CN115505646B - Ban osteoblast/cartilage precursor cell characteristic surface molecular marker, screening method and application thereof - Google Patents

Abstract

The present invention discloses characteristic surface molecular markers of scleral plate osteoblasts/chondrogenic mesenchymal precursor cells, screening methods and applications thereof, belonging to the field of biotechnology, and solving the problem that the scleral plate osteoblasts/chondrogenic mesenchymal precursor cells prepared by in vitro differentiation cannot be separated and enriched in the prior art, which affects clinical safety. The characteristic surface molecular markers of the present invention include ITGA9, which is used in the separation and enrichment of scleral plate osteoblasts/chondrogenic mesenchymal precursor cells derived from human pluripotent stem cells. The ITGA9 of the present invention can well distinguish target cells from non-target cells in the mixture obtained by differentiation; at the same time, the ITGA9 positive cells obtained by sorting express molecular markers related to scleral plate mesenchymal precursor cells, and have the potential for chondrogenic and osteogenic differentiation, while the negative cells do not have such ability.

Description

Characteristic surface molecular markers of scleral osteoblasts/chondrogenic mesenchymal precursor cells, their screening methods and applications

Technical Field

The invention belongs to the field of biotechnology, and specifically relates to characteristic surface molecular markers of scleral osteoblast/chondrogenic mesenchymal precursor cells derived from human pluripotent stem cells, and a screening method and application thereof.

Background Art

The induction of pluripotent stem cells (iPSCs) from somatic cells is a revolutionary discovery in the field of biology and medicine. Induced pluripotent stem cells have multidirectional differentiation potential and can achieve self-renewal. Starting from pluripotent stem cells, through recombinant proteins, growth factors, small molecule induction and other technical means, the in vitro differentiation and large-scale preparation of a variety of human functional cells have been achieved, including cardiomyocytes, insulin-secreting cells, neurons, etc., which is a research field with important transformation value in regenerative medicine research.

At present, the use of pluripotent stem cells to prepare functional cells for cell therapy still faces some key technical/process problems that need to be solved urgently: Taking the mesenchymal precursor cells derived from the sclera as an example, first of all, their preparation must undergo the induction of multiple germ layers/multiple differentiation steps. Even in a culture medium environment with clear chemical composition, there is still a certain degree of heterogeneity. Among the cells obtained by in vitro differentiation preparation, there are potential undifferentiated iPSCs, which will bring potential tumorigenic risks to cell products and affect the safety of their clinical transformation. In the cell preparation process, it is necessary to consider the specific purification and enrichment of target cells and the removal of potential contaminating iPSCs. These technical processes all rely on the identification of specific surface markers for the target cells. Secondly, the current preparation process of sclera mesenchymal precursor cells still relies on the expression of reporter genes, that is, the in vitro induction differentiation of iPSCs with the Sox9 reporter gene knocked in is used. This preparation process cannot be directly used for the development of clinical-grade cell therapy drugs, and it is urgent to develop cell induction differentiation and preparation methods that do not rely on reporter genes. Therefore, the discovery of surface markers that can effectively enrich sclera mesenchymal precursor cells is a key technical bottleneck that needs to be solved in the current cell preparation process.

Summary of the invention

One of the purposes of the present invention is to provide characteristic surface molecular markers of scleral plate osteoblasts/chondrocyte mesenchymal precursor cells derived from human pluripotent stem cells, so as to solve the problem in the prior art that the scleral plate osteoblasts/chondrocyte mesenchymal precursor cells obtained by in vitro differentiation cannot be separated and enriched, thus affecting clinical safety.

The second object of the present invention is to provide a method for screening characteristic surface molecular markers of scleral osteoblasts/chondrogenic mesenchymal precursor cells derived from human pluripotent stem cells.

The third object of the present invention is to provide the application of the surface molecular marker.

To achieve the above purpose, the technical solution adopted by the present invention is as follows:

The present invention provides characteristic surface molecular markers of scleral plate osteogenic/chondrogenic mesenchymal precursor cells derived from human pluripotent stem cells, characterized in that they include ITGA9.

The present invention provides a method for screening characteristic surface molecular markers of scleral osteoblasts/chondrogenic mesenchymal precursor cells derived from human pluripotent stem cells, characterized in that it comprises the following steps:

Step 1. SOX9-positive cells obtained by sorting scleral osteoblasts/chondrogenic mesenchymal precursor cells derived from human pluripotent stem cells are used as positive cells, and SOX9-negative cells are used as negative cells, and differential gene analysis is performed, and genes that are upregulated at least twice in positive cells relative to negative cells (the logarithm of Fold Change is greater than or equal to 1) are used as differential genes;

Step 2. Compare the differentially expressed genes with known surface molecular proteins and take the intersection to obtain all the surface molecular markers that are upregulated in positive cells;

Step 3. Calculate the standardized expression amount (TPM value) of the upregulated surface molecular marker in positive cells and negative cells, delete the surface molecular markers with low expression in positive cells and high expression in negative cells, and obtain candidate surface molecular markers;

Step 4. Based on the standardized gene expression level, an optimal marker is selected as a characteristic surface molecular marker of scleral osteoblast/chondrogenic mesenchymal precursor cells derived from human pluripotent stem cells.

In some embodiments of the present invention, the optimal marker is ITGA9.

In some embodiments of the present invention, in step 1, the edgeR operating package of the R language platform is used to perform differential gene analysis.

In some embodiments of the present invention, in step 2, R Studio software is used to compare with known surface molecule proteins and then take the intersection.

In one embodiment of the present invention, in step 2, the cell surface protein molecule library http://wlab.ethz.ch/cspa established by Damaris Bausc-Fluck et al. using mass spectrometry for 41 types of human cells and 31 types of mouse cells is compared and then the intersection is taken.

In some embodiments of the present invention, in step 3, the standardized expression levels of the upregulated surface molecular markers in positive cells and negative cells are calculated using R Studio software.

In some embodiments of the present invention, in step 3, surface molecular markers with standardized expression levels less than 50 in positive cells and greater than 50 in negative cells are deleted to obtain candidate surface molecular markers.

Some embodiments of the present invention further include a verification step, and based on the verification results, the characteristic surface molecular markers of scleral osteoblast/chondrogenic mesenchymal precursor cells derived from human pluripotent stem cells are determined.

In some embodiments of the present invention, the verification step comprises:

S1. Use ITGA9 antibody to stain and sort scleral osteoblasts/chondrogenic mesenchymal precursor cells;

S2. Collecting the sorted ITGA9-positive cells and ITGA9-negative cells;

S3. Inducing chondrogenic differentiation and osteogenic differentiation of ITGA9-positive cells and ITGA9-negative cells in vitro;

S4. Based on whether the ITGA9-positive cells after induced differentiation have the function of promoting and/or repairing cartilage growth and promoting and/or repairing osteogenesis, it is expected to determine whether they are characteristic surface molecular markers of scleral osteoblasts/chondrocyte mesenchymal precursor cells derived from human pluripotent stem cells.

The invention provides an application of characteristic surface molecular markers of scleral plate osteoblasts/chondrocyte mesenchymal precursor cells derived from human pluripotent stem cells in separating and enriching scleral plate osteoblasts/chondrocyte mesenchymal precursor cells derived from human pluripotent stem cells.

The scleral plate osteoblast/chondrocyte mesenchymal precursor cells derived from human pluripotent stem cells described in the present invention refer to scleral plate osteoblast/chondrocyte mesenchymal precursor cells differentiated from human pluripotent stem cells by induction.

Methods for inducing human pluripotent stem cells into scleral osteoblasts/chondrogenic mesenchymal precursor cells are known in the art.

Or follow the steps below:

Step A. Primitive streak differentiation: adding the human pluripotent stem cells after inoculation and passage into differentiation medium I for induction culture; the differentiation medium I is a CDMi basal medium containing activin A, CHIR99021, and bFGF;

Step B. Presomitic mesoderm differentiation: remove differentiation medium I from the cells cultured in step A, rinse the cells, add differentiation medium II, and induce culture; the differentiation medium II is a CDMi basal medium containing SB431542, LDN193189, CHIR99021, and bFGF;

Step C. somitogenesis: remove differentiation medium II from the cells cultured in step B, rinse the cells, add differentiation medium III, and induce culture; the differentiation medium III is a CDMi basal medium containing PD0325901 and XAV939;

Step D. Scleral plate mesenchymal stem cell differentiation: The cells cultured in step C are aspirated to remove differentiation medium III, the cells are rinsed, and differentiation medium IV is added to induce culture; the differentiation medium IV is CDMi basal medium containing SAG and LDN193189.

The induction culture condition in step A is culturing at 37±1°C for 12-48 hours, preferably 24 hours;

or/and the induction culture condition in step B is culturing at 37±1° C. for 12-48 hours, preferably for 24 hours;

Or/and the induction culture condition in step C is culturing at 37±1°C for 12-48 hours; preferably 24 hours;

or/and the induction culture condition in step D is culturing at 37±1° C. for 48-72 hours; preferably 72 hours;

In the differentiation medium, the content of each component is activin A 10-40ng/mL, CHIR99021 3-10μM, bFGF 10-30ng/mL; preferably activin A 30ng/mL, CHIR99021 7μM, bFGF 20ng/mL;

Or/and in the differentiation medium II, the content of each component is: SB431542 10-30 μM, LDN193189100-200 nM, CHIR99021 1-6 μM, bFGF 20-80 ng/mL; preferably SB431542 20 μM, LDN193189125 nM, CHIR99021 3 μM, bFGF 40 ng/mL;

or/and in the differentiation medium III, the content of each component is: XAV939 1-3 μM, PD0325901 0.2-0.8 μM; preferably XAV939 1 μM, PD0325901 0.5 μM;

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

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

The present invention is scientifically designed and ingeniously conceived. It uses bulk transcriptome sequencing technology to extensively and comprehensively screen surface molecular markers of scleral plate osteoblasts/chondrogenic mesenchymal precursor cells. ITGA9 screened by the present invention is used as a surface molecular marker of scleral plate osteoblasts/chondrogenic mesenchymal precursor cells, which can well distinguish target cells from non-target cells in the differentiated mixture. The coincidence rate of ITGA9-positive cells and SOX9-positive cells is as high as more than 97%; at the same time, the ITGA9-positive cells obtained by sorting express scleral plate mesenchymal precursor cell-related molecular markers, which form a sharp contrast with negative cells; in vitro induction experiments have proved that ITGA9-positive cells have the potential for chondrogenic and osteogenic differentiation, while negative cells have almost no such ability. It shows that the surface molecules of the present invention can be used to separate and enrich scleral plate osteoblasts/chondrogenic mesenchymal precursor cells derived from human pluripotent stem cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagram showing the results of qPCR detection and verification of the enrichment expression of surface molecular markers in scleral lamina mesenchymal precursor cells in Table 1;

Figure 2 is a flow cytometric analysis result of staining the scleral mesenchymal precursor cell differentiation mixture with ITGA9 antibody: Figure 2 shows that ITGA9-positive cells are enriched with a large number of SOX9-positive cells;

Figure 3 is a graph showing the qPCR identification results of molecular markers of scleral plate osteoblasts/chondrocyte mesenchymal precursor cells of ITGA9 positive cells and negative cells: Figure 3 shows that scleral plate osteoblasts/chondrocyte mesenchymal precursor cell marker genes are enriched in ITGA9 positive cells;

FIG4 is a diagram showing the results of the verification of the in vitro chondrogenic and osteogenic induction potentials of ITGA9-positive and -negative cells. FIG4 shows that cells with in vitro chondrogenic and osteogenic differentiation are enriched in ITGA9-positive cells.

DETAILED DESCRIPTION

In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

The human pluripotent stem cells used for inducing differentiation in the embodiment of the present invention are SOX9-tdTomato fluorescent reporter cell lines, which use gene editing technology to insert tdTomato expression elements in the 3'UTR region of the SOX9 gene locus, and their transcription is controlled by the SOX9 promoter. After transcription is completed, the IRES sequence in the self-expression element is used to mediate translation.

Example 1

This example discloses a method for inducing human pluripotent stem cells into scleral osteoblasts/chondrogenic mesenchymal precursor cells, wherein the reagents used are:

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

Matrigel working solution: Take the bottled Matrigel solution out of the -80℃ refrigerator, immerse the bottle completely in ice, and place the ice box in a 4℃ refrigerator overnight. The next day, place the pipette tip and EP tube used to absorb the stock solution in a -80℃ refrigerator for more than 10 minutes. Use a -20℃ pre-cooled pipette tip to take 1mg of Matrigel stock solution (Corning, USA) and add it to 12mL of DMEM/F12 (Gibco, USA) pre-cooled on ice. The working solution can be stored in a 4-degree refrigerator for two weeks.

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

Rh-insulin working solution: weigh 2 mg of rh-insulin (Solarbio, China) powder, add 1 mL of 10 mM HCL, and filter sterilize;

Polyvinyl alcohol working solution: Weigh 500 mg of polyvinyl alcohol powder, add 18 mL of UP water (Invitrogen, USA), stir until a granular suspension is formed, heat in a water bath at 85°C for about 20 minutes to dissolve, dilute to 20 mL, and filter to sterilize;

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

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;

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);

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);

Differentiation medium III: CDMi basal medium supplemented with PD0325901 (MCE, USA, 0.5 μM) and XΑV939 (MCE, USA, 1 μM);

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

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

1. A method for inoculating and subculturing human pluripotent stem cells for differentiation, comprising the following steps:

1. Take out the human pluripotent stem cells cultured in six-well plates from the 37°C incubator and observe the cell density under an ordinary optical microscope. The cell aggregation degree is 70%-90%;

2. Aspirate 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 under a microscope. If cracks begin to appear in the cell clones and the surrounding of the clones begin to shrink, the digestion solution can be aspirated to terminate the digestion. Otherwise, place them in a 37℃ incubator to continue digestion.

3. After discarding the digestion solution, add an appropriate amount of PGM1 medium, blow the cells off the bottom of the plate evenly, slowly and gently, and subculture them to another six-well plate treated with Matrigel working solution at a ratio of 1:8 corresponding to a polymerization degree of 70%-90% before subculture. Shake the culture plate left and right to evenly distribute the cells 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 the form of clones and the cell clones are not connected into pieces, they can be used for differentiation.

2. The method of inducing human pluripotent stem cells into scleral osteoblasts/chondrogenic mesenchymal precursor cells is as follows:

1. PS differentiation

On the second day of subculturing, remove the cells from the 37°C incubator and discard the PGM1 medium. Add 2 mL of Washbuffer to the wells to wash away dead cells and residual PGM1 medium, discard the Wash buffer, add 2 mL of Differentiation Medium I, and then culture in a 37°C incubator;

2. PSM differentiation

After 24 hours, take out the cells from the 37°C incubator and discard the 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 culture in a 37°C incubator;

3. SM differentiation

After 24 hours, remove the cells from the 37°C incubator and discard the differentiation medium II. Add 2 mL of Wash buffer to the wells to wash away dead cells and residual differentiation medium II, discard the Wash buffer, add 2 mL of differentiation medium III, and then culture in a 37°C incubator;

4. SCL differentiation

After 24 hours, remove the cells from the 37°C incubator and discard the differentiation medium III. Add 2 mL of Washbuffer to the wells to wash away dead cells and residual differentiation medium III, discard the Wash buffer, add 2 mL of differentiation medium IV, and then culture in a 37°C incubator;

After 72 hours, scleral osteoblasts/chondrogenic mesenchymal precursor cells can be collected.

Example 2

This example discloses a method for screening characteristic surface molecular markers of scleral plate osteoblasts/chondrocyte mesenchymal precursor cells of the present invention. The scleral plate osteoblasts/chondrocyte mesenchymal precursor cells used in this example are obtained by induction and differentiation according to the method of Example 1.

The specific screening methods are:

1. Sorting of SOX9-positive and -negative cells

1. Take out the culture plate after adding differentiation medium IV in Example 2 and culturing in a 37°C incubator for 24 hours, discard the culture medium by suction, add 500 μL of TrypLE digestion solution to the wells, and digest in a 37°C incubator for 5 minutes;

After 2.5 minutes, take out the cells, dissociate them from the bottom of the plate by blowing, and leave them at room temperature for two minutes. After two minutes, add 2 mL of Wash buffer and blow to neutralize;

3. Centrifuge at 300g for 3 minutes at room temperature;

4. Resuspend the cells with PBS and filter with a 70 μm cell mesh;

5. Prepare the flow sorter as required, calibrate the laser and adjust the side liquid flow to make it suitable for sorting;

6. Use undifferentiated pluripotent stem cells as negative controls to adjust the negative gate, and collect the sorted cells in a collection tube containing CDMi basal culture medium;

7. After cell sorting, centrifuge at 300g for 5 minutes;

8. Add 700 μL Trizol reagent to the tube, blow it thoroughly and store it in a -80°C refrigerator. When sending the sample, extract RNA with chloroform, isopropanol, etc. and send it to a sequencing company for library construction and sequencing.

2. Screening and identification of surface molecular markers

The specific steps of surface molecular marker screening in this implementation are:

Step 1. SOX9-positive cells in scleral osteoblasts/chondrogenic progenitor cells derived from human pluripotent stem cells were taken as positive cells, and SOX9-negative cells were taken as negative cells. The edgeR package of the R language platform was used to perform differential gene analysis, and genes that were upregulated at least twice in positive cells relative to negative cells (the logarithm of Fold Change was greater than or equal to 1) were taken as differential genes:

The sequence information is compared with the reference genome to obtain an expression matrix containing information such as gene name and gene length; the sequence information refers to the offline data of the sequencing company, including all sequence information obtained by sequencing the transcriptome library of positive cells and negative cells.

Use the library function to load the edgeR package in R Studio:

library(edgeR);

Import the expression matrix into R Studio software:

For example: count_all<-read.table("/Users/xxx/Desktop/bulk/count_sample.tsv")

Use the DGEList function to create a DGEList object:

group<-factor(c(rep("D",3),rep("P",3)))

d=DGEList(counts=count_all,group=group)

Normalize the counts obtained from sequencing and use the calcNormFactors function to estimate the normalization factor:

d = calcNormFactors(d)

The topTags function was used to obtain differentially expressed genes and logFC (logarithm of the gene expression fold ratio between two groups) and FDR values;

de＝exactTest(d,pair＝c("D","P"))

tt＝topTags(de,n＝nrow(d))

head(tt$table)

deg = rn[tt$table$FDR<.05]

Use the filter function (e.g., count.filter<-tt[apply(tt[,1],1,function(x){all(x>1)}),] to remove genes with logFC less than or equal to 1.

Step 2. Using R Studio software, compare the differentially expressed genes with known surface molecular proteins and take the intersection to obtain all the surface molecular markers that are upregulated in positive cells;

Step 3. Using R Studio software, calculate the standardized expression levels (TPM values) of the upregulated surface molecular markers in positive cells and negative cells, delete the surface molecular markers with low expression in positive cells and high expression in negative cells, and obtain candidate surface molecular markers.

The specific operations of step 2 and step 3 are as follows: in the expression matrix, divide the gene length by 1000 and assign it to kb; then divide the count of each gene by the kb value of the corresponding gene to obtain the RPK (reads per kilobase) value; then perform sequencing depth correction, that is, divide each RPK value by the sum of the RPK values in the column to obtain the TPM value:

kb<-data$length/1000

countdata<-data[,3:8]

rpk<-countdata/kb

rpk<-as.matrix(rpk)

tpm<-t(t(rpk)/colSums(rpk)*1000000)

Among the surface molecular markers that are differentially upregulated in the screened SOX9-positive cells, the merge function is used to capture the surface molecular markers and merge them with the expression matrix containing the TPM value:

Surface.marker<-merge(countdata,sm,by=c("ensembl_id"))

In the merged table, the filter function was used to extract genes whose TPM values of SOX9-negative cells were greater than 50 and whose TPM values of SOX9-positive cells were less than 50. The results are shown in Table 1:

Table 1

Gene SOX9Negativr-1 SOX9Negative-2 SOX9Negative-3 Sox9Positive-1 SOX9Positive-2 SOX9Positive-3 logFc logCPM PValue FDR FAT4 12.89 11.80 13.36 57.20 57.95 52.01 2.11 7.51 0.00E+00 0.00E+00 FLRT2 14.07 13.75 15.12 63.66 62.77 59.28 2.09 8.50 0.00E+00 0.00E+00 ITGA9 33.81 30.76 32.61 104.38 107.02 99.51 1.66 7.14 0.00E+00 0.00E+00 DCHS1 38.18 37.70 39.10 117.20 116.65 107.46 1.55 7.68 0.00E+00 0.00E+00 NCAM1 23.23 22.76 24.17 51.03 50.67 50.80 1.34 6.89 2.33E-203 1.63E-201 PDGFRA 37.47 35.93 38.61 77.51 80.19 74.35 1.16 7.10 9.47E-167 5.51E-165 PLXNA2 30.35 29.28 31.20 79.61 79.21 74.06 1.10 7.55 1.09E-302 1.25E-300 SCARF2 29.95 24.27 27.66 64.01 62.00 58.92 1.03 5.31 1.53E-77 4.25E-76

Step 4. Sort by TPM*logFC and obtain ITGA9 as a potential surface molecular marker.

Example 3

This example discloses a validation experiment of ITGA9 as a characteristic surface molecular marker of human pluripotent stem cells to osteogenic/chondrogenic precursor cells of the scleral plate.

1. Verification of Chondrogenic Differentiation

Reagents:

0.1 mM dexamethasone working solution: weigh 1 μmol equivalent of dexamethasone (Sigma, USA) powder, add 10 mL DMSO (Sigma, USA) to it, and filter and sterilize;

50 mM sodium ascorbate working solution: weigh 50 μmol equivalent sodium ascorbate (Sigma, USA) powder, add 1 mL UP water, and filter sterilize;

50 mg/mL proline working solution: Weigh 50 mg of proline powder, add 1 mL of UP water (Invitrogen, USA), and filter sterilize;

20 μg/mL TGFβ3 working solution: weigh 20 μg TGFβ3 powder (Pepprotech, USA), add it into 1 mL UP water, and filter sterilize;

20 μg/mL BMP2 working solution: weigh 20 μg BMP2 powder (Pepprotech, USA), add it into 1 mL UP water, and filter and sterilize;

10 μg/mL bFGF working solution: weigh 10 μg bFGF powder (Pepprotech, USA), add to 1 mL UP water, and filter sterilize;

20 mg/mL poly-HEMA working solution: weigh 2 g of poly-HEMA solid into a 100 mL filter bottle, add 100 mL of 95% ethanol, dissolve in a 37°C constant temperature shaker overnight, and filter and sterilize as soon as possible after dissolution;

Preparation of low-adhesion culture plates (poly-HEMA treated culture plates): Add an appropriate amount of the above poly-HEMA working solution to the wells and shake the culture plate to spread the liquid evenly. Open the cover of the culture plate and place it in a clean bench (laminar flow), turn off the fan, and leave it at room temperature overnight to allow the liquid in the plate to evaporate. It can be used the next day. The treated culture plate can be placed at room temperature for at least two months.

Chondrogenic induction medium: DMEM supplemented with 1% ITS, 0.1 μM dexamethasone, 50 μM sodium ascorbate, 50 μg/mL proline, 20 ng/mL TGFβ3, 20 ng/mL BMP2, and 20 ng/mL FGF2.

The specific steps for in vitro chondrogenic differentiation and identification of ITGA9 positive cells are as follows:

1. Staining, labeling and sorting of scleral osteoblasts/chondrocyte mesenchymal precursor cells were performed according to the instructions of ITGA9 antibody;

2. Centrifuge at 300 g to collect and sort ITGA9 positive and negative cells;

3. Resuspend the cells in Wash buffer and count them using a hemocytometer;

4. According to the counting results, resuspend the cells in differentiation medium IV containing 10 μM ROCKi at a volume of 10,000 cells/20 μL, and seed them on the lid of a 100 mm culture plate at intervals. Add more than 5 mL of PBS (Gibco, USA) to the culture dish, carefully cover the lid, and place it in a 37°C incubator overnight to culture into spheres;

5. Take out the cells cultured overnight from the incubator, carefully suck the pellet on the lid of the dish with a pipette, and transfer the pellet to an EP tube;

6. After the pellets sink to the bottom, discard the culture medium, wash the pellets once with Wash buffer, rinse the pellets once with chondrogenic induction solution, and discard the rinse solution;

7. Resuspend the pellet with an appropriate amount of cartilage induction solution and transfer it to a low-adhesion culture plate treated with poly-HEMA overnight and culture for 30 days, replacing the induction solution every two days.

8. After 30 days, the induced pellets were taken for routine embedding of frozen sections. After sectioning, the morphology was examined under a microscope, and cartilage-specific staining (Alcian blue staining, safranin-fast green staining, COL II immunohistochemical staining) was performed to confirm the differentiation results.

2. Verification of Osteogenic Differentiation

Reagents: Osteogenic induction solution: Weigh 10 μM equivalent of β-glycerophosphate sodium, add it to 1 mL of α-MEM containing 0.1 μM dexamethasone, 50 μM sodium ascorbate, and 10% fetal bovine serum, and filter and sterilize.

The specific steps of in vitro osteogenic differentiation of ITGA9 positive cells are as follows:

1. Staining, labeling and sorting of scleral osteoblasts/chondrocyte mesenchymal precursor cells were performed according to the instructions of ITGA9 antibody;

2. Centrifuge at 300 g to collect and sort ITGA9 positive and negative cells;

3. Resuspend the cells in Wash buffer and count them using a hemocytometer;

4. Based on the counting results, resuspend the cells in differentiation medium IV containing 10 μM ROCKi at a volume of 150,000 cells/100 μL and inoculate them into a 96-well cell culture plate;

5. Take out the cells cultured overnight from the incubator, discard the culture medium, and add 200 μL Washbuffer to rinse once;

6. Aspirate and discard the rinsing solution, add 150 μL of osteogenic induction solution and place in a 37°C incubator for 30 days, changing the induction solution every two days.

7. After 30 days of osteogenic induction, discard the induction solution, stain with Alizarin Red solution for 2 minutes, and observe the number of calcium nodules.

The experimental results of chondrogenic differentiation verification and osteogenic differentiation verification are shown in Figure 4: The in vitro induction experiment proved that ITGA9 positive cells have the potential for chondrogenic and osteogenic differentiation, while negative cells have almost no such ability.

Example 4

This example discloses a method for identifying ITGA9 positive cells by fluorescent quantitative PCR, specifically:

1. RNA Extraction

Take out the culture plate inoculated with scleral osteoblasts/chondrogenic mesenchymal precursor cells from the 37°C incubator, remove the original culture medium, add an appropriate amount of TRI reagent to the EP tube, blow continuously, and let it stand at room temperature for 5-10 minutes until the cells are fully lysed;

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

Centrifuge at 12000g for 15 minutes (2-8℃ environment). After centrifugation, three phases can be seen, and the top layer (colorless and transparent) contains RNA;

Precipitate RNA. Add isopropanol to a new EP tube at a ratio of isopropanol:TRI reagent = 1:2, and add an equal amount of transparent supernatant containing RNA; vortex on a vortex oscillator for 10 seconds and then let stand at room temperature for 10 minutes;

Centrifuge at 12000g for 10 minutes (2-8°C environment) and discard the supernatant;

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 upside down, centrifuge at 12000g for 4 minutes (2-8℃ environment), discard the supernatant; repeat this step once.

Place the EP tube in the air at room temperature and add an appropriate amount of 56°C enzyme-free water to dissolve the RNA.

RNA concentration was measured using a quantitative device such as Nanodrop;

2. Reverse transcription and real-time fluorescence quantitative PCR

Take 100-1000 ng of RNA and reverse transcribe it into cDNA using a commercial reverse transcription reagent;

The cDNA was diluted 1:4-1:10 with enzyme-free water;

ACTB (encoding β-actin) was used as the internal reference gene, and the expression of the surface markers shown in Table 1 was quantified using a commercial qPCR kit (Vazyme, China); the results are shown in Figure 1.

ACTB (encoding β-actin) was used as the internal reference gene, and the marker genes of scleral lamina mesenchymal precursor cells were quantified using a commercial qPCR kit (Vazyme, China), and their expression in ITGA9 positive cells and negative cells was compared. The results are shown in Figure 3.

As shown in Figures 1 and 3, the surface molecular markers shown in Table 1 are enriched in SOX9-positive cells, which is consistent with the RNA-seq data; the marker genes related to scleral plate mesenchymal precursor cells are enriched in ITGA9-positive cells. This shows that ITGA9 of the present invention can be used as a characteristic surface molecular marker of scleral plate osteogenic/chondrogenic mesenchymal precursor cells derived from human pluripotent stem cells.

Example 5

This example discloses a flow cytometric analysis method for staining a mixture of scleral mesenchymal precursor cells with ITGA9 antibodies, specifically:

The cultured scleral osteoblasts/chondrogenic mesenchymal precursor cells were taken out from the 37°C incubator and the supernatant was removed. The suspended dead cells were washed with PBS, the supernatant was discarded, and the cells were digested with TryPLE digestion solution at 37°C for 3 minutes;

The digestion was terminated with 4 volumes of Wash buffer, and the cell suspension was collected and centrifuged at 300 g for 3 min at 2-8°C;

Aspirate and discard the supernatant, and resuspend the cells in pre-chilled PBS;

The staining of scleral osteoblasts/chondrogenic mesenchymal precursor cells was performed according to the instructions of the ITGA9 antibody;

Use a 70μm cell sieve to remove cell clumps before analysis.

As shown in FIG2 , ITGA9-positive cells are enriched with a large number of SOX9-positive cells, indicating that ITGA9 of the present invention can be used as a characteristic surface molecular marker of scleral osteoblast/chondrogenic mesenchymal precursor cells derived from human pluripotent stem cells.

The embodiments described above are part of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims (1)

1. Application of ITGA9, a characteristic surface molecular marker of scleral plate osteoblast/chondrocyte mesenchymal precursor cells derived from human pluripotent stem cells, in the separation and enrichment of scleral plate osteoblast/chondrocyte mesenchymal precursor cells derived from human pluripotent stem cells.