Human pluripotent stem cell-derived sclera-derived osteochondral mesenchymal precursor cells, methods of making and uses thereof

Abstract

The application discloses human pluripotent stem cell-derived sclerotome osteogenic / chondrogenic mesenchymal precursor cells, a preparation method and application thereof, and belongs to the technical field of biological medicines, and solves the problems of poor repeatability, low differentiation efficiency and long differentiation time in the prior art. The preparation method comprises the following steps: step 1, original strip differentiation; step 2, precursor segment mesoderm differentiation; and step 3, subculture and sclerotome mesenchymal precursor cell differentiation. The application has the advantages of scientific design, ingenious concept, simple, specific and easy-to-implement starting cell density control. The differentiation process of the application is simple and short. The cells differentiated by the application express related molecular markers of sclerotome osteogenic / chondrogenic mesenchymal precursor cells and have in-vitro chondrogenic and osteogenic differentiation capabilities. The method has high differentiation efficiency and high repeatability. The differentiation efficiency is stably controlled at 99.2% 0±0.7%.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to lamina osteoblast / chondrocyte mesenchymal precursor cells derived from human pluripotent stem cells, their preparation method and application. Background Technology

[0002] The development of the mammalian skeletal system originates from three different germ layers during embryonic development. The bones and cartilage of the head and face originate from the mesenchymal cells of the neural crest; the axial bones (excluding the sternum, such as the spine, ribs, and costal cartilage) originate from specialized mesenchymal cells of the sclera within the somites; and the bones of the limbs and sternum originate from the mesoderm of the lateral plates and the specialized mesenchymal cells of the limb buds. On the other hand, the induction of pluripotent stem cells (iPSCs) from somatic cells is a revolutionary discovery in the fields of biology and medicine. Induced pluripotent stem cells (iPSCs) not only maintain self-renewal but also possess multipotent differentiation potential. Using pluripotent stem cells as material, various human cells have been prepared in vitro through transgenic or chemical induction methods, and under appropriate induction conditions, they can efficiently differentiate into neurons, cardiomyocytes, and insulin-secreting cells, among others.

[0003] Currently, scleral osteoblast / chondrogenic mesenchymal progenitor cells derived from human pluripotent stem cells are all induced stepwise from four different germ layer lineages. For example, Matsuda et al. first induced pluripotent stem cells in a single-cell state (8x10⁸ cells per cell). 4 Human induced pluripotent stem cells (i.e., 100 cells / 100 mm cell culture dish) were seeded and expanded for 4-5 days. Then, by regulating the signaling pathways related to the differentiation of each germ layer during the formation of scleral osteoblasts / chondral mesenchymal progenitor cells, induced pluripotent stem cells were gradually induced into primitive streaks (PS), prosomal mesoderm (PSM), somites (SM), and scleral osteoblasts / chondral mesenchymal progenitor cells (SCL) over 6 days. Except for the SM to SCL induction, which required 72 hours, the induction time for the other germ layers was 24 hours. In addition, Loh et al. first passaged human embryonic stem cells and human induced pluripotent stem cells with 90% aggregation at a ratio of 1:12 to 1:20 into culture dishes. After overnight adhesion / expansion, similar pathways were regulated, and the pluripotent stem cells were gradually induced into primitive streaks (PS), prosomal mesoderm (PSM), somites (SM), and scleral osteoblasts / chondral mesenchymal progenitor cells (SCL) over 6 days. Except for the SM to SCL induction, which required 72 hours, the induction time for the other germ layers was 24 hours. However, both of the above methods currently have obvious drawbacks, including:

[0004] (1) Poor reproducibility and low differentiation efficiency. In current preparation methods, all processes from pluripotent stem cell seeding to differentiation completion are carried out in the same culture dish. This leads to excessive cell proliferation, resulting in low differentiation efficiency. Furthermore, the differentiation efficiency varies between batches depending on the appropriate density control. Although Loh et al. described their control of initial cell density in their literature as "passage to new culture dishes at a ratio of 1:12 to 1:20, followed by overnight adhesion and differentiation," such a broad density range makes it difficult to guarantee reproducibility between batches in actual operation. Testing showed that the proportion of SOX9+ lamina reticulata mesenchymal cells differentiated using traditional methods was only 84.9% ± 9.2% (mean ± standard deviation, based on the proportion of SOX9-positive cells analyzed by flow cytometry). This means that more than 15% of the differentiated cells were contaminated with impurity cells.

[0005] (2) Long differentiation time. Although Matsuda et al. improved Loh et al.'s method by seeding cells in single-cell form to control the initial cell density, thus limiting the low differentiation efficiency caused by excessively high cell density during differentiation, human pluripotent stem cells have poor survival rate in single-cell state. Even with the addition of apoptosis inhibitors, the loss of initial cells is inevitable. In addition, after seeding cells in single-cell form at the recommended cell density, a 4-5 day expansion period is required before differentiation can begin, resulting in an excessively long preparation time, that is, at least about 10 days to complete the preparation of target cells.

[0006] Therefore, providing a human pluripotent stem cell-derived osteoblast / chondrogenic mesenchymal precursor cell that can be prepared in high purity in a short time has become a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] One of the objectives of this invention is to provide a method for preparing osteoblastic / chondrogenic mesenchymal progenitor cells derived from human pluripotent stem cells, thereby solving the problems of poor differentiation reproducibility, low differentiation efficiency, and long differentiation time in the prior art.

[0008] A second objective of this invention is to provide scleral osteoblast / chondrocyte mesenchymal precursor cells derived from human pluripotent stem cells obtained by this method.

[0009] A third objective of this invention is to provide the application of scleral osteoblast / chondrocyte mesenchymal precursor cells derived from human pluripotent stem cells.

[0010] A fourth objective of this invention is to provide a pharmaceutical composition comprising osteoblastic / chondrogenic mesenchymal precursor cells derived from human pluripotent stem cells.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] This invention provides a method for preparing scleral osteoblast / chondrogenic mesenchymal progenitor cells (hereinafter referred to as scleral mesenchymal progenitor cells) derived from human pluripotent stem cells, comprising the following steps:

[0013] Step 1. Primary stem differentiation: Differentiation medium I is added to human pluripotent stem cells and cultured induction culture; the differentiation medium I is CDMi basal medium containing activin A, CHIR99021 and bFGF;

[0014] Step 2. Pre-segmental mesoderm differentiation: After culturing the cells in Step 1, remove differentiation medium I, rinse the cells, add differentiation medium II, and induce culture; the differentiation medium II is CDMi basal medium containing SB431542, LDN193189, CHIR99021, and bFGF.

[0015] Step 3. Passage and differentiation of lamina brachii mesenchymal precursor cells: After culturing in step 2, remove differentiation medium II from the cells, then digest, neutralize, and pipette the cells to obtain a single-cell suspension. Centrifuge, resuspend, and seed the cell resuspended in a culture dish. Add differentiation medium III and induce culture for a period of time. Then replace the medium with differentiation medium IV.

[0016] The differentiation medium III is a CDMi basal medium containing SAG, LDN193189, XAV939, and ROCKi;

[0017] The differentiation medium IV is a CDMi basal medium containing SAG, LDN193189, and XAV939.

[0018] In some embodiments of the present invention, the induction culture conditions in step 1 are 37±1℃ for 12-48 hours; preferably 24 hours.

[0019] Or / and the induction culture conditions in step 2 are 37±1℃ for 12-48 hours; preferably 24 hours;

[0020] Or / and the induction culture conditions using differentiation medium III in step 3 are 37±1℃ for 12-48 hours; preferably 24 hours;

[0021] Or / and use differentiation medium IV for induction culture at 37±1℃ for 12-48 hours; preferably 24 hours.

[0022] In some embodiments of the present invention, the content of each component in the differentiation culture medium I is activin A 10-40 ng / mL, CHIR99021 3-10 μM, and bFGF 10-30 ng / mL; preferably activin A 30 ng / mL, CHIR99021 7 μM, and bFGF 20 ng / mL.

[0023] Or / and in the differentiation medium II, the content of each component is: SB431542 10-30 μM, LDN193189 100-200 nM, CHIR99021 1-6 μM, bFGF 20-80 ng / mL; preferably SB431542 20 μM, LDN193189 125 nM, CHIR99021 3 μM, bFGF 40 ng / mL;

[0024] Or / and in the differentiation medium III, the content of each component is: SAG 100-300 nM, LDN193189 400-800 nM, XAV939 0.1-0.8 μM, ROCKi 5-20 μM; preferably SAG 200 nM, LDN193189 600 nM, XAV939 0.5 μM, ROCKi 10 μM;

[0025] Or / and in the differentiation medium IV, the content of each component is: SAG 100-300 nM, LDN193189 400-800 nM, XAV939 0.1-0.8 μM; preferably SAG 200 nM, LDN193189 600 nM, XAV939 0.5 μM.

[0026] In some embodiments of the present invention, in step 3, the cell resuspension is seeded into a culture dish at a seeding density of 1×10⁻⁶. 5 ~1.5×10 5 The preferred value is 1.18 × 10⁻⁶. 5 cells / cm 2 .

[0027] In some embodiments of the present invention, prior to the differentiation of the original stem cells in step 1, a seeding step of human pluripotent stem cells is further included, specifically comprising the following steps:

[0028] S1. Human pluripotent stem cells were routinely cultured in culture plates containing Matrigel working solution;

[0029] S2. When the cell density reaches 70%-90%, remove the culture medium and digest the cells at 37℃ for 3-5 minutes;

[0030] S3. After aspirating the digestion solution, the culture is transferred to another culture plate containing Matrigel working solution for culture, preferably inoculated at a ratio of 1:7 to 1:8.

[0031] In some embodiments of the present invention, the Matrigel working solution is prepared by diluting 1 mg of Matrigel stock solution in 8 to 15 mL of DMEM / F12, preferably by diluting 1 mg of Matrigel stock solution in 12 mL of DMEM / F12.

[0032] The above-described preparation method provided by this invention yields scleral osteoblast / chondrocyte mesenchymal precursor cells derived from human pluripotent stem cells.

[0033] The application of human pluripotent stem cell-derived osteoblast / chondrocyte mesenchymal precursor cells obtained by the above-described preparation method provided by this invention in the preparation of drugs for repairing articular cartilage.

[0034] The application of human pluripotent stem cell-derived osteoblast / chondrocyte mesenchymal precursor cells obtained by the above-described preparation method provided by this invention in the preparation of drugs that promote the regeneration of articular cartilage tissue.

[0035] The present invention provides a pharmaceutical composition comprising lamina osteoblast / chondrocyte mesenchymal progenitor cells derived from human pluripotent stem cells prepared according to the above-described preparation method, and a pharmaceutically acceptable carrier.

[0036] The pharmaceutically acceptable carriers described in this invention refer to diluents, excipients, formulations, or mediators that are administered co-administered with a therapeutic agent and are suitable, to the extent of reasonable medical judgment, for contact with human and / or other animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0037] Preferably, the pharmaceutically acceptable carrier includes hyaluronic acid and collagen.

[0038] The human pluripotent stem cells described in this invention are human embryonic stem cells or human induced pluripotent stem cells.

[0039] In embodiments of the present invention, the human pluripotent stem cells are SOX9-tdTomato fluorescent reporter cell lines, which utilize gene editing technology to insert tdTomato expression elements into the 3'UTR region of the SOX9 gene locus. Transcription is controlled by the SOX9 promoter, and after transcription is completed, translation is mediated by the IRES sequence in its own expression elements.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] This invention is scientifically designed and ingeniously conceived. The control of initial cell density is simple, specific, and easy to implement. For conventionally cultured human pluripotent stem cells, differentiation can begin the next day simply by passage at a ratio of 1:7 to 1:8 when cell aggregation reaches 70%-80%.

[0042] The differentiation process of this invention is simple and quick. From the start of differentiation to completion of preparation, it only takes 4 days.

[0043] The cells differentiated by this invention express molecular markers related to osteoblastic / chondrogenic mesenchymal precursor cells of the sclera and have the ability to differentiate into cartilage and bone in vitro.

[0044] The method of this invention exhibits high differentiation efficiency and high reproducibility. Differentiation efficiency (as measured by flow cytometry for SOX9) is [data missing]. + (%) Stable control at 99.2% ± 0.7%. Attached Figure Description

[0045] Appendix Figure 1 A diagram illustrating the morphological changes during the differentiation of pluripotent stem cells into osteoblasts / chondrocyte mesenchymal precursor cells.

[0046] Appendix Figure 2 The image shows the results of immunofluorescence staining of SOX9 and TWIST1, marker molecules for osteoblasts / chondrocytes prepared by the method of this invention.

[0047] Appendix Figure 3 The image shows the verification results of in vitro chondrogenic (COLII immunohistochemical staining, Alixin blue staining, Safranin-Fix Green staining) / bone (Alizarin Red staining) differentiation of the scleral osteoblast / chondrogenic mesenchymal precursor cells prepared by the method of the present invention.

[0048] Appendix Figure 4 Figure showing the efficiency results of this method in preparing induced pluripotent stem cells into osteoblastic / chondrogenic mesenchymal precursor cells of the sclera.

[0049] Appendix Figure 5 The image shows the results of real-time quantitative PCR identification of differentiated cells.

[0050] Appendix Figure 6 This is a graph showing the results of flow cytometry analysis for the identification of differentiated cells.

[0051] Appendix Figure 7 Flowchart of mouse bone and articular cartilage repair;

[0052] Appendix Figure 8 Comparison of results showing that osteoblasts / chondrocyte mesenchymal precursor cells can efficiently repair articular cartilage damage. Detailed Implementation

[0053] The human pluripotent stem cells used in the embodiments of the present invention are the SOX9-tdTomato fluorescent reporter cell line. The line uses gene editing technology to insert the tdTomato expression element into the 3'UTR region of the SOX9 gene locus. Its transcription is controlled by the SOX9 promoter. After transcription is completed, translation is mediated by the IRES sequence in its own expression element.

[0054] Example 1

[0055] This embodiment discloses the seeding and passage method of human pluripotent stem cells for differentiation according to the present invention, and the reagents used are:

[0056] PGM1 culture medium: Add 5mL L Pen-strep (100X stock solution, Gibco, USA) to 500mL of human pluripotent stem cell culture medium PGM1 (China Saibei Company) and then filter to sterilize.

[0057] Matrigel working solution: Remove the bottled Matrigel solution from the -80°C freezer and completely immerse the bottle in ice. Place the ice pack in a 4°C freezer overnight. The next day, place the pipette tip and EP tube used to draw the stock solution in a -80°C freezer for at least 10 minutes. Using a pipette tip pre-chilled at -20°C, add 1 mg of Matrigel stock solution (Corning, USA) to 12 mL of pre-chilled DMEM / F12 (Gibco, USA) on ice. This working solution can be stored at 4°C for two weeks.

[0058] The seeding and passage method for differentiation-use human pluripotent stem cells in this embodiment specifically includes the following steps:

[0059] 1. Human pluripotent stem cells cultured in six-well plates were removed from the 37°C incubator and observed under a regular optical microscope to determine cell density, with a cell aggregation degree of 70%-90%.

[0060] 2. Remove the PGM1 medium, add 0.5mM EDTA (2mL / 60mm culture plate), digest the cells at 37℃ for 5 minutes, then take them out and observe them under a microscope. If cracks begin to appear in the cell clone clusters and the area around the clone clusters begins to shrink, the digestion solution can be aspirated to stop digestion. Otherwise, the cells should be placed in a 37℃ incubator to continue digestion.

[0061] 3. After aspirating the digestion solution, add an appropriate amount of PGM1 medium. Gently and slowly blow the cells from the bottom of the plate. Passage the cells to another six-well plate treated with Matrigel working solution at a ratio of 1:8, corresponding to a pre-passage polymerization degree of 70%-90%. Shake the culture plate from side to side to distribute the cells evenly in the growth area of ​​the culture plate. After overnight culture, observe the cells the next day. If the cells adhere to the culture plate in a clonal form and the cell clones do not merge into sheets, they can be used for differentiation.

[0062] Example 2

[0063] This embodiment discloses a method for preparing scleral osteoblast / chondrogenic mesenchymal precursor cells derived from human pluripotent stem cells according to the present invention. The reagents used in this embodiment are:

[0064] Holo-transferrin working solution: Weigh 15mg of Holo-transferrin (Sigma, USA) powder and add 1mL of UP water (Invitrogen, USA);

[0065] Rh-insulin working solution: Weigh 2 mg of rh-insulin (Solarbio, China) powder, add 1 mL of 10 mM HCl, and filter to sterilize;

[0066] Polyvinyl alcohol working solution: Weigh 500mg of polyvinyl alcohol powder, add 18mL of UP water (Invitrogen, USA), stir until a granular suspension is formed, heat in an 85℃ water bath for about 20 minutes to dissolve, make up to 20mL, and filter to sterilize.

[0067] Monothioglycerol working solution: Take 450 μmol equivalent of monothioglycerol stock solution (Sigma, USA), add it to UP water to make the total volume of the solution 1 mL, vortex to mix, and filter to sterilize;

[0068] CDMi basal medium: IMDM (Gibco, USA) 234.384 mL, Ham's F12 (Gibco, USA) 234.384 mL, Chemically Defined Lipid Concentrate (Gibco, USA) 5 mL, Holo-transferrin working solution 500 μL, rh-insulin working solution 234 μL, Polyvinyl alcohol working solution 20 mL, Pen-strep (Gibco, USA) 5 mL, monothioglycerol working solution 500 μL;

[0069] Differentiation medium I: CDMi basal medium supplemented with activin A (Solarbio, China, 30 ng / mL), CHIR99021 (MCE, USA, 7 μM), and bFGF (20 ng / mL, Peprotech, USA);

[0070] Differentiation medium II: CDMi basal medium supplemented with SB431542 (MCE, USA, 20 μM), LDN193189 (MCE, USA, 125 nM), CHIR99021 (MCE, USA, 3 μM), and bFGF (Peprotech, USA, 40 ng / mL);

[0071] Differentiation medium III: CDMi basal medium supplemented with SAG (MCE, USA, 200 nM), LDN193189 (MCE, USA, 600 nM), XAV939 (MCE, USA, 0.5 μM), and ROCKi (MCE, USA, 10 μM);

[0072] Differentiation medium IV: CDMi medium supplemented with SAG (MCE, 200 nM), LDN193189 (MCE, 600 nM), and XAV939 (MCE, 0.5 μM);

[0073] Wash buffer: Weigh 1.5g bovine serum albumin (BSA, Sigma, USA) and add it to 500mL DMEM / F12 medium (Gibco, USA), mix well and filter to sterilize;

[0074] The preparation method in this embodiment is as follows:

[0075] 1. Differentiation of original streaks (PS)

[0076] On the second day of passage, cells were removed from the 37°C incubator and PGM1 medium was discarded. 2 mL of Washbuffer was added to the wells to wash away dead cells and residual PGM1 medium, the Washbuffer was discarded, and 2 mL of differentiation medium I was added. The cells were then incubated at 37°C.

[0077] 2. Differentiation of the precursor mesodermal layer (PSM)

[0078] After 24 hours, remove the cells from the 37°C incubator and discard differentiation medium I. Add 2 mL of wash buffer to the wells to wash away dead cells and residual differentiation medium I, discard the wash buffer, add 2 mL of differentiation medium II, and then incubate at 37°C.

[0079] 3. Passage and differentiation of scleral lamina cortex mesenchymal precursor cells (SCL)

[0080] 3.1 Digestion: After 24 hours, remove the cells from the 37°C incubator and discard differentiation medium II. Add 500 μL of LypLE Express digestion solution to the wells and place the cells in the 37°C incubator for 3 minutes to digest.

[0081] 3.2 Neutralization and Resuspension: After digestion, add 4 times the volume (2 mL) of Wash buffer to the wells for neutralization, and pipette evenly to form a single-cell suspension. Transfer the cells to a 15 mL centrifuge tube, centrifuge at 300 g for 3 minutes, and resuspend the cells with an appropriate amount of Wash buffer after centrifugation.

[0082] 3.3 Count the blood cells using a hemocytometer;

[0083] 3.4 Take 1.18 x 10 5 cells / cm 2 The equivalent number of cells were placed in a 15 mL centrifuge tube and centrifuged at 300 g for 3 minutes; the supernatant was discarded, 4 mL of differentiation medium III was added and mixed by pipetting, the cells were transferred to a 60 mm culture plate and then cultured in an incubator at 37 °C.

[0084] 3.5 After 24 hours, remove the cells from the 37°C incubator and discard differentiation medium III. Add 2 mL of Washbuffer to the wells to wash away dead cells and residual differentiation medium III, discard the Washbuffer, add 2 mL of differentiation medium IV, and then incubate at 37°C.

[0085] After 24 hours, osteoblastic / chondrogenic mesenchymal precursor cells of the sclera can be collected.

[0086] Experimental Example 1

[0087] 1. Cells differentiated from pseudoblastic stem cells (PS), presomnotic mesoderm (PSM), passaged cells, and cells differentiated from scleral lamina cortex precursor cells (SCL) were observed under a light microscope. The results are shown in the appendix. Figure 1 As shown.

[0088] The left image shows PS (protoplasts), the middle image shows PSM (prostomy mesoderm), and the right image shows SCL (scleral lamina osteoblasts / chondroblastic mesenchymal precursor cells), which are attached to the periosteal tissue. Figure 1 It is evident that the method of this invention successfully achieved primary streak differentiation, precursor mesoderm differentiation, and SCL differentiation of human pluripotent stem cells.

[0089] 2. Differentiated cells were analyzed by immunofluorescence staining with SOX9 and TWIST1, markers for osteoblastic / chondrogenic precursor cells of the sclera. The results are shown in the attached figure. Figure 2 As shown, the left image is the SOX9 staining result, and the right image is the TWIST1 staining result. Figure 2 It can be seen that the progenitor cells differentiated from human pluripotent stem cells express molecular markers related to scleral cells with good uniformity.

[0090] The specific steps for immunofluorescence staining detection are as follows:

[0091] Remove the cultured osteoblast / chondrocyte mesenchymal precursor cells (seeded on a slide) from the 37°C incubator and aspirate the supernatant. Wash away the suspended dead cells with PBS, discard the supernatant, and immediately add an appropriate amount of 4% paraformaldehyde solution to fix the cells at room temperature for 8-10 minutes.

[0092] Remove the fixative, add an appropriate amount of PBS to the wells, and wash away any residual fixative.

[0093] Treat with 0.3% PBST (a 0.3% Triton solution prepared with PBS) at room temperature for 10 minutes;

[0094] Remove the PBST, add 5% bovine serum albumin solution (prepared with PBS) to the wells, and block at room temperature for 1 hour;

[0095] Remove the blocking solution, add an appropriate amount of primary antibody (dilution ratio 1:200, diluted with blocking solution) to the wells, and stain overnight at 4°C.

[0096] Aspirate the primary antibody and wash three times with PBS for three minutes each time.

[0097] Remove the PBS, add an appropriate amount of secondary antibody (diluted at a ratio of 1:400, diluted with blocking buffer) to the wells, and stain at room temperature for 1 hour.

[0098] Remove the secondary antibody and wash three times with PBS, three minutes each time.

[0099] Remove the PBS, stain the nuclei with a mounting medium containing DAPI, mount the slide, and take a picture.

[0100] 3. Validation of differentiated cells after in vitro chondrogenic (COLII immunohistochemical staining, Alcian blue staining, Safranin-Fix Green staining) / bone (Alizarin Red staining) differentiation was performed, and the results are attached. Figure 3 As shown, the top left image is the COLII immunohistochemical staining result, the top right image is the alexandrite blue staining result, the bottom left image is the safranin-fast green staining result, and the bottom right image is the alizarin red staining result. (See attached image.) Figure 3 It is known that osteoblasts / chondrocyte precursor cells of the sclera have the ability to differentiate into cartilage in vitro, and after induction, they can form tissues expressing various cartilage-specific molecular markers.

[0101] 3.1 Staining for in vitro chondrogenic differentiation

[0102] 3.1.1 COLII Immunohistochemical Staining

[0103] After the chondrogenic differentiation of the samples was induced, the culture medium was removed from the 37℃ incubator, the cartilage balls were placed directly in the OCT gel, and they were immediately placed in the -80℃ freezer for embedding. After the gel solidified, the samples were sectioned using a cryostat with a section thickness of 6μm.

[0104] After sectioning, fix the sections in an appropriate amount of 4% paraformaldehyde solution for 8-10 minutes before staining.

[0105] Discard the fixative and wash away any residual fixative in an appropriate amount of PBS;

[0106] Use an immunohistochemistry pen to draw a circle around the location of the sample;

[0107] Antigen retrieval was performed using pepsin (Sigma, USA) and the sample was incubated at 37°C for 30 minutes.

[0108] Aspirate the refolding solution and rinse the sections twice in PBS for 5-10 seconds each time.

[0109] Blocking. Block with 5% bovine serum albumin solution (prepared with PBS) at room temperature for 1 hour;

[0110] Remove the blocking solution, add an appropriate amount of primary antibody (dilution ratio 1:200, diluted with blocking solution), and stain overnight at 4°C.

[0111] Aspirate the primary antibody and wash three times with PBS for three minutes each time.

[0112] Wipe PSB dry, add an appropriate amount of secondary antibody (dilution ratio 1:400, diluted with blocking buffer), and stain at room temperature for 1 hour;

[0113] Remove the secondary antibody and wash three times with PBS, three minutes each time.

[0114] Wipe the PBS dry, stain the nuclei with mounting medium containing DAPI, and then mount the slide.

[0115] 3.1.2 Alixin Blue Staining

[0116] Take one of the above-mentioned fixed sections and treat the rehydrated section with 0.1N HCl for 2 minutes;

[0117] Stain directly with 1% albicane blue solution for 30 minutes;

[0118] Immerse the slides twice in distilled water for 5-10 seconds each time, then add 0.1N N HCl to the sample area and treat for 2 minutes to remove non-specific staining.

[0119] Wash away residual hydrochloric acid with distilled water, add hematoxylin (commercial H&E staining kit) for nucleus development, and stain for 4 minutes;

[0120] Rinse the slices under tap water for 4 minutes to promote the blueing of the hematoxylin;

[0121] Dehydrate with 95% ethanol for 1 minute, dehydrate with 100% ethanol for 1 minute, clear with xylene, and seal with neutral resin.

[0122] 3.1.3 Safranin-Fix Green Staining

[0123] Take one of the above-preserved sections and stain it with hematoxylin (commercial H&E staining kit) for 2 minutes;

[0124] Rinse the slices under tap water for 4 minutes to promote the blueing of the hematoxylin;

[0125] Stain with 1% Fast Green solution (Sigma, USA) for 1 minute and 30 seconds;

[0126] Differentiate with 1% glacial acetic acid solution for 15 seconds, then wash away the non-specific staining of Fast Green;

[0127] Remove the glacial acetic acid with a pipette, and stain with 5% safranin (Sigma, USA) for 1 minute;

[0128] Immerse in 95% ethanol for 15 seconds, then dehydrate the sections in 100% ethanol for 1 minute. Clear with xylene and mount with neutral resin.

[0129] 3.2 Staining for in vitro osteogenic differentiation

[0130] Osteogenically induced differentiated cells were removed from the 37°C incubator and fixed with 4% paraformaldehyde solution at room temperature for 8-10 minutes.

[0131] Aspirate the fixative and rinse the well plate with PBS to remove any residual fixative.

[0132] Discard the PBS and add alizarin red staining solution directly to stain at room temperature for 2 minutes.

[0133] Remove the staining solution and rinse the cells three times with PBS for five minutes each time to remove non-specific staining.

[0134] 4. The efficiency test of preparing induced pluripotent stem cells into osteoblastic / chondrogenic mesenchymal precursor cells using the method in Example 2, the specific steps are as follows:

[0135] Remove the cultured osteoblast / chondrocyte mesenchymal precursor cells from the 37°C incubator and aspirate the supernatant. Wash away the suspended dead cells with PBS, discard the supernatant, and then digest with TryPLE digestion solution at 37°C for 3 minutes.

[0136] The digestion was terminated with 4 times the volume of Wash buffer, the cell suspension was collected, and centrifuged at 300g at 2-8℃ for 3 minutes.

[0137] Discard the supernatant and resuspend the cells in pre-cooled PBS;

[0138] Flow cytometry analysis can be performed after removing cell clumps using a 70μm cell sieve.

[0139] The results were analyzed by flow cytometry to determine the proportion of SOX9-expressing cells, as shown in the attached figure. Figure 4 As shown. (From the appendix) Figure 4 It can be seen that the proportion of SOX9-expressing cells in different batches of differentiated lamina cortex precursor cells was above 99%, indicating good uniformity.

[0140] Experimental Example 2

[0141] This experimental example discloses the quantitative real-time PCR identification of the scleral chondrocytes / osteoprogenitor cells prepared in Example 2, specifically as follows:

[0142] 1. RNA extraction

[0143] Remove the differentiation medium, add an appropriate amount of TRI reagent to the culture dish / well, and continuously pipette to dissociate the cells from the culture dish / well;

[0144] Homogenized cells were aspirated into a 1.5 mL EP tube and allowed to stand at room temperature for 5-10 minutes to allow for complete cell lysis.

[0145] Phase separation. Add chloroform to the tube at a ratio of chloroform:TRI reagent = 1:5, shake vigorously for 15 seconds, and then let stand at room temperature for 4-10 minutes.

[0146] Centrifuge at 12000g for 15 minutes (2-8℃). After centrifugation, three phases will be visible; the top layer (colorless and transparent) contains RNA.

[0147] Precipitate RNA. Add isopropanol to a new EP tube at a ratio of isopropanol:TRI reagent = 1:2, and add an equal volume of clear supernatant containing RNA; vortex for 10 seconds and let stand at room temperature for 10 minutes; centrifuge at 12000g for 10 minutes (2-8℃) and discard the supernatant;

[0148] Wash with ethanol. Prepare 75% ethanol with enzyme-free water. Add anhydrous ethanol at a ratio of 75% ethanol:TRI reagent = 1.5:1, gently invert the EP tube, centrifuge at 12000g for 4 minutes (2-8℃), and discard the supernatant;

[0149] Repeat step 8;

[0150] Allow the EP tube to air dry at room temperature, then add an appropriate amount of enzyme-free water at 56°C to dissolve the RNA.

[0151] RNA concentration was determined using quantitative instruments such as Nanodrop.

[0152] 2. Reverse transcription and real-time quantitative PCR

[0153] Take 100-1000 ng of RNA and reverse transcribe it into cDNA using commercial reverse transcription reagents;

[0154] Dilute the cDNA with enzyme-free water at a ratio of 1:4 to 1:10.

[0155] Using undifferentiated pluripotent stem cells from day 0 as a negative control and ACTB (encoding β-actin) as an internal reference gene, the expression of characteristic markers of scleral stromal progenitor cells was quantified using a commercially available qPCR kit (Vazyme, China). Among the genes that must be quantified, SOX9, PAX1, and TWIST1 were included.

[0156] The results are attached. Figure 5 As shown, the expression of characteristic molecular markers SOX9, PAX1, and TWIST1 in scleral chondrocytes / osteoprogenitor cells was significantly upregulated compared with undifferentiated pluripotent stem cells.

[0157] Experimental Example 3

[0158] This experimental example discloses an experiment using human pluripotent stem cells induced by the present invention to repair mouse femoral articular cartilage, specifically as follows:

[0159] 1. Graft preparation

[0160] The transplantation process is as follows Figure 7 As shown. Differentiation of cells must be identified before transplantation. At the RNA level, characteristic molecular markers of scleral chondrocytes / osteoma precursor cells are identified using real-time quantitative PCR. The expression of SOX9, PAX1, and TWIST1 should be significantly upregulated compared to undifferentiated pluripotent stem cells, such as... Figure 5 As shown; at the protein level, the proportion of SOX9-positive cells in differentiated cells, detected by flow cytometry, should be above 99.0%. Figure 6 As shown (left: undifferentiated pluripotent stem cells; right: differentiated scleral chondrocyte / osteomyotrophic progenitor cells).

[0161] After induction in Example 2, the culture medium was aspirated from the cells, and 500 μL of TrypLE digestion solution was added to the wells. The cells were then digested in a 37°C incubator for 3 minutes. The cells were then removed, and 2 mL of Wash buffer was added for neutralization. The cells were centrifuged at 300 g at room temperature for 3 minutes. After centrifugation, the cells were resuspended in Wash buffer.

[0162] Cell counting was performed using a hemocytometer.

[0163] Resuspend the cells in collagen I solution at a density of 150,000 cells / 3 μL, seed them on a 60 mm bacterial culture plate, and incubate at 37 °C for 30 minutes to allow the collagen gel to solidify.

[0164] After 30 minutes, remove the cell culture plate from the incubator, add 4 mL of PBS to the plate and gently shake it to allow the collagen aggregate containing cells to separate from the bottom of the plate. Return the plate to the 37°C cell culture incubator and culture it overnight with differentiation medium III to obtain the cells to be transplanted.

[0165] 2. Cartilage lesioning and cell transplantation in mouse femoral articular cartilage

[0166] Reagents: Aphrodine stock solution: Weigh 25 g of 2,2,2-tribromoethanol (Sigma-Aldrich, USA) into a filter flask, and add 15.5 mL of tert-amyl alcohol (Sigma-Aldrich, USA) to prepare an 80x stock solution. Before use, dilute to 1x with 0.9% sodium chloride (i.e., add 0.5 mL of stock solution to 39.5 mL of physiological saline).

[0167] 2.1 Establishment of a mouse femoral articular cartilage defect model

[0168] Non-obese diabetic / severe immunodeficiency (NOD / SCID) mice were anesthetized via intraperitoneal injection with Afodin working solution at a dose of 20 μL / g body weight. The mice were placed supine on a clean bench, and lubricant was used to prevent eye dryness. Hair near the surgical site was shaved. The skin at the knee joint was grasped with pointed forceps, and a 1 cm incision was made from the grasped area down to the lower abdomen. The subcutaneous tissue was exposed, and scissors were inserted into the subcutaneous tissue and continuously opened and closed horizontally to examine the skin and subcutaneous tissue. Blunt dissection was performed between the tissues. A small incision was made in the muscle on the right side of the patella with a scalpel, and the muscle was then cut upwards along the incision with the back of the scalpel to facilitate exposure of the patella. The patella was then dislocated to the left with the back of the scalpel, exposing the femoral articular surface. A hole was drilled into the femoral articular surface using an 18G needle. The purpose of drilling was to create an opening. Drilling should be gentle and slow, avoiding force towards the subchondral bone as much as possible. The detached articular cartilage tissue was continuously aspirated with PBS until a 0.5mm notch was created, at which point drilling was stopped. A 26G needle was inserted deeper into the opening and drilling continued. The purpose of drilling was to expose the blood vessels of the subchondral bone to provide nutrition for the transplanted cells. Drilling was stopped when an opening of about 0.5mm wide and 1mm deep was created. At this point, blood from the subchondral bone should flow upwards. The opening was cleaned with PBS, and the PBS was wiped off with a dry cloth to facilitate transplantation.

[0169] The prepared collagen gel graft is placed on a hydrophobic semi-permeable membrane to facilitate graft transfer. The semi-permeable membrane is then picked up with tweezers and transferred to the defect site and inserted into the opening.

[0170] After the transplant, the left hand holds the pointed forceps to grasp the muscle at the defect site and pulls it to the right, while the right hand holds a cotton swab to push the femur to the left to reposition the patella; the muscle is sutured with 8-0 absorbable sutures with needles, and then the skin is sutured with 4-0 absorbable sutures with needles.

[0171] Experimental Example 4: Identification of the graft

[0172] Reagent: 4% PFA fixative: Weigh 4g of paraformaldehyde powder (Sigma, USA) into a filter flask, add 90mL of ultrapure water, and dissolve by shaking overnight at 37℃. After dissolution, bring the volume to 100mL with ultrapure water and store at 4℃ protected from light.

[0173] 19% EDTA Decalcification Solution: Weigh 19g of EDTA powder into a filter flask, add 90mL of ultrapure water, and stir to dissolve on a magnetic stirrer. When the solution turns white, gradually add NaOH powder, continuing to stir until dissolved. Test the pH value of the solution with precision pH paper, adjust the pH to 7.0 with concentrated hydrochloric acid, and finally bring the volume to 100mL with ultrapure water.

[0174] 15% sucrose solution: Weigh 15g of sucrose crystals into a filter flask, add 90mL of ultrapure water, dissolve at room temperature, and then dilute to 100mL with ultrapure water.

[0175] 30% sucrose solution: Weigh 15g of sucrose crystals into a filter flask, add 90mL of ultrapure water, dissolve at room temperature, and then dilute to 100mL with ultrapure water.

[0176] 0.1N HCl solution: Take 95mL of concentrated hydrochloric acid into a filter flask and dilute to 100mL with ultrapure water;

[0177] 1% Alecid Blue Staining Solution: First, prepare a 5% Alecid Blue solution. Weigh 5g of Alecid Blue powder into a filter flask, add 90mL of ultrapure water and mix well. Make up to 100mL with ultrapure water. Dilute the 5% Alecid Blue solution to 1% using 0.1N HCl.

[0178] 1. Tissue sampling and embedding

[0179] 1) Harvest tissue 4-8 weeks after transplantation. Euthanize the mice using the cervical dislocation method;

[0180] 2) Use pointed tweezers to grasp the skin at the knee joint, and use scissors to make a 1cm incision along the grasped area down to the lower abdomen;

[0181] 3) Expose the subcutaneous tissue of the incision, insert scissors into the subcutaneous area and continuously open and close them horizontally to bluntly separate the skin and subcutaneous tissue;

[0182] 4) Make a small incision in the muscle on the right side of the patella with a scalpel, and use the back of the scalpel to cut the muscle upwards along the incision to make the patella easier to expose;

[0183] 5) Use the back of a scalpel to move the patella to the left, causing it to dislocate to the left and exposing the femoral articular surface;

[0184] 6) Use a scalpel to cut downwards at the knee joint connection to free the femur, use scissors to cut away the muscles on both sides of the femoral joint surface, and use scissors to cut it off 5mm upstream of the distal femoral joint. Immediately immerse it in fixation solution and fix it overnight at 4℃.

[0185] 7) Discard the fixative and wash away any remaining fixative with PBS;

[0186] 8) Immerse the tissue in decalcification solution and decalcify it on a shaker at room temperature for 2 days, changing the decalcification solution daily;

[0187] 9) Discard the decalcification solution, immerse the tissue in a 15% sucrose solution, dehydrate at room temperature (4°C), and replace with a 30% sucrose solution after the tissue has settled to the bottom. Once the tissue has settled to the bottom, the dehydration is complete.

[0188] 10) Immerse the tissue in OCT embedding gel. At this point, the tissue can be frozen and sectioned directly. If it is not to be sectioned immediately, the embedded tissue can be frozen and stored at -80°C.

[0189] 2. Chemical staining identification and analysis

[0190] Frozen sections can be stored at room temperature in the slide box. For staining, rehydrate the sections by immersing them in PBS to remove the OCT gel from the tissue surface. Stain the frozen sections according to the instructions of the commercial kit. Staining identification methods include: H&E, safranin-fast green staining, Masson's staining, and alcine blue staining.

[0191] 1) Allicin Blue staining

[0192] Treat the rehydrated sections with 0.1N HCl for 2 minutes; stain with 1% Alcian Blue solution for 30 minutes;

[0193] First, wash the slide with distilled water, then wash away non-specific binding with 0.1N HCl; wash away residual hydrochloric acid with distilled water, add hematoxylin (H&E staining kit) to show nuclei, and stain for 4 minutes; rinse under tap water for 4 minutes to return to blue; dehydrate with 95% ethanol for 1 minute, dehydrate with 100% ethanol for 1 minute, clear with xylene, and mount with neutral resin.

[0194] 2) Safranin-Fast Green staining

[0195] Frozen sections were rehydrated using standard procedures. They were stained with hematoxylin (using a commercial H&E staining kit) for 2 minutes; rinsed under tap water for 4 minutes to promote hematoxylin blue reversion; stained with 1% Fast Green solution (Sigma-Aldrich, USA) for 1 minute 30 seconds; differentiated with 1% glacial acetic acid solution for 15 seconds, washing away non-specific staining from Fast Green; the glacial acetic acid was removed by pipette, and the sections were stained with 5% safranin (Sigma-Aldrich, USA) for 1 minute.

[0196] Immerse in 95% ethanol for 15 seconds, then dehydrate the sections in 100% ethanol for 1 minute. Clear with xylene and mount with neutral resin.

[0197] 3) Masson staining and H&E staining

[0198] Frozen sections were routinely rehydrated. Staining was performed using modified Masson's trichrome staining solution (Solepro Corporation, China) and H&E staining kit (Solepro Corporation, China), following the kit instructions.

[0199] The results are attached. Figure 8 As shown, analysis of the staining results reveals that the H&E map shows the damaged area is filled with obvious cartilage-like tissue. Masson staining shows abundant collagen tissue (deep blue staining) in the filled area. Alcian blue and Safranin O staining further indicate that in addition to abundant collagen, this region also produces a large amount of polysaccharides (the cartilage portion is stained blue by Alcian blue and red by Safranin O), consistent with the staining pattern of normal articular cartilage and showing a significant contrast to the untransplanted cell group. This demonstrates that the scleral chondrocytes / bone progenitor cells induced by human pluripotent stem cells of this invention can repair femoral articular cartilage in mice and promote articular cartilage regeneration.

[0200] Finally, it should be noted that the above embodiments are merely preferred embodiments of the present invention used to illustrate the technical solutions of the present invention, and are not intended to limit the invention, nor are they intended to limit the patent scope of the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. That is to say, any changes or refinements made to the main design concept and spirit of the present invention that are not of substantial significance, but whose technical problems are still consistent with the present invention, should be included within the protection scope of the present invention. In addition, the direct or indirect application of the technical solutions of the present invention to other related technical fields are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing scleral osteoblast / chondrocyte mesenchymal progenitor cells derived from human pluripotent stem cells, characterized in that, Includes the following steps: Step 1. Differentiation of primary stem cells: Differentiation medium I was added to human pluripotent stem cells and cultured at 37±1℃ for 24 hours; the differentiation medium I was CDMi basal medium containing 30 ng / mL activin A, 7 μM CHIR99021, and 20 ng / mL bFGF. Step 2. Pre-segmental mesoderm differentiation: After culturing the cells in Step 1, remove differentiation medium I, rinse the cells, add differentiation medium II, and culture at 37±1℃ for 24 hours; the differentiation medium II is CDMi basal medium containing 20μM SB431542, 125nML DN193189, 3μM CHIR99021, and 40ng / mL bFGF. Step 3. Passaging and differentiation of mesenchymal progenitor cells: After culture in Step 2, the cells were aspirated from differentiation medium II, then digested, neutralized, and pipetted to obtain a single-cell suspension. The suspension was centrifuged, resuspended, and seeded into culture dishes at a density of 1×10⁶ cells / mL. 5 ~1.5×10 5 cells / cm 2 Add differentiation medium III and incubate at 37±1℃ for 24 hours, then replace the medium with differentiation medium IV and incubate at 37±1℃ for 24 hours. The differentiation medium III is a CDMi basal medium containing 200 nM SAG, 600 nM LDN193189, 0.5 μM XAV939, and 10 μM ROCKi; The differentiation medium IV is a CDMi basal medium containing 200 nM SAG, 600 nM LDN193189, and 0.5 μM XAV939.

2. The method for preparing scleral osteoblast / chondrocyte mesenchymal progenitor cells derived from human pluripotent stem cells according to claim 1, characterized in that, In step 3, the cell resuspension is seeded into a culture dish at a density of 1.18 × 10⁻⁶. 5 cells / cm 2 .

3. The method for preparing scleral osteoblast / chondrocyte mesenchymal progenitor cells derived from human pluripotent stem cells according to claim 1, characterized in that, Before the differentiation of the original stem in step 1, there is also a seeding step for human pluripotent stem cells, which specifically includes the following steps: S1. Human pluripotent stem cells were routinely cultured in culture plates containing Matrigel working medium; S2. When the cell density reaches 70%-90%, remove the culture medium and digest the cells at 37℃ for 3-5 minutes; S3. After aspirating the digestion solution, passage the culture medium at a ratio of 1:7 to 1:8 into another culture plate containing Matrigel working solution.

4. The method for preparing scleral osteoblast / chondrocyte mesenchymal progenitor cells derived from human pluripotent stem cells according to claim 3, characterized in that, The Matrigel working solution is prepared by diluting 1 mg of Matrigel stock solution in 8-15 mL of DMEM / F12.

5. A method for preparing scleral osteoblast / chondrocyte mesenchymal progenitor cells derived from human pluripotent stem cells according to claim 4, characterized in that, The Matrigel working solution was prepared by diluting 1 mg of Matrigel stock solution in 12 mL of DMEM / F12.

Images