Method for inducing alum plate interstitial precursor cells to be differentiated into osteoblast progenitor cells, differentiated osteoblast progenitor cells and application of differentiated osteoblast progenitor cells

Abstract

The invention discloses a method for inducing alumba interstitial precursor cells to be differentiated into osteoblast progenitor cells, the differentiated osteoblast progenitor cells and application, and belongs to the technical field of biological medicine. The method disclosed by the invention comprises the following steps: S1, treating a cell culture container by using a Matrigel working solution; s2, absorbing and abandoning the Matrigel working solution, dripping the alum interstitial precursor cell suspension into each area treated by the Matrigel working solution, and culturing the cell culture container at 37 DEG C; s3, after the cells are adhered to the wall, supplementing a transition culture medium into the cell culture container, and continuing to culture; s4, absorbing and abandoning the transition culture medium, and adding the osteogenic progenitor cell induction culture medium for continuous culture to obtain the osteogenic progenitor cells. The invention also discloses the osteoblast progenitor cell obtained by induction of the method, and application of the osteoblast progenitor cell in preparation of a medicine for promoting bone growth. According to the method, the osteogenic progenitor cells capable of simulating the osteogenic process in cartilage can be obtained through rapid and efficient differentiation.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a method for inducing scleral stromal progenitor cells to differentiate into osteoprogenitor cells, the differentiated osteoprogenitor cells and applications thereof. Background Art

[0002] The skeletal system of mammals is composed of bone tissue and cartilage tissue. Among them, bone tissue is one of the hardest tissues in the human body, which can not only support the weight of the human body, but also has a motor function. Cartilage tissue is regularly distributed in the cavity parts and joint surfaces of the body, playing a role in shock absorption and buffering. The important functions of the skeletal system have attracted extensive attention to related developmental disorders and tissue function injury diseases. For example, the acquired mutation of the FGFR3 gene will lead to endochondral ossification defects and cause abnormal bone development throughout the body. In this case, symptoms such as disproportionate trunk and limbs, large head and short limbs can be observed at birth in children. In addition to developmental disorders caused by gene mutations, various injury diseases, such as osteochondritis dissecans, large segment bone fractures and nonunion, have also become important challenges in the field of regenerative medicine due to the lack of sufficient seed cells and poor prognosis.

[0003] The isolation and in vitro culture of embryonic stem cells, as well as the induction of somatic cells into pluripotent stem cells, are revolutionary discoveries in the fields of biology and medicine. In recent years, the research on differentiating pluripotent stem cells into functional seed cells has gradually come into the view of developmental related diseases and tissue engineering research. The differentiation of embryonic / pluripotent stem cells into functional cells is a simulation of the embryonic development process. The development of the skeletal system originates from the convergence of mesenchymal cells in different parts (such as the craniofacial region, axial region and limb buds). For the axial region and limb buds, mesenchymal cells usually develop into bone through endochondral ossification, that is, mesenchymal cells first develop into chondrocytes, and then form bone tissue through processes such as hypertrophy, apoptosis, vascular invasion and osteoblast recruitment.

[0004] At present, multiple laboratories have successfully constructed osteogenic / chondrogenic progenitor cells (i.e., mesenchymal cells), and established a differentiation system from mesenchymal cells to chondrogenic progenitor cells. However, there is still a lack of a differentiation method that can simulate the in vivo endochondral ossification process and directionally induce osteogenic / chondrogenic mesenchymal progenitor cells into osteoprogenitor cells. Although Craft et al. have tried to induce the differentiation of paraxial mesoderm-derived mesenchymal progenitor cells into osteoblasts, the differentiation of the progenitor cells and osteoprogenitor cells involved in this method is very lengthy, and the in vitro induction time of osteoprogenitor cells even requires several weeks. In addition, after transplanting the osteoprogenitor cells differentiated by this method under the skin of immunodeficient mice, no typical growth plate and mature bone tissue were observed, only a large amount of calcified hypertrophic cartilage was seen.

[0005] Therefore, it has become an urgent problem for those skilled in the art to quickly and efficiently differentiate osteoprogenitor cells that can mimic the process of endochondral ossification. Summary of the Invention

[0006] The object of the present invention is to provide a method for directing the differentiation of scleral osteogenic / chondrogenic mesenchymal progenitor cells derived from human pluripotent stem cells into osteoprogenitor cells, and to direct the differentiation of scleral mesenchymal progenitor cells into osteoprogenitor cells in the form of micromass. The method is simple, easy to operate, and has a short induction and differentiation time.

[0007] Another object of the present invention is to provide osteoprogenitor cells obtained by directed differentiation using this method.

[0008] The third object of the present invention is to provide the application of the osteoprogenitor cells.

[0009] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] In the first aspect, the present invention discloses a method for inducing the differentiation of scleral mesenchymal progenitor cells into osteoprogenitor cells, comprising the following steps:

[0011] S1. Treat the cell culture container with Matrigel working solution: Add Matrigel working solution to the cell culture container, and then incubate it at 37°C.

[0012] S2. Take out the cell culture container treated with Matrigel, aspirate the Matrigel working solution, drop the scleral mesenchymal progenitor cell suspension on each area treated with Matrigel working solution, and then place the cell culture container at 37°C for incubation.

[0013] S3. After the cells adhere, supplement the transition medium to the cell culture container, and then continue to incubate at 37°C.

[0014] S4. Take out the cell culture container cultured in step S3, aspirate the transition medium, and then add osteoprogenitor cell induction medium to continue culturing to obtain osteoprogenitor cells.

[0015] In some embodiments of the present invention, in step S1, the Matrigel working solution is composed of a DMEM / F12 solution and a Matrigel stock solution; preferably, the volume-mass ratio of the DMEM / F12 solution to the Matrigel stock solution is 10-14:1; preferably 12:1;

[0016] Preferably, add Matrigel working solution to the cell culture container so that the liquid forms a domed arch and the contact surface part is circular;

[0017] Preferably, after adding the Matrigel working solution to the cell culture container, it is cultured at 37°C for 0.5 to 2 hours; preferably 1 hour.

[0018] In some embodiments of the present invention, in step S2, when dropping the suspension of scleral stromal progenitor cells on each area treated with the Matrigel working solution, the liquid is arched in a dome shape.

[0019] Further, the cell culture container seeded with the suspension of scleral stromal progenitor cells is placed at 37°C for 10 to 60 minutes, preferably 25 minutes.

[0020] In some embodiments of the present invention, in step S3, after the cells adhere, transitional medium is supplemented to the cell culture container and cultured for 12 to 48 hours, preferably 12 to 24 hours.

[0021] In some embodiments of the present invention, the osteogenic progenitor cell induction medium is composed of CDMi basal medium and SAG, BMP2, and KSR added to the CDMi basal medium.

[0022] Preferably, in the osteogenic progenitor cell induction medium, 100 to 300 nmol of SAG, 50 to 200 μg of BMP2 are added to each liter of CDMi basal medium, and the volume content of KSR in the osteogenic progenitor cell induction medium is 0.5 to 2%.

[0023] In some embodiments of the present invention, in step S4, osteogenic progenitor cell induction medium is added and cultured for 6 to 24 days, preferably 12 days.

[0024] In some embodiments of the present invention, the method for preparing the suspension of scleral stromal progenitor cells includes the following steps:

[0025] The scleral stromal progenitor cells are digested, neutralized, repeatedly pipetted, centrifuged, and the supernatant is aspirated; then transitional medium is added to resuspend the cells to obtain the suspension of scleral stromal progenitor cells.

[0026] Preferably, the scleral stromal progenitor cells are added to a digestive enzyme to digest the cells, Wash buffer is added to neutralize, the cells are pipetted into a single cell suspension, centrifuged, the supernatant is aspirated, the cells are resuspended with Wash buffer, pipetted and mixed evenly, counted, and then centrifuged again, and the supernatant is aspirated; then transitional medium is added to resuspend the cells to obtain the suspension of scleral stromal progenitor cells.

[0027] In some embodiments of the present invention, 200,000 to 1,200,000 cells are resuspended with 5 to 30 μL of transitional medium; preferably, 600,000 cells are resuspended with 15 μL of transitional medium.

[0028] In some embodiments of the present invention, the transition medium consists of CDMi basal medium and SAG, LDN193189, and ROCKi added to the CDMi basal medium;

[0029] 50 - 250 nmol of SAG, 150 - 800 nmol of LDN193189, and 5 - 20 μmol of ROCKi are added to each liter of the CDMi basal medium.

[0030] Second, the present invention discloses an osteogenic progenitor cell, which is induced by the above method.

[0031] Third, the present invention discloses the application of an osteogenic progenitor cell in the preparation of a drug for promoting bone growth.

[0032] The scleral stromal progenitor cells described in the present invention are human pluripotent stem cell-derived scleral osteogenic / chondrogenic stromal progenitor cells prepared by the method described in the patent with the publication number CN 115612664 A.

[0033] The volume-mass ratio of the DMEM / F12 solution to the Matrigel mother liquor in the present invention is 10 - 14:1; it means that when the volume of the DMEM / F12 solution is 10 - 14 parts, the mass of the Matrigel mother liquor is 1 part. When the mass unit is mg, the volume unit is mL.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The differentiation process of the present invention is simple and easy to repeat; the components of the used medium are clear and do not contain serum components. The induction and differentiation time using the method of the present invention is short, and directed osteogenic progenitor cells can be obtained in only 12 days.

[0036] The osteogenic progenitor cells induced by the method of the present invention express early chondrocyte-related marker genes Sox9, Col2a1, Sox5, Sox6, Col9a1, Acan, and Epyc; after the osteogenic progenitor cells are transplanted subcutaneously into immunodeficient mice, a uniform cartilage tissue can be formed after 1 month, which contains calcified hypertrophic cartilage and no other tissues are formed; after the osteogenic progenitor cells are transplanted subcutaneously into immunodeficient mice, a structure similar to the growth plate and mature calcified bone tissue can be formed after 2 months. The method of the present invention can quickly and efficiently differentiate into osteogenic progenitor cells that can simulate the process of endochondral ossification. Description of the Drawings

[0037] Figure 1 It is a graph of the real-time fluorescence quantitative PCR detection results of Example 2;

[0038] Figure 2Bright field, human-specific protein Ku80 immunofluorescence staining (human KU80 DAPI), Col II immunofluorescence staining (Col II DAPI), Col X immunofluorescence staining (Col X DAPI), Safranin O staining, and Alizarin red staining images of the sampled tissues one month (4-week timepoint) after the transplantation of osteoprogenitor cells into the subcutaneous tissue of immunodeficient mice;

[0039] Figure 3 Bright field, human-specific protein Ku80 immunofluorescence staining (human KU80 DAPI), and Safranin O staining images of the sampled tissues two months (8-week timepoint) after the transplantation of osteoprogenitor cells into the subcutaneous tissue of immunodeficient mice, where Figure 3 v is Figure 3 a partial enlarged view of iv, Figure 3 vi and Figure 3 vii are Figure 3 Col II immunofluorescence staining (Col II DAPI) and Col X immunofluorescence staining (Col X DAPI) images of the same region as v;

[0040] Figure 4 HE staining pictures of the sampled tissues two months after the transplantation of osteoprogenitor cells into the subcutaneous tissue of immunodeficient mice, where Figure 4 i, 4ii are Figure 4 partial enlarged views of the left figure. Detailed implementation manners

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0042] The reagents and drugs involved in the embodiments of the present invention are as follows:

[0043] Holo-transferrin working solution: Weigh 15 mg of Holo-transferrin (Sigma, USA) powder and add 1 mL of UP water (Invitrogen, USA) to it;

[0044] Rh-insμLin working solution: Weigh 2 mg of rh-insμLin (from Solarbio, China) powder, add 1 mL of 10 mM HCL to it, and filter to sterilize.

[0045] Polyvinyl alcohol working solution: Weigh 500 mg of Polyvinyl alcohol powder, add 18 mL of UP water (from Invitrogen, USA), stir until a granular suspension is formed, then heat and dissolve in a water bath at 85 °C for about 20 minutes, make up the volume to 20 mL, and filter to sterilize.

[0046] monothioglycerol working solution: Pipette 450 μmol equivalent of monothioglycerol stock solution (from Sigma, USA), add it to UP water to make the total volume of the solution 1 mL, vortex and mix well, and filter to sterilize.

[0047] CDMi basal medium: 234.384 mL of IMDM (from Gibco, USA), 234.384 mL of Ham’s F12 (from Gibco, USA), 5 mL of Chemically Defined Lipid Concentrate (from Gibco, USA), 500 μL of Holo-transferrin working solution, 234 μL of rh-insμLin working solution, 20 mL of Polyvinyl alcohol working solution, 5 mL of Pen-strep (from Gibco, USA), 500 μL of monothioglycerol working solution.

[0048] Transition medium: CDMi basal medium supplemented with SAG (from MCE, USA, 200 nM), LDN193189 (from MCE, USA, 600 nM), and ROCKi (from MCE, USA, 10 μM).

[0049] Osteoprogenitor cell medium: CDMi basal medium supplemented with SAG (from MCE, USA, 200 nM), BMP2 (from Preprotech, USA, 100 ng / mL), and KSR (from Thermo Fisher, USA, 1%).

[0050] Wash buffer: Weigh 1.5 g of bovine serum albumin (BSA, from Sigma, USA) and add it to 500 mL of DMEM / F12 medium (from Gibco, USA), mix well and filter to sterilize.

[0051] Matrigel working solution: Add 1 mg of Matrigel stock solution (Corning, USA) to 12 mL of DMEM / F12 solution (Gibco, USA) and mix well.

[0052] The scleral stromal progenitor cells in Example 1 of the present invention are human pluripotent stem cell-derived scleral osteogenic / chondrogenic stromal progenitor cells prepared by the method of Patent Example 2 with the publication number CN 115612664 A.

[0053] Example 1

[0054] This example discloses a method for the directed differentiation of scleral stromal progenitor cells into osteoprogenitor cells in the form of micromass.

[0055] 1) Treat the culture plate with Matrigel working solution. Pipette 5 μL of Matrigel working solution and seed 5 μL of Matrigel working solution in the six-well plate, ensuring that the liquid forms a dome-shaped prototypic droplet at the bottom. Place the culture plate in a 37 °C incubator for 1 hour;

[0056] 2) Take out the scleral stromal progenitor cells from the 37 °C incubator. Aspirate and discard the culture medium. Add 500 μL of TrypLE digestive solution (Thermofisher, USA) to the well and place the cells in a 37 °C incubator for digestion for 3 minutes;

[0057] 3) After digestion, add 4 times the volume (2 mL) of Wash buffer to the well for neutralization, and gently pipette to make a single-cell suspension. Aspirate the cells into a 15 mL centrifuge tube, centrifuge at 300 g for 3 minutes, and resuspend the cells with an appropriate amount of Wash buffer (usually 1 mL) after centrifugation;

[0058] 4) Count the cells using a hemocytometer;

[0059] 5) According to the counting results, take an appropriate amount of cell suspension into a new 15 mL centrifuge tube. The number of cells taken should be based on 200,000 cells / sample (micromass). For example, when the suspension contains 600,000 cells, all can be taken into a 15 mL centrifuge tube, and the number of cells contained can be used for 3 samples (micromass). Supplement the liquid in the tube with Wash buffer to 2.5 mL or more and centrifuge at 300 g for 3 minutes;

[0060] 6) Aspirate and discard the supernatant, and add the transition medium to it at a volume of 5 μL / sample according to the sampling quantity and pipette to mix well to form a scleral stromal progenitor cell suspension for inoculation.

[0061] 7) Remove the culture dish (well plate) treated with Matrigel from the incubator, aspirate the Matrigel, and add the scleral stromal progenitor cell suspension dropwise to the Matrigel-treated area at 5 μL / drop. Similarly, ensure that the liquid forms a dome-like arch, and then place the culture dish (or well plate) back into the 37°C incubator for 25 minutes;

[0062] 8) After 25 minutes, take out the culture dish (well plate) from the incubator. Observation under an optical microscope shows that the cells adhere to the Matrigel-treated area, forming round cell clusters of uniform size, and each cell cluster is a micromass;

[0063] 9) Supplement an appropriate amount of transitional medium to the culture dish (well plate), and the amount supplemented depends on the size of the culture dish (well plate). For example, a 24-well plate usually requires 450 μL - 500 μL of medium. Place the culture dish (or well plate) back into the 37°C incubator for overnight culture;

[0064] 10) The next day, aspirate the transitional medium. Add an equal volume of Washbuffer to the culture dish (or well plate) to wash the cells;

[0065] 11) Aspirate the Wash buffer, add an equal volume of osteoblast progenitor cell induction medium to the culture dish (or well plate), and then place it back into the 37°C incubator for continuous culture for 12 days. Replace the fresh osteoblast progenitor cell induction medium every day to induce the directed differentiation of osteoblast progenitor cells.

[0066] Example 2

[0067] This example discloses the identification test of RNA extraction and fluorescence quantitative PCR of osteoblast progenitor cells. The osteoblast progenitor cells in this example are obtained by directed differentiation according to the method of Example 1.

[0068] 1) Take out the uninduced scleral stromal progenitor cells, the micromass cells induced in the osteoblast progenitor cell induction medium for 6 days and 12 days from the 37°C incubator, aspirate the original medium, add 700 μL of TRI reagent to the culture plate, pipette continuously, and let it stand at room temperature for 5 - 10 minutes until the cells are fully lysed;

[0069] 2) Phase separation. Add chloroform to the tube at a ratio of chloroform:TRI reagent = 1:5 (v / v), shake vigorously for 15 seconds, and then let it stand at room temperature for 4 - 10 minutes;

[0070] 3) Centrifuge at 12000g for 15 minutes (2 - 8°C). Take the top layer (colorless and transparent) for RNA precipitation;

[0071] 4) Add isopropanol to a new EP tube at a ratio of isopropanol: TRI reagent = 1:2 (v / v), and add an equal amount of clear supernatant containing RNA; vortex for 10 seconds and then let stand at room temperature for 10 minutes;

[0072] 5) Centrifuge at 12000g for 10 minutes (2 - 8 °C), and discard the supernatant;

[0073] 6) Wash with ethanol. Prepare 75% ethanol with enzyme - free water. Add absolute ethanol at a ratio of 75% ethanol: TRI reagent = 1.5:1 (v / v), gently invert the EP tube up and down, centrifuge at 12000g for 4 minutes (in an environment of 2 - 8 °C), and discard the supernatant;

[0074] 7) Repeat step 6);

[0075] 8) Let the EP tube dry at room temperature, and add an appropriate amount of enzyme - free water at 56 °C to dissolve the RNA;

[0076] 9) Measure the RNA concentration using a quantitative device such as Nanodrop;

[0077] 10) Reverse - transcribe 100 - 1000 ng of the extracted RNA into cDNA using a commercial reverse - transcription kit;

[0078] 11) Dilute the cDNA with enzyme - free water at a ratio of 1:4 - 1:10 (v / v);

[0079] 12) Using ACTB (encoding β - actin) as the internal reference gene, quantitatively analyze the genes related to scleral stromal progenitor cells (Sox9, Col2a1, Sox5, Sox6, Col9a1, Acan, Epyc) using a commercial qPCR kit (Vazyme Company, China). The specific operation is carried out according to the instructions of the qPCR kit. The results are as shown in the appendix Figure 1 as follows.

[0080] As can be seen from the appendix Figure 1 it can be known that during the induction process, scleral stromal progenitor cells gradually express genes related to early chondrocytes, indicating that the osteoprogenitor cells of the present invention are differentiating downward from stromal progenitor cells in the way of endochondral ossification.

[0081] Experimental Example 1

[0082] This experimental example discloses the subcutaneous transplantation of osteoprogenitor cells and the identification test of the graft. The reagents involved are as follows:

[0083] Avertin stock solution: Weigh 25 g of 2,2,2-tribromoethanol (Sigma, USA) into a filter flask, and add 15.5 mL of tert-amyl alcohol (Sigma, USA) to it to prepare an 80x stock solution. When in use, dilute it to 1x with 0.9% sodium chloride (i.e., add 0.5 mL of the stock solution to 39.5 mL of normal saline);

[0084] 4% PFA fixative: Weigh 4 g of paraformaldehyde powder (Sigma, USA) into a filter flask, add 90 mL of ultrapure water, and dissolve it by shaking overnight in a shaker at 37 °C. After dissolution, make up the volume to 100 mL with ultrapure water and store it in the dark at 4 °C;

[0085] 19% EDTA decalcifying solution: Weigh 19 g of EDTA powder into a filter flask, add 90 mL of ultrapure water to it, and stir to dissolve it on a magnetic stirrer. When the solution turns white, gradually add NaOH powder to it and continue stirring until dissolved. Use a precision pH test paper to detect the pH value of the solution, and adjust the pH to 7.0 with concentrated hydrochloric acid. Finally, make up the volume to 100 mL with ultrapure water;

[0086] 15% sucrose solution: Weigh 15 g of sucrose crystal particles into a filter flask, add 90 mL of ultrapure water to it, and dissolve it at room temperature. Then make up the volume to 100 mL with ultrapure water;

[0087] 30% sucrose solution: Weigh 15 g of sucrose crystal particles into a filter flask, add 90 mL of ultrapure water to it, and dissolve it at room temperature. Then make up the volume to 100 mL with ultrapure water;

[0088] 1. Subcutaneous transplantation of osteoprogenitor cells

[0089] This test example discloses a subcutaneous transplantation test of osteoprogenitor cells. The osteoprogenitor cells in this example were directionally differentiated according to the method of Example 1.

[0090] 1) Take out the osteoprogenitor cells from the 37 °C incubator, add Accutase digestive enzyme (Innovative cell technologies, USA) to the culture dish (or well plate) at a ratio of medium: digestive enzyme of 2:1 (v / v), and digest at 37 °C for 1 minute;

[0091] 2) Take out the digested cells from the incubator, use a 200 μL pipette to blow off the micromass from the culture dish (or well plate), and continue to blow 10 times. At this time, the micromass can be seen floating in the digestive solution in a flocculent state. Put the culture dish (well plate) back into the incubator for continued digestion;

[0092] 3) After 3 minutes, take out the culture dish (well plate) from the incubator and blow it 15 times with a 200 μL pipette;

[0093] 4) After repeating step 3 two to three times, it can be seen that micromass is digested into a single-cell suspension. Add 4 times the volume of Wash buffer to it, transfer the cells to a centrifuge tube, and centrifuge at 300 g for 3 minutes;

[0094] 5) Aspirate and discard the supernatant. Resuspend the cells according to the standard of 1 - 1.5 micromass / 50 μL of Matrigel stock solution;

[0095] 6) Anesthetize non-obese diabetic / severe combined immunodeficient (NOD / SCID) mice by intraperitoneal injection with avertin working solution, and the injection dose is 20 μL / g body weight;

[0096] 7) Place the mice on a laminar flow bench, and use lubricating fluid to prevent their eyeballs from drying; Use a hair clipper to shave the hair near the surgical site;

[0097] 8) Use an insulin syringe (Bayer, Germany) vertically buried in an ice box to aspirate 50 μL of osteoprogenitor cell suspension (suspended in Matrigel stock solution), and horizontally inject it subcutaneously at the disinfected site. It can be seen that the injected substance forms a bulge in the subcutaneous area;

[0098] 9) After transplantation, wait for 15 minutes while the mice are placed on a 30 °C constant temperature table, and then put them back into the cage.

[0099] 2. Identification of the graft: Immunofluorescence staining and chemical staining of graft sections

[0100] 2.1 Embedding and frozen sectioning of the graft

[0101] 1) Harvest the samples 4 - 8 weeks after transplantation. Sacrifice the mice by cervical dislocation;

[0102] 2) Cut open the skin around the transplantation site, remove the excess tissue with scissors, and then use forceps to take out the graft. Immediately immerse it in the fixative and fix it overnight at 4 °C;

[0103] 3) Pour out the fixative and wash away the residual fixative with PBS;

[0104] 4) Immerse the tissue in the decalcifying solution and decalcify it at room temperature on a shaker for 2 days, and change the decalcifying solution every day;

[0105] 5) Pour out the decalcifying solution, immerse the tissue in 15% sucrose solution, dehydrate it at 4 °C at room temperature. After the tissue sinks to the bottom, change it to 30% sucrose solution, and the dehydration is completed after the tissue sinks to the bottom;

[0106] 6) Immerse the tissue in OCT embedding medium. At this time, tissue frozen sections can be directly made, and the section thickness is 6 μm. If not sectioned immediately, the embedded tissue can be stored frozen at -80 °C.

[0107] 2.2 Chemical staining identification

[0108] 1) Take the sections and soak them in PBS for rehydration to remove the OCT compound on the tissue surface; stain the frozen sections according to the method described in the commercial kit instructions. For example: H&E, safranin-fast green staining

[0109] 2) Safranin-fast green staining:

[0110] a. Routinely rehydrate the frozen sections. Stain with hematoxylin (commercial H&E staining kit) for 2 minutes; rinse the sections under tap water for 4 minutes to promote the blueing of hematoxylin; stain with 1% fast green solution (Sigma, USA) for 1 minute and 30 seconds; differentiate with 1% glacial acetic acid solution for 15 seconds to wash away the non-specific staining of fast green; aspirate the glacial acetic acid with a pipette and stain with 5% safranin (Sigma, USA) for 1 minute;

[0111] b. Wash with 95% ethanol for 15 seconds, dehydrate the sections in 100% ethanol for 1 minute, clear with xylene, and mount with neutral resin.

[0112] 3) H&E staining:

[0113] Routinely rehydrate the frozen sections. Stain with an H&E staining kit (Solarbio, China), and the method follows the kit instructions.

[0114] 2.3 Immunofluorescence staining identification: Taking Col II staining as an example

[0115] 1) Routinely rehydrate the frozen sections. Repair the antigen with pepsin (Sigma, USA) and incubate in an incubator at 37°C for 30 minutes;

[0116] 2) Block with 5% bovine serum albumin solution (prepared with PBS) at room temperature for 1 hour;

[0117] 3) Aspirate the blocking solution and stain with COL II antibody (Invitrogen, USA, dilution ratio 1:200), incubate overnight in a 4°C refrigerator;

[0118] 4) Wash three times with PBS, 3 minutes each time;

[0119] 5) Add an appropriate amount of secondary antibody (dilution ratio 1:400, diluted with the blocking solution) and stain at room temperature for 1 hour;

[0120] 6) Aspirate the secondary antibody and wash 3 times with PBS, 3 minutes each time;

[0121] 7) Dry the PBS, stain the nuclei and mount with a mounting medium containing DAPI.

[0122] The results are as Figures 2 to 4As shown in the figure. Analysis of the staining results showed that uniform cartilage tissue was formed one month after the transplantation of osteoprogenitor cells, and this tissue was formed by human cells (positive staining with Ku80 antibody); two months after transplantation, a growth plate-like tissue was formed, and the cartilage tissue was arranged directionally like coins and developed into hypertrophic cartilage (positive staining with Col X). At the same time, bone tissue with deep eosin staining in H&E staining was formed. It indicates that osteoprogenitor cells have experienced the whole process of development from cartilage to bone, forming a growth plate-like tissue, and well simulating the endochondral ossification process in vivo.

[0123] The above description is only the preferred embodiment of the present invention, which is merely illustrative of the present invention rather than limiting the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solution of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for inducing scleral mesenchymal precursor cells to differentiate into osteoblastic progenitor cells, characterized in that: The steps include: S1. Treat the cell culture container with Matrigel working solution: Add Matrigel working solution to the cell culture container and then culture it at 37°C; S2. Take out the cell culture container treated with Matrigel, discard the Matrigel working solution, drop the suspension of scleral plate mesenchymal precursor cells in each area treated with Matrigel working solution, and then culture the cell culture container at 37°C; S3. After the cells adhere to the wall, add transition medium to the cell culture container, and then continue to culture at 37°C; S4. Take out the cell culture container after culture in step S3, discard the transition culture medium, and then add osteoprogenitor cell induction medium to continue culture to obtain osteoprogenitor cells.

2. The method of inducing scleral mesenchymal precursor cells to differentiate into osteoblastic progenitor cells according to claim 1, characterized in that: In step S1, the Matrigel working solution is prepared by mixing a DMEM / F12 solution with a Matrigel mother solution; preferably, the volume mass ratio of the DMEM / F12 solution to the Matrigel mother solution is 10 to 14:1; preferably 12:1; Preferably, Matrigel working solution is added to the cell culture container so that the liquid is arched in a dome shape and is divided into a circle with the contact surface; Preferably, the Matrigel working solution is added to the cell culture container and then cultured at 37° C. for 0.5 to 2 hours, preferably 1 hour.

3. The method of inducing scleral mesenchymal precursor cells to differentiate into osteoblastic progenitor cells according to claim 1, characterized in that: In step S2, when the suspension of scleral plate mesenchymal precursor cells is dropped on each area treated with Matrigel working solution, the liquid is made to bulge in a dome shape; Furthermore, the cell culture container seeded with the scleral plate mesenchymal precursor cell suspension is placed at 37° C. for 10 to 60 minutes, preferably for 25 minutes.

4. The method of inducing scleral mesenchymal precursor cells to differentiate into osteoblastic progenitor cells according to claim 1, characterized in that: In step S3, after the cells adhere to the wall, a transition medium is added to the cell culture container and the culture is continued for 12 to 48 hours, preferably 12 to 24 hours.

5. The method of inducing scleral mesenchymal precursor cells to differentiate into osteoblastic progenitor cells according to claim 1, characterized in that: The induction medium consists of a CDMi basal medium and SAG, BMP2 and KSR added to the CDMi basal medium; Preferably, in the induction medium, 100-300 nmol of SAG and 50-200 μg of BMP2 are added to each liter of CDMi basal medium, and the volume content of KSR in the induction medium is 0.5-2%.

6. The method of inducing scleral mesenchymal precursor cells to differentiate into osteoblastic progenitor cells according to claim 1, characterized in that: In step S4, osteoprogenitor cell induction medium is added and the culture is continued for 6 to 24 days, preferably 12 days.

7. The method of inducing scleral mesenchymal precursor cells to differentiate into osteoblastic progenitor cells according to claim 1, characterized in that: The method for preparing the suspension of scleral mesenchymal precursor cells comprises the following steps: The scleral plate mesenchymal precursor cells are digested, neutralized, repeatedly blown and centrifuged, and the supernatant is discarded; then the transition culture medium is added to resuspend the cells to obtain a scleral plate mesenchymal precursor cell suspension.

8. A method for inducing scleral mesenchymal precursor cells to differentiate into osteoblastic progenitor cells according to any one of claims 1 to 7, characterized in that: The transition medium consists of a CDMi basal medium and SAG, LDN193189, and ROCKi added to the CDMi basal medium; Add 50-250 nmol of SAG, 150-800 nmol of LDN193189, and 5-20 μmol of ROCKi per liter of CDMi basal culture medium.

9. An osteoblast progenitor cell, characterized in that: The method is induced by any one of claims 1 to 8.

10. Use of the osteoblast progenitor cells according to claim 9 in the preparation of a drug for promoting bone growth.

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