2) Inoculating the subcultured cells in step 1) into the culture medium for culture to obtain ectoderm chondrogenic progenitor cells.

[0040] In the method for inducing embryonic stem cells to differentiate into ectodermal chondrogenic progenitor cells in vitro provided by the present application, in step 1), the embryonic stem cells are selected from one of Hues9, Hues 7, H9, and reprogrammed cells (ihPSCs). The recovery refers to the process in a medium coated with matrix gel. The passaging refers to the process in which cells stop growing due to contact inhibition when they grow to a certain density in a culture bottle, after which they need to be diluted and divided before the cells can continue to grow.

[0041] In the method for inducing embryonic stem cells to differentiate into ectodermal chondrogenic progenitor cells in vitro provided in the present application, in step 2), the cells are first cultured in a neural crest induction medium in the culture medium for 2 to 10 days, specifically, 2 to 5 days, 5 to 7 days, 7 to 9 days, or 9 to 10 days; and then cultured in a CSSEDF medium in the culture medium until the target cells are obtained. The CSSEDF culture medium can be used for a long time during cell passage. In a preferred embodiment, the cells are first cultured in a neural crest induction medium in the culture medium for 7 days, and then cultured in a CSSEDF medium in the culture medium for 23 days. The step-by-step induction method can simulate the developmental pathway of condylar chondrocytes of the temporomandibular joint, and realize the differentiation of embryonic stem cells into chondrogenic stem cells through the neural ectodermal pathway.

[0042] The present application also provides an ectodermal chondrocyte, which is prepared by the method described above. Compared with traditional mesoderm-derived chondrocytes, the ectodermal chondrocytes differentiated by the present invention are closer to temporomandibular joint condylar chondrocytes in terms of the entire expression spectrum.

[0043] The present application also provides the use of ectodermal chondrogenic cells in the preparation of products for repairing cartilage defects, which is helpful in providing new ideas and cell sources for the repair of damaged condylar cartilage of the temporomandibular joint.

[0044] The present application is further described below by way of examples, but the scope of the present application is not limited thereby.

[0045] The types of reagents used in the examples are shown in Table 1:

[0046] Table 1 Reagent information

[0047]

[0048]

[0049] Example 1

[0050] Step-by-step induction of embryonic stem cells into ectodermal chondrogenic progenitor cells

[0051] (A) In Essential 8 TM Four commonly used embryonic stem cell lines (Hues9, Hues7, H9 and H1) and reprogrammed cells (ihPSCs) were revived and expanded in culture medium, and the medium was changed regularly, and the cells were counted and passaged.

[0052] (B) Hues9, Hues 7, H9, H1 and ihPSCs were cultured at a rate of 1*10 5The cells were seeded at a density of 10 cells / dish, and the culture medium was changed on the next day: 20 ng / ml BMP4 and 2 μM SB431542 were added to the DMEM / F12 basal culture medium containing N2 and B27, and the cells were cultured in this culture medium for 7 days.

[0053] (C) Subsequently, the culture medium was replaced with CSSEDF medium, the specific components of which are as follows: 1 μM CHIR99021, 2 μM SB431542, 0.2 μM SAG, 20 ng / ml EGF, 20 ng / ml FGF2 and 1 μM DMH1 were added to the DMEM / F12 basal medium containing N2 and B27. The various cell lines were cultured in this medium for 23 days to obtain cells differentiated from Hues9, Hues7, H9, H1 and ihPSCs.

[0054] (D) Cells were collected on days 0, 3, 7, 16, and 30, and intracellular RNA was extracted for transcriptome sequencing. The steps for RNA extraction are as follows:

[0055] (1) Discard the culture medium in the cells and add the RNA extraction reagent Trizol;

[0056] (2) Centrifuge the Trizol-lysed cells at 12,000 rpm for 10 min at 4°C and transfer the supernatant to a DEPC-Treated EP tube;

[0057] (3) Add chloroform (1 / 5 the volume of Trizol), shake to mix, and centrifuge at 8000 rpm for 15 min at 4°C;

[0058] (4) Carefully transfer about 400 μl of the supernatant to a new DEPC-Treated EP tube, add an equal volume of isopropanol, mix well, and place at -20°C for half an hour;

[0059] (5) Take out the EP tube and centrifuge at 4°C, 8000 rpm, for 15 min;

[0060] (6) Discard the supernatant, being careful not to pour out the precipitate. Wash the precipitate with 75% alcohol (prepared with DEPC water), centrifuge at 4°C, 7500 rpm, 10 min, and repeat once.

[0061] (7) Air dry in a clean bench to completely remove all alcohol, and dissolve the RNA in about 20 μl of DEPC water;

[0062] (8) Take 2 μl of the obtained RNA to measure the concentration and pay attention to the purity. Store the rest at -80°C.

[0063] The process diagram is as follows Figure 1a shown. Figure 1bThe PCA graph of the transcriptome data of different cell lines at each time point. As shown in Figure 1, the induction method has good consistency and repeatability among different cell lines.

[0064] Example 2

[0065] Verification of the differentiation pathway of embryonic stem cells into ectodermal chondrogenic progenitor cells

[0066] The data of transcriptome sequencing at each time point in step (D) of Hues9, Hues7, H9, H1 and ihPSCs obtained in Example 1 were analyzed, with reference to, for example, Chia-Lung Wu et al., Single cell transcriptomic analysis of human pluripotent stem cell chondrogenesis, Nature Communications, Vol. 13, No. 12 (1), 2021, and the analysis content is as follows:

[0067] (A) Cluster analysis of the expression of key ectoderm markers in each cell line during the induction process.

[0068] (B) Cluster analysis of the expression of key neural crest markers in each cell line during the induction process.

[0069] (C) GO enrichment analysis compared the enrichment of differentially expressed genes in the bone-cartilage system.

[0070] (D) Cluster analysis of the expression of key markers of cartilage development in each cell line during the induction process.

[0071] The test results are shown in Figure 2. Figure 2a It can be seen that ectoderm-related markers FOXC1, FOXD1, TWIST1, SIX1, SIX2, ETS1, ZIC1, VIM and ZBTB17 are highly expressed in the induced cells; Figure 2b It can be seen that neural crest-related marker genes are highly expressed in cells during and after induction; Figure 2c It can be seen that the bone-cartilage development pathway, especially craniofacial bone development, is enriched in the induced cells; Figure 2d It can be seen that the key genes for cartilage development are highly expressed in the induction process and in the cells after induction.

[0072] Example 3

[0073] Identify the self-renewal capacity of induced cells

[0074] (A) Clone formation experiment: Hues9 chondrogenic progenitor cells (ECC) in Example 1 that were growing logarithmically after CSSEDF induction were digested with trypsin into single cells and the cells were counted. According to the counting results, the cells were diluted to 1×10 3 / mL, then inoculate into 6-well plates at 100, 200, and 400 / well, and shake the culture plate crosswise to disperse the cells evenly. Culture in a cell culture incubator at 37°C, 5% CO2, and saturated humidity for 2 to 3 weeks. Observe every day, and terminate the culture when visible clones appear in the culture dish. Count the number of clones greater than 10 cells under a microscope.

[0075] (B) Cell immunofluorescence staining:

[0076] (1) Take the cell slide out of the 4°C refrigerator, place it on a tray to warm up, attach a label (experiment name, antibody name, secondary antibody fluorescent name and date), and let it dry.

[0077] (2) Dilute the primary antibody with light microscopy antibody diluent at a dilution of 1:200-2000. Mix thoroughly and add to the slide, 50-100 μl per slide, and incubate at 4°C for 16-24 hours.

[0078] (3) After rewarming, discard the primary antibody working solution (see Table 1), add 100 μl 0.01M PBS, and change the PBS every 5 minutes for a total of 3 times. Be careful not to rinse the slide when changing the solution. (Keep away from light during the following steps)

[0079] (4) Dilute the fluorescent secondary antibody (see Table 1) with 0.01M PBS. The secondary antibody is a fluorescent antibody labeled with FITC or AlexFluo594, and the antibody dilution is 1:100.

[0080] (5) Aspirate the nail polish and surrounding water, add fluorescent secondary antibody, and incubate at 37°C for 45 min.

[0081] (6) Aspirate and discard the primary antibody working solution, add 100 μl of 0.01 M PBS, and change PBS every 5 minutes for a total of 3 times.

[0082] (7) Sealing: Scrape off the nail polish, seal the slide with fluorescent mounting medium, and apply nail polish around the coverslip to fix it. The slices can be observed immediately or stored at -20℃. After taking the slices out of the -20℃ refrigerator, they need to be placed in a dark place to warm up.

[0083] (C) Flow cytometry was used to detect the expression of key cartilage markers in different generations of cells after induction:

[0084] (1) The cultured cells were trypsinized into single cells and filtered through a BD 40 μm filter.

[0085] (2) Add the single cell suspension to a 2 mL round-bottom centrifuge tube and centrifuge at 1500 rpm for 5 min. Discard the supernatant. Wash once with 1 mL of PBS and centrifuge. Repeat three times.

[0086] (3) Add 200 μl of fluorescein-labeled antibody diluted with PBA, gently pipette and mix, incubate at 4°C or on ice for 30 min to 1 h. Centrifuge and discard the supernatant. Add 1 mL of cold PBS and centrifuge twice to remove excess unbound antibody components.

[0087] (4) Add 500 μl of cold PBS to the cells, pipette to mix, place in a flow cytometry tube, and store at 4°C in the dark until assayed.

[0088] (D) PCR detection of the expression of cartilage markers in cells of different generations after induction: Total RNA was extracted, reverse transcribed into cDNA, and detected by PCR instrument.

[0089] Test results Figure 3 As shown. Figure 3 a As can be seen, the induced single cell can form clone-like growth; Figure 3 b~3d, it can be seen that the induced cells express ectoderm markers SIX1, NESTIN and ETS1; Figure 3 e~3l show that the induced cells express cartilage markers SOX9, RUNX2, SOX5, TWIST1, CD29 and key maxillofacial development markers FOXC1, FOXC2 and MSX1; Figure 3 mIt can be seen that the positive cell rates of key cartilage markers SOX5, SOX9, TWIST1, CD29 and key cell proliferation marker Ki67 were relatively consistent in the early and late passages of cells after induction. Figure 3 Similarly, there was no significant difference in the expression of key cartilage markers between different generations.

[0090] Example 4

[0091] Evaluation of the self-differentiation ability of induced cells

[0092] (A) The collagen-coated gel sponge was cut into 1×1×0.5 cm pieces using a sterilized surgical blade and placed in a low-adhesion 6-well plate, with 2 to 3 pieces placed in each well; the Hues9 chondrocyte progenitor cells (ECC) induced by CSSEDF in Example 1 were digested into single cells, counted, and resuspended in CSSEDF medium to 5×10 6 / mL concentration; add 100μl of cell suspension to each gel sponge, place it in the incubator for 15-20 minutes, then add 1.5mL of CSSEDF culture medium to each well; replace with fresh expansion medium every other day until N2 and B27 medium are replaced after 1 week.

[0093] (B) After 8 weeks of culture, the scaffolds cultured on the scaffolds were fixed, dehydrated, embedded, and sectioned for HE staining, toluidine blue, alcian blue, and red O staining, and immunohistochemical staining was used to detect the expression of Col2, AGG, RUNX2, Col1, and lubricin.

[0094] The specific steps of immunohistochemical staining are as follows:

[0095] (1) Take the slices (paraffin slices) and dewax them into water: xylene I (15 min) → xylene II (15 min) → 100% alcohol (5 min) → 100% alcohol (3 min) → 90% alcohol (3 min) → 80% alcohol (3 min) → 70% alcohol (3 min) → distilled water (3 min). Note: In winter, put xylene in a warm box and raise the temperature before use.

[0096] (2) Wash with PBS three times, 3 min each time.

[0097] (3) Microwave repair: Microwave the sodium citrate repair solution on medium-high heat for 7-8 minutes, cool for 2 minutes, repair for another 1-2 minutes, and naturally cool to room temperature for 30 minutes.

[0098] (4) Wash with PBS three times, 3 min each time.

[0099] (5) Incubate with 3% H2O2 at room temperature for 10 min.

[0100] (6) Wash twice with PBS, 3 min each time.

[0101] (7) 10% goat serum (solution A) blocking at 37°C for 30 min;

[0102] (8) Remove the serum and add the primary antibody (see Table 1) and incubate at 4°C overnight or at 37°C for 2 h.

[0103] (9) If incubated at 4°C overnight, rewarm at 37°C for 30 min the next day.

[0104] (10) Wash with PBS three times, 3 min each time.

[0105] (11) Add biotinylated secondary antibody (Solution B) (see Table 1) and incubate at 37°C for 30 min.

[0106] (12) Wash with PBS three times, 3 min each time.

[0107] (13) Add horseradish enzyme-labeled streptavidin working solution (solution C) and incubate at 37°C for 30 min.

[0108] (14) Wash with PBS 5 times, 5 min each time.

[0109] (15) DAB solution was used for color development and controlled under a microscope.

[0110] (16) Rinse thoroughly with distilled water to terminate the reaction.

[0111] (17) Restain with hematoxylin for 5 min and rinse with running water for 15 min.

[0112] (18) Dehydrate with alcohol and make transparent with xylene for about 40 minutes.

[0113] (19) Seal the slides with neutral gum, dry them, and observe them under a microscope.

[0114] Test results such as Figure 4 As shown in Figure a, the scaffold material was translucent, shiny, and had a certain elasticity after 8 weeks of induction. Figure 4 HE staining in b shows that colony-like cartilage masses can be seen in the scaffold material; the cartilage masses are strongly positive for toluidine blue, alcian blue and red O staining ( Figure 4 c~4e); Immunohistochemical staining showed that spontaneously differentiated cartilage masses secreted Col2, AGG, RUNX2, Col1 and lubricin ( Figure 4 f~4k).

[0115] Example 5

[0116] Comparison of single-cell transcriptome expression between induced chondroprogenitor cells, classical mesodermal chondroprogenitor cells, and human ectodermal condylar chondrocytes

[0117] Refer to, for example, Chia-Lung Wu et al., Single cell transcriptomic analysis of human pluripotent stem cell chondrogenesis, Nature Communications, Vol. 13, No. 12(1), 2021. The specific steps are:

[0118] (A) The chondrogenic progenitor cells (ECC) and human TMJ condylar chondrocytes (TMJ-CC) induced by Hues9 in Example 1 were collected for single-cell transcriptional sequencing.

[0119] (B) Comparison of induced chondroprogenitor cells (ECC), human TMJ condylar chondrocytes (TMJ-CC), and classical mesodermal chondroprogenitor cells (CP) from the literature.

[0120] The results are shown in Figure 5. Figure 5a It can be seen that both ECC and TMJ-CC express key ectoderm markers, such as col1a, BARX1, SIX1, MSX1, TWIST, and FOXC1. Figure 5b It can be seen that compared with CP, ECC and TMJ-CC are closer at the transcriptome level. Functional clustering analysis shows that ( Figure 5c ), ECC and TMJ-CC are both enriched for ectoderm markers, while CP is enriched for mesoderm markers.

[0121] Example 6

[0122] In vivo experiments were used to evaluate the effect of ectodermal chondrogenic progenitor cells in repairing cartilage defects.

[0123] (A) Ectodermal chondrogenic progenitor cells are labeled with green fluorescent protein (GFP), and proteins with the GPF label are implanted into the sponge scaffold material. The specific steps are as follows:

[0124] (1) Day 1, prepare cells: inoculate several wells of a 24-well culture plate with Hues9 chondrocyte progenitor cells (ECC) in the logarithmic growth phase after CSSEDF induction in Example 1 and parallel control 293T cells. When plating, the cell fusion rate is about 50%. Add 100 μL of culture medium to each well and culture in an incubator until the cells adhere to the wall. The optimal cell fusion rate for virus infection is about 70%.

[0125] (2) Day 2, prepare the virus: Take out the virus stored at 4°C and gently mix it with a pipette. Thaw the virus frozen at -80°C on ice before use.

[0126] Then infect the target cells: remove the old culture medium from the culture vessel, wash twice with PBS, and replace with fresh culture medium. Use a pipette to draw an accurate volume of virus solution and add it to the target cells and control cells. Mix the virus solution and culture medium, and incubate in a carbon dioxide incubator (37°C, 5% CO2).

[0127] (3) On Day 3 (24 hours after adding the virus solution), discard the virus-containing culture medium, replace it with fresh complete culture medium, and continue culturing in the incubator.

[0128] (4) Day 4 (48 to 72 hours after adding the virus). For viruses carrying the GFP reporter gene, the GFP fluorescence intensity can be observed under a fluorescence microscope. For viruses carrying the puromycin resistance gene, the culture medium is replaced with complete culture medium containing an appropriate concentration of puromycin (the standard final concentration of puromycin is 1 to 10 μg / mL) to screen for cell lines with stable expression.

[0129] (B) A rat knee defect model was constructed and the sponge scaffold material loaded with ectodermal chondrogenic progenitor cells was placed in the knee cartilage defect. The knee joint specimens were removed 8 weeks later for histological evaluation.

[0130] (C) Stereoscopic direct observation of GFP green fluorescence, histological staining to evaluate the repaired cartilage tissue, the specific steps are:

[0131] (1) Take the slices out of the oven and dry them at 37°C, then rinse them with PBS three times for 3 min each time.

[0132] (2) Block with 10% goat serum at 37°C for 30 min;

[0133] (3) Discard the blocking solution, add two concentrated primary antibodies of appropriate dilution (see Table 1), and place in a 4°C refrigerator overnight or at 37°C for 2 h;

[0134] (4) If the sample has been kept in a 4°C refrigerator overnight, take it out on the second day and rewarm it at 37°C for 30 min.

[0135] (5) Wash with PBS three times, 3 min each time;

[0136] (6) Add two fluorescently labeled secondary antibodies from different sources at appropriate dilutions (see Table 1), protect from light, and incubate at room temperature for 2 h;

[0137] (7) Wash with PBS three times, 5 min each time, in dark;

[0138] (8) Add appropriate dilution of 4',6-diamidino-2-phenylindole (DAPI) for staining and discard after 10 min at room temperature;

[0139] (9) Wash with PBS three times, 5 min each time;

[0140] (10) Seal the slides with 80% glycerol, observe under a fluorescence microscope, and take photos.

[0141] Test results such as Figure 6 shown. Figure 6 a shows the knee cartilage defect and repair model; Figure 6 b~6c show that green fluorescence can be seen in the repaired cartilage tissue of the experimental group under stereomicroscope, but no green fluorescence can be seen in the control group; histological observation shows ( Figure 6 d~6e), green fluorescence was observed in the repaired cartilage tissue in the experimental group, but not in the control group. Figure 6 f~6k show that the cartilage tissue formed in the experimental group was positive for red O, Col2, Col1, Col10, RUNX2, lubricin and AGG staining.

[0142] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present application. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed by the present invention shall still be covered by the claims of the present application.

Claims

1. A culture medium for inducing embryonic stem cells to differentiate into ectodermal chondrogenic progenitor cells in vitro, characterized in that: The culture medium comprises a neural crest induction medium and a CSSEDF medium. The components of the neural crest induction medium comprise a basal culture medium, BMP4 and a TGF-β receptor inhibitor. Based on the total volume of the basal culture medium, the concentration of the BMP4 is 20 ng / ml, and the amount of TGF-β added is 2 μM. The components of the CSSEDF culture medium comprise a basal culture medium, a GSK-3 inhibitor, a TGF-β receptor inhibitor, a smoothened agonist, EGF, FGF2 and a BMP receptor inhibitor. The smoothened agonist is selected from From SAG, the TGF-β receptor inhibitor is selected from SB431542, the BMP receptor inhibitor is selected from DMH1, and the GSK-3 inhibitor is selected from CHIR99021; based on the total volume of the basal culture medium, the added amount of the GSK-3 inhibitor is 1 μM, the added amount of the TGF-β receptor inhibitor is 2 μM, the added amount of the smoothened agonist is 0.2 μM, the concentration of the EGF is 20 ng / ml, the concentration of the FGF2 is 20 ng / ml, and the added amount of the BMP receptor inhibitor is 1 μM.

2. The culture medium for inducing in vitro differentiation of embryonic stem cells into ectoderm chondrogenic progenitor cells according to claim 1, characterized in that: The basic culture medium is selected from one or more of MEM, DEM, DEM / F12, RPMI1640 or F12.

3. The culture medium for inducing in vitro differentiation of embryonic stem cells into ectoderm chondrogenic progenitor cells according to claim 2, characterized in that: The basic culture medium is DEM / F12 containing N2 and B27.

4. The culture medium for inducing in vitro directed differentiation of embryonic stem cells into ectoderm chondrogenic progenitor cells according to claim 3, characterized in that: Based on the volume of the basic culture medium, the concentration of N2 is 0.1-10% (v / v), and the concentration of B27 is 0.1-10% (v / v).

5. Use of the culture medium according to any one of claims 1 to 4 for inducing embryonic stem cells to differentiate into ectoderm chondrogenic progenitor cells in vitro.

6. A method for inducing embryonic stem cells to differentiate into ectodermal chondrogenic progenitor cells in vitro, characterized in that: The following steps are involved: 1) Resuscitating and expanding embryonic stem cells to obtain cells for subculture; 2) The subcultured cells of step 1) are inoculated into the culture medium of any one of claims 1 to 4 to obtain ectoderm chondrogenic progenitor cells.

7. The method for inducing in vitro directed differentiation of embryonic stem cells into ectodermal chondrogenic progenitor cells according to claim 6, characterized in that: In step 2), the cells are first cultured in a neural crest induction medium in the culture medium as claimed in any one of claims 1 to 4 for 2 to 10 days; and then cultured in a CSSEDF medium in the culture medium as claimed in any one of claims 1 to 4 until target cells are obtained.

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