WO2021248675A1

WO2021248675A1 - Inducer for differentiation of stem cells into chondrocytes and application thereof

Info

Publication number: WO2021248675A1
Application number: PCT/CN2020/108478
Authority: WO
Inventors: 徐洪杰; 秦大江
Original assignee: 生物岛实验室
Priority date: 2020-06-09
Filing date: 2020-08-11
Publication date: 2021-12-16
Also published as: CN111676190A; CN111676190B

Abstract

Provided are an inducer for the differentiation of stem cells into chondrocytes and an application thereof. The inducer comprises PDGF, bFGF and TGF-β, and further comprises insulin and/or IGF. Under the condition of a high-concentration ratio, PDGF, bFGF, TGF-β, and insulin or IGF induce the differentiation of mesenchymal stem cells into chondrocytes within three days, shortening the production cycle of chondrocytes.

Description

Inducer for differentiation of stem cells to chondrogenesis and application thereof

Technical field

This application belongs to the field of biomedicine technology, and relates to an inducer for the differentiation of stem cells into chondrogenicity and its application.

Background technique

The damage of articular cartilage seriously affects the quality of life of patients. Due to the limited ability of self-repair, the damaged articular cartilage cannot repair itself. Clinical treatment usually adopts conservative strategies, or replaces damaged articular cartilage with biomaterial substitutes, but still faces many problems and cannot achieve the goal of complete repair.

In recent years, the development of stem cell therapy technology has provided unlimited possibilities for the treatment of degenerative or traumatic diseases. Mesenchymal stem cells (MSCs) are a type of cells with self-renewal and differentiation capabilities, and more and more studies have confirmed their potential for application in the treatment of various diseases. There are many sources of MSCs, such as umbilical cord, bone marrow, skin, and peripheral blood. The perinatal tissues are at an early stage of development and have stronger cell viability, making them an ideal source of stem cells. Tissues derived from the perinatal period include umbilical cord and placenta. Human umbilical cord MSCs (hUC-MSCs) have high cell purity, strong proliferation and secretion activity, low immune risk, no matching, no ethical controversy, and are ideal for future clinical applications. Seed cells. The anatomical structure of the umbilical cord mainly includes the outer layer of amniotic membrane, the middle three blood vessels and the Huatong glue between them. Huatong glue is the main structure. In existing studies, umbilical cord MSCs are isolated from umbilical cord Huatong glue and amniotic membrane, usually called umbilical cord MSCs and amniotic membrane MSCs. Both MSCs have adherent growth, expression of specific cell surface marker molecules, and osteogenesis, adipogenesis, and growth. The characteristics of cartilage three-way differentiation ability meet the internationally accepted MSCs identification standards.

The potential of MSCs to differentiate into cartilage and the promotion of cartilage differentiation make it an unparalleled advantage in the treatment of cartilage damage, and it is expected to be used as a therapeutic drug to completely cure cartilage damage. However, whether it is to directly transplant MSCs to induce cartilage differentiation in situ or to construct cartilage in vitro through cartilage tissue engineering and then transplant and replace, both ideas cannot avoid a key step, which is to induce MSCs to differentiate into cartilage.

At present, MSCs induce differentiation into cartilage generally using inducers containing dexamethasone, transfer growth factor β1 (TGF-β1), ascorbic acid, sodium pyruvate, etc. and supplement other auxiliary components. For example, CN105039247A discloses a small molecule polypeptide, Transplant growth factor β (TGF-β), dexamethasone and vitamin C induce stem cells to differentiate into chondrogenic preparations. CN109825469A discloses a cartilage containing TGF-β1, ferritin, ascorbic acid, dexamethasone, and sodium pyruvate Differentiation inducers, there are many kinds of factors added, which can easily lead to inaccurate preparation ratios and increase costs. In addition, MSCs need to be induced for 14 to 21 days before MSCs have obvious morphological characteristics and positive staining results; CN110468097A, CN108753700A, etc., can differentiate cartilage The inducer or method has been improved, and the induction time has been shortened to 8 days, but there are still problems such as a wide variety of added factors and complicated operation procedures.

The prior art has not been able to shorten the differentiation cycle of MSCs to cartilage to less than one week.

Summary of the invention

This application provides an inducer for chondrogenic differentiation of stem cells and its application. The inducer has a simple formula and low cost. It can efficiently induce the differentiation of mesenchymal stem cells to chondrogenic within 3 days, which significantly shortens the research period.

In the first aspect, the present application provides an inducer for the differentiation of stem cells into chondrogenicity, and the inducer includes PDGF, bFGF, and TGF-β;

The inducer also includes insulin and/or IGF.

Platelet-derived factor (PDGF) is an important mitogenic factor that has the ability to stimulate the division and proliferation of specific cell populations, promote the survival and differentiation of osteoblasts, and guide the migration of osteoblasts. Basic fibroblast growth factor (bFGF) is a family of fibroblast factors. FGFs and their receptors (FGFRs) play an important role in the process of MSCs differentiation into cartilage and cartilage formation. FGFs can promote the proliferation of chondrocytes. Inhibiting its hypertrophy and inhibiting FGF signaling can lead to achondroplasia; when MSCs are cultured in vitro, FGF is the main cytokine that promotes their proliferation and maintains their differentiation potential. The FGF signaling pathway is not only related to the development of the skeletal system, but also closely related to cartilage formation. Studies have used FGF9 and FGF18 knockout mice to prove that FGFs are the upstream cytokine of Runt-related transcription factor 2 (RUNX2) and can promote Immature chondrocytes proliferate and differentiate into chondrocytes; they can up-regulate the transcription factor Sox2 and inhibit Wnt signaling, which leads to a decline in the osteogenic differentiation of MSCs and promotes the differentiation of MSCs into cartilage; FGF can increase the concentration of cartilage microspheres when inducing MSCs to differentiate into cartilage. The production of proteoglycans. TGF-β has a stimulating effect on cells of mesenchymal origin. The TGF-β pathway has been proven to play an important role in the osteogenic and chondrogenic differentiation system; TGF-β can inhibit the differentiation of MSCs into fat through the Smad3 pathway and block Smad 3 mediated TGF-β signaling leads to adipogenic differentiation. TGF-β can also activate the MAPKs (mitogen-activated protein kinase) pathway. MAPKs include JNK, p38 and ERK, which belong to serine/threonine protein kinases, and participate in a large number of cell activities, such as proliferation, inflammation, migration and differentiation. JNK , P38 and ERK can regulate TGF-β through different signal pathways and stimulate mesenchymal stem cells to differentiate into cartilage and cartilage formation. A large number of studies have shown that a small amount of TGF-β can make proteoglycan and type Ⅱ The synthesis of collagen is significantly increased; TGF-β can increase the expression level of SOX-9, a gene related to early chondrogenic differentiation, and at the same time increase the gene expression level of type II collagen in the extracellular matrix of chondrocytes, and type II collagen is hyaluronic acid An important biomarker of cartilage synthesis; the expression of aggrecan, another component of the extracellular matrix of chondrocytes, also has similar changes; the expression level of the osteogenic-related gene type I collagen is further significantly decreased, which shows Another function of TGF-β is to maintain the hyaluronic acid cartilage phenotype of chondrocytes induced by MSCs. Insulin can significantly increase the degree of chondrogenic differentiation of MSCs, and can significantly increase the synthesis of the main components of chondrocyte extracellular matrix such as proteoglycan, type II collagen and mucopolysaccharide, and has a synergistic effect with TGF-β. The combination of the two can be added separately. It can maximize the proliferation and chondrogenic differentiation of MSCs. Insulin-like growth factor (IGF) can promote cartilage formation by stimulating cell proliferation, regulating cell apoptosis and inducing the expression of chondrocyte-related marker genes.

In the study of the proliferation process of mesenchymal stem cells in this application, it was unexpectedly discovered that the combination of PDGF, bFGF, TGF-β and insulin/IGF can be used as an inducer to induce the differentiation of stem cells into chondrogenesis. Without the addition of other cofactors, it can be The mesenchymal stem cells were efficiently induced to differentiate into chondrocytes within 3 days, and the induced cell aggregation formed the classic tissue cluster pattern of chondrogenic differentiation. The results of alician blue staining confirmed that the cells had typical chondrogenic differentiation.

In a specific solution, the PDGF includes any one or a combination of at least two of PDGF-AA, PDGF-AB, or PDGF-BB.

In this application, PDGF-AA, PDGF-AB and PDGF-BB have similar effects in promoting the survival and differentiation of osteoblasts and guiding the migration of osteoblasts. They are similar to bFGF, TGF-β and insulin/IGF. Cooperate with each other to induce the differentiation of stem cells into cartilage.

In a specific embodiment, the TGF-β includes any one or a combination of at least two of TGF-β1, TGF-β2, or TGF-β3.

In this application, TGF-β1, TGF-β2 and TGF-β3 all have a stimulating effect on cells of mesenchymal origin, and can maintain the hyaluronic acid cartilage phenotype of chondrocytes induced by MSCs.

In a specific solution, the IGF includes IGF-1 and/or IGF-2.

In this application, IGF-1 and IGF-2, especially IGF-1, are considered to be one of the important growth factors for cartilage formation. According to reports, IGF-1 can recruit chondrocytes in the joints to repair articular cartilage damage. IGF-1 can enhance the metabolism of chondrocytes and regulate the differentiation of mesenchymal stem cells into cartilage, while maintaining the potential of mesenchymal stem cells to differentiate into cartilage.

In a specific solution, the mass ratio of the PDGF and the bFGF is (0.8-4):3, for example, it may be 0.8:3, 1:3, 2:3, 3:3 or 4:3.

In a specific solution, the mass ratio of the PDGF and the TGF-β is (0.8-4):3, for example, it can be 0.8:3, 1:3, 2:3, 3:3, or 4:3.

In a specific solution, the mass ratio of the PDGF and the insulin is (0.6-8): 1000, for example, it can be 0.6: 1000, 1: 1000, 2: 1000, 3: 1000, 4: 1000, 5: 1000, 6:1000, 7:1000 or 8:1000.

In a specific solution, the mass ratio of the PDGF and the IGF is (0.6-8):1000, for example, it can be 0.6:1000, 1:1000, 2:1000, 3:1000, 4:1000, 5: 1000, 6:1000, 7:1000 or 8:1000.

In this application, PDGF, bFGF, TGF-β and insulin or IGF within a reasonable mass ratio range can efficiently induce mesenchymal stem cells to differentiate into chondrocytes. If they are not within this reasonable mass ratio range, the induction efficiency will be affected. .

In the second aspect, the present application provides a chondrogenic differentiation induction medium, which includes a basic medium and the inducer of the first aspect added to the basic medium.

In a specific solution, the concentration of the PDGF in the culture medium is 30 to 80 ng/mL, for example, it can be 30 ng/mL, 40 ng/mL, 50 ng/mL, 60 ng/mL, 70 ng/mL or 80 ng/mL , Preferably 40-50ng/mL.

In a specific solution, the concentration of the bFGF in the culture medium is 60-110 ng/mL, for example, it can be 60 ng/mL, 70 ng/mL, 80 ng/mL, 90 ng/mL, 100 ng/mL or 110 ng/mL , Preferably 70 to 90 ng/mL.

In a specific solution, the concentration of the TGF-β in the culture medium is 60-110 ng/mL, for example, it may be 60 ng/mL, 70 ng/mL, 80 ng/mL, 90 ng/mL, 100 ng/mL or 110 ng. /mL, preferably 70 to 90 ng/mL.

In a specific solution, the concentration of the insulin in the culture medium is 10-50 μg/mL, for example, it may be 10 μg/mL, 20 μg/mL, 30 μg/mL, 40 μg/mL or 50 μg/mL, preferably 10 μg/mL. ~20μg/mL.

In a specific solution, the concentration of the IGF in the culture medium is 10-50 μg/mL, for example, it may be 10 μg/mL, 20 μg/mL, 30 μg/mL, 40 μg/mL or 50 μg/mL, preferably 10 μg/mL. ~20μg/mL.

In this application, PDGF, bFGF, TGF-β and insulin/IGF play a multi-factor network regulation effect under the condition of high concentration ratio, and synergistically induce the differentiation of mesenchymal stem cells into chondrocytes. The formula is simple, There is no need to add other auxiliary factors, the preparation process is simple, and the influence of cell contamination and other conditions on the experimental results is avoided.

In a specific solution, the basic medium is a serum-free basic medium.

In a specific solution, the basal medium includes any one of DMEM/F12, α-MEM or DMEM high sugar, and DMEM is the abbreviation of Dulbecco's Modified Eagle Medium.

The medium of the present application has a significant effect in inducing the differentiation of mesenchymal stem cells into chondrogenic cells. The mesenchymal stem cells are cultured for 3 days with a preferred medium formula, and then they aggregate to form chondrogenic differentiation tissue masses.

In the third aspect, the present application provides a method for inducing stem cells to differentiate into chondrogenicity, the method comprising culturing mesenchymal stem cells using the medium described in the second aspect.

In a specific scheme, the culture time does not exceed 3 days.

In this application, a serum-free basal medium culture room containing 30～80ng/mL PDGF, 60～110ng/mL bFGF, 60～110ng/mL TGF-β and 10～50μg/mL insulin or 10～50μg/mL IGF Mesenchymal stem cells can be induced to form chondrocytes within 3 days, which significantly shortens the stem cell research cycle.

In a specific solution, the passage number of the mesenchymal stem cells is 4 to 8 generations.

In a specific solution, before culturing the mesenchymal stem cells, the method further includes the step of obtaining the mesenchymal stem cells from umbilical cord Huatong glue and/or umbilical cord amniotic membrane.

In a fourth aspect, the present application provides a pharmaceutical composition comprising chondrogenic cells prepared by inducing and culturing mesenchymal stem cells using the medium described in the second aspect.

Optionally, the pharmaceutical composition further includes any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

In the fifth aspect, the present application provides a medicine for treating cartilage injury, the medicine includes the pharmaceutical composition of the fourth aspect.

In the sixth aspect, the present application provides a treatment method, which includes the step of administering to an individual the chondrogenic cells prepared by inducing and culturing mesenchymal stem cells using the medium described in the second aspect, and the The amount is sufficient to reconstruct articular cartilage.

Compared with the prior art, this application has the following beneficial effects:

(1) This application uses a combination of PDGF, bFGF, TGF-β and insulin or a combination of PDGF, bFGF, TGF-β and IGF as an inducer to induce stem cells to differentiate into chondrogenesis. Without the addition of other cofactors, four These factors play a role in network regulation, and synergistically induce mesenchymal stem cells to differentiate into cartilage within 3 days;

(2) The induction agent and induction medium of the application have a simple formula, no additional auxiliary factors are required, and the preparation process is simple, avoiding the influence of cell contamination and other conditions on the experimental results;

(3) The induction method of the present application is simple and efficient, significantly shortens the chondrogenic cell culture cycle, and is of great significance in the field of cartilage injury treatment.

Description of the drawings

Figure 1A shows the cell morphology of hUMSCs in Example 1 after 3 days of induction, and Figure 1B and Figure 1C show the staining of Alcian Blue cells in different visual fields;

Figure 2 shows the cell morphology of hUMSCs in Example 2 after 3 days of induction;

Figure 3 shows the cell morphology of hBMSCs in Example 3 after 3 days of induction;

Figure 4 shows the cell morphology of hAMSCs in Example 4 after 3 days of induction;

Figure 5 shows the cell morphology of hUMSCs in Example 5 after 3 days of induction;

Figure 6 shows the cell morphology of hUMSCs in Example 6 after 3 days of induction;

Figure 7 shows the cell morphology of hUMSCs of Comparative Example 1 after 3 days of induction;

Figure 8 shows the cell morphology of hUMSCs of Comparative Example 2 after 3 days of induction;

Figure 9 shows the cell morphology of hUMSCs of Comparative Example 3 after 3 days of induction;

Figure 10 shows the cell morphology of hUMSCs of Comparative Example 4 after 3 days of induction;

Figure 11 shows the cell morphology of hUMSCs of Comparative Example 5 after 3 days of induction;

Figure 12 shows the cell morphology of hUMSCs of Comparative Example 6 after 3 days of induction;

Figure 13 shows the cell morphology of hUMSCs of Comparative Example 7 after 3 days of induction;

Figure 14 shows the cell morphology of hUMSCs of Comparative Example 8 after 3 days of induction;

Figure 15 shows the cell morphology of hUMSCs of Comparative Example 9 after 3 days of induction;

Figure 16 shows the cell morphology of hUMSCs of Comparative Example 10 after 3 days of induction;

Figure 17 shows the cell morphology of hUMSCs of Comparative Example 11 after 3 days of induction;

Figure 18 shows the cell morphology of hUMSCs of Comparative Example 12 after 3 days of induction;

Figure 19 shows the cell morphology of hUMSCs of Comparative Example 13 after 3 days of induction;

Figure 20 shows the cell morphology of hUMSCs of Comparative Example 14 after 3 days of induction.

detailed description

In order to further illustrate the technical means adopted by this application and its effects, the application will be further described below in conjunction with embodiments and drawings. It can be understood that the specific implementations described here are only used to explain the application, but not to limit the application.

If the specific technology or conditions are not indicated in the examples, it shall be carried out in accordance with the technology or conditions described in the literature in the field, or in accordance with the product specification. The reagents or instruments used without the manufacturer's indication are all conventional products that can be purchased through formal channels.

Example 1

In this example, human umbilical cord mesenchymal stem cells (hUMSCs) are used as the cell type, and DMEM/F12 is selected as the basal medium to induce chondrogenic differentiation. The steps are as follows:

(1) Collect the umbilical cord of a full-term healthy fetus delivered by cesarean section, immerse it in PBS or saline containing 1% penicillin and streptomycin, and place on ice;

(2) Cut the umbilical cord into small sections about 3cm in length on the ultra-clean table, cut them apart longitudinally, rinse them repeatedly with sterile PBS until the liquid is free of blood stains, separate the arteries and veins (total 3 pieces), and the remaining tissue is Huatong glue And amniotic membrane;

(3) Cut the remaining tissue into ^{small pieces smaller than 2mm 3} , rinse the tissue pieces with PBS and place them in DMEM/F12 medium containing 15% fetal bovine serum, and culture them in a 37°C, 5% CO ₂ incubator ；

(4) Observe the cell crawling out of the tissue block under an inverted phase-contrast microscope. Change the culture medium for the first time after 5 days, and then change the medium every 4 days. When the cells expand to occupy 80%-90% of the bottom area of the cell flask, use 0.05 % Trypsin-EDTA digestion passage (first passage 1:2, subsequent passages 1:3 or 1:4 every 3 days);

(5) Take 1×10 ⁶ third-generation cells, add 100 μL cell staining buffer, and add corresponding volume of antibody (PE, APC or FITC-labeled mouse anti-human CD105, CD44, CD90, CD29, CD34, CD45 and human leucocyte antigen (HLA)-DR monoclonal antibody), incubate at room temperature for 30 minutes, centrifuge at 1000 r/min for 5 minutes, discard the supernatant, and add 500 μL PBS for computer testing. The results are compared with the international standard that the cell surface expresses CD105, CD73 and CD90 (≥95%), do not express CD45, CD34, CD14 or CD11b, CD79α or CD19, HLA-DR (≤2%), the experimental results meet and exceed this standard, indicating that umbilical cord MSCs and amniotic membrane MSCs have been successfully isolated;

(6) When the cells are passaged to 4 to 8 passages, inoculate a 12-well plate, and use DMEM/F12 medium containing 80ng/mL PDGF-AB, 110ng/mL bFGF, 110ng/mL TGF-β1 and 50μg/mL insulin for culture ；

(7) After culturing for three days, observe the cell morphology under a microscope.

As shown in Figure 1A, the hUMSCs cell morphology induced by the differentiation medium of the present application changed significantly, and the cells aggregated to form a classic tissue-like pattern of chondrogenic differentiation; in order to confirm the occurrence of osteogenic differentiation, the cells were stained with alicin blue After washing twice with distilled water for 30 minutes, observe the staining effect under the microscope. Figure 1B and Figure 1C show the staining of cells in different fields of view. The alician blue staining part shows the internal acid mucopolysaccharide in cartilage tissue. The staining result It can be seen that all cells have undergone chondrogenic differentiation, with a high degree of differentiation and high maturity, and 6 to 15 chondrocyte clusters can be seen in each hole of the petri dish.

Flow cytometric analysis of MSCs pluripotency marker molecules was performed on the cells. The results are shown in Table 1, indicating that the cell clusters are indeed differentiated from MSCs.

Table 1 Molecular flow analysis results of MSCs surface markers

表面标志分子Surface marker molecule	阳性表达(％)Positive expression (%)
CD73CD73	99.699.6
CD90CD90	99.999.9
CD105CD105	100100
CD14CD14	0.0250.025
CD19CD19	00
HLA-DRHLA-DR	0.0380.038
CD34CD34	0.0120.012
CD45CD45	00

Example 2

In this example, human umbilical cord mesenchymal stem cells (hUMSCs) are used as the cell type. DMEM high glucose is selected as the basic medium to induce chondrogenic differentiation. The differentiation medium contains 30ng/mL PDGF-AB, 60ng/mL bFGF, 60ng/mL. mL TGF-β1 and 10μg/mL insulin DMEM high glucose medium, other conditions and experimental procedures are the same as in Example 1.

As shown in Figure 2, after 3 days of culture, the hUMSCs cell morphology changed significantly. The cells aggregated to form the classic tissue-like pattern of chondrocyte differentiation, and differentiated into chondrocyte-like tissue masses efficiently, indicating that the differentiation medium of the present application can be used within 3 days. Efficiently induce MSCs to differentiate into chondrocyte clusters.

Example 3

In this example, human bone marrow mesenchymal stem cells (hBMSCs) are used as the cell type. α-MEM is selected as the basal medium to induce chondrogenic differentiation. The differentiation medium contains 40ng/mL PDGF-AB, 70 ng/mL bFGF, 70ng /mL TGF-β1 and 10μg/mL insulin α-MEM medium, other conditions and experimental procedures are the same as in Example 1.

As shown in Figure 3, hBMSCs cell morphology changed significantly after 3 days of culture. The cells aggregated to form a classic tissue-like pattern of chondrocyte differentiation, and differentiated into chondrocyte-like tissue clusters efficiently, indicating that the differentiation medium of the present application can be used within 3 days. Efficiently induce MSCs to differentiate into chondrocyte clusters.

Example 4

In this example, human amniotic membrane mesenchymal stem cells (hAMSCs) are used as the cell type. α-MEM is selected as the basic medium to induce chondrogenic differentiation. The differentiation medium contains 50ng/mL PDGF-AA, 90ng/mL bFGF, 90ng/ mL TGF-β1 and 10μg/mL insulin α-MEM medium, other conditions and experimental procedures are the same as in Example 1.

The results are shown in Figure 4, hAMSCs efficiently induced differentiation into chondrocyte clusters within 3 days of induction.

Example 5

In this example, human umbilical cord mesenchymal stem cells (hUMSCs) were used as the cell type. DMEM/F12 was selected as the basal medium to induce cartilage differentiation. The differentiation medium containing 80ng/mL PDGF-BB, 110ng/mL bFGF, 110ng/mL mL TGF-β1 and 50μg/mL insulin DMEM/F12 medium were cultured, and other conditions and experimental procedures were the same as in Example 1.

The results are shown in Figure 5, the cells can also undergo significant chondrocyte differentiation. The staining results confirm that the degree of differentiation is high, but there are fewer chondrocyte clusters.

Example 6

In this example, human umbilical cord mesenchymal stem cells (hUMSCs) are used as the cell type. DMEM/F12 is selected as the basal medium to induce chondrogenic differentiation. The differentiation medium contains 80ng/mL PDGF-AB, 110ng/mL bFGF, 110ng/mL. mL TGF-β1 and 50 μg/mL IGF-1 DMEM/F12 medium were cultured, and other conditions and experimental procedures were the same as in Example 1.

The results are shown in Figure 6, the cells can efficiently aggregate into cartilage balls, and the staining of arizin blue is obvious.

Comparative example 1

Compared with Example 1, a commercial cartilage differentiation inducing medium (Guangzhou Saiye Catalog No. HUXUC-90041) was used as the induction differentiation medium, and other conditions and experimental procedures were the same as those in Example 1.

As shown in Figure 7, after 3 days of induction of hUMSCs with a commercial chondrogenic differentiation induction medium, the cells proliferated in a large amount and the number increased significantly, but the cell morphology did not change significantly. According to its instructions, the chondrocyte spheroids need to be continuously induced for 21 to 28 days before they can be detected by arizin blue staining.

Comparative example 2

Compared with Example 2, the differentiation induction medium adopts the formula of WO2016147005A1, and other conditions and experimental procedures are the same as Example 2.

As shown in Figure 8, after 3 days of inducing hUMSCs with the formula of WO2016147005A1, the cells proliferated in a large amount and the number increased significantly, but the cell morphology did not change significantly. After 14 days of continued culture, hUMSCs gradually differentiated into chondrocyte clusters.

It can be seen that the existing technical solutions cannot achieve the effect of shortening the differentiation cycle of MSCs to cartilage to less than one week, and it cannot achieve the effect of shortening the differentiation induction cycle to 3 days.

Comparative example 3

Compared with Example 1, PDGF-AB was omitted from the differentiation medium, and other conditions and experimental procedures were the same as Example 1.

The results are shown in Figure 9. The induced differentiation of the cells was significantly delayed, the arrangement pattern of the cells only showed signs of local aggregation, and no chondrocyte cluster-like structure was observed.

Comparative example 4

Compared with Example 1, bFGF was omitted from the differentiation medium, and other conditions and experimental procedures were the same as Example 1.

The results are shown in Figure 10, the induced differentiation of the cells was significantly delayed, the arrangement pattern of the cells only showed signs of local aggregation, and no chondrocyte cluster-like structure was observed.

Comparative example 5

Compared with Example 1, TGF-β1 was omitted from the differentiation medium, and other conditions and experimental procedures were the same as Example 1.

The results are shown in Figure 11, the induced differentiation of the cells was significantly delayed, and the arrangement pattern of the cells only showed signs of local aggregation, and no chondrocyte cluster-like structure was observed.

Comparative example 6

Compared with Example 1, insulin was omitted from the differentiation medium, and other conditions and experimental procedures were the same as Example 1.

The results are shown in Figure 12, the induced differentiation of the cells was significantly delayed, the arrangement pattern of the cells only showed signs of local aggregation, and no chondrocyte cluster-like structure was observed.

Comparative example 7

Compared with Example 1, the concentration of PDGF-AB in the differentiation medium was 10 ng/mL, and the other conditions and experimental procedures were the same as in Example 1.

The results are shown in Figure 13, the cells did not observe a distinctly differentiated morphology, and the cells proliferated to a certain extent.

Comparative example 8

Compared with Example 1, the concentration of PDGF-AB in the differentiation medium was 100 ng/mL, and other conditions and experimental procedures were the same as Example 1.

The results are shown in Figure 14. The cells did not observe a distinctly differentiated morphology, and the cells proliferated to a certain extent, but at the same time, increased apoptosis and excessive secretion accumulation could be seen. Rapid hyperproliferation and apoptosis may occur. The cell state Getting worse.

Comparative example 9

Compared with Example 1, the concentration of bFGF in the differentiation medium was 30 ng/mL, and other conditions and experimental procedures were the same as Example 1.

The results are shown in Figure 15. The cells did not observe a distinctly differentiated morphology, and the cells proliferated to a certain extent.

Comparative example 10

Compared with Example 1, the concentration of bFGF in the differentiation medium was 150 ng/mL, and other conditions and experimental procedures were the same as Example 1.

The results are shown in Figure 16. The cells did not observe a distinctly differentiated morphology, and the cells proliferated to a certain extent, but at the same time, increased apoptosis and excessive secretion accumulation could be seen. Rapid hyperproliferation and apoptosis may occur. The cell state Getting worse.

Comparative example 11

Compared with Example 1, the concentration of TGF-β1 in the differentiation medium was 30 ng/mL, and other conditions and experimental procedures were the same as Example 1.

The results are shown in Figure 17, the cells did not observe a distinctly differentiated morphology, and the cells proliferated to a certain extent.

Comparative example 12

Compared with Example 1, the concentration of TGF-β1 in the differentiation induction medium was 150 ng/mL, and other conditions and experimental procedures were the same as in Example 1.

The results are shown in Figure 18. The cells did not observe a distinctly differentiated morphology, and the cells proliferated to a certain extent, but at the same time, increased apoptosis and excessive accumulation of secretions could be seen. Rapid hyperproliferation and apoptosis may occur. The cell state Getting worse.

Comparative example 13

Compared with Example 1, the concentration of insulin in the differentiation medium was 1 ng/mL, and other conditions and experimental procedures were the same as Example 1.

The results are shown in Figure 19, the cells did not observe a distinctly differentiated morphology, and the cells proliferated to a certain extent.

Comparative example 14

Compared with Example 1, the concentration of insulin in the differentiation medium was 60 ng/mL, and other conditions and experimental procedures were the same as Example 1.

The results are shown in Figure 20. No obvious differentiation of the cells was observed, the cells proliferated to a certain extent, and there were signs of increased apoptosis.

In summary, this application uses a combination of PDGF, bFGF, TGF-β and insulin or a combination of PDGF, bFGF, TGF-β and IGF as an inducer to induce stem cells to differentiate into chondrogenesis, without adding other cofactors. , The four factors play a role in network regulation, and synergistically induce the differentiation of mesenchymal stem cells into chondroblasts within 3 days, which has important application prospects in the field of cartilage injury treatment.

The applicant declares that this application uses the above-mentioned embodiments to illustrate the detailed methods of this application, but this application is not limited to the above-mentioned detailed methods, which does not mean that this application must rely on the above-mentioned detailed methods to be implemented. Those skilled in the art should understand that any improvement to this application, the equivalent replacement of each raw material of the product of this application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of this application.

Claims

An inducer of stem cell differentiation into chondrogenesis, which includes PDGF, bFGF and TGF-β;

Wherein the inducer also includes insulin and/or IGF.
The inducer according to claim 1, wherein the PDGF comprises any one or a combination of at least two of PDGF-AA, PDGF-AB, or PDGF-BB.
The inducer according to claim 1 or 2, wherein the TGF-β comprises any one or a combination of at least two of TGF-β1, TGF-β2, or TGF-β3.
The inducer according to any one of claims 1-3, wherein the IGF comprises IGF-1 and/or IGF-2.
The inducer according to any one of claims 1 to 4, wherein the mass ratio of the PDGF and the bFGF is (0.8-4):3.
The inducer according to any one of claims 1 to 5, wherein the mass ratio of the PDGF and the TGF-β is (0.8-4):3.
The inducer according to any one of claims 1 to 6, wherein the mass ratio of the PDGF and the insulin is (0.6-8):1000.
The inducer according to any one of claims 1-7, wherein the mass ratio of the PDGF and the IGF is (0.6-8):1000.
A chondrogenic differentiation induction medium, comprising a basic medium and the inducer according to any one of claims 1 to 8 added to the basic medium.
The medium according to claim 9, wherein the concentration of the PDGF in the medium is 30 to 80 ng/mL, preferably 40 to 50 ng/mL;

Optionally, the concentration of the bFGF in the culture medium is 60-110 ng/mL, preferably 70-90 ng/mL;

Optionally, the concentration of the TGF-β in the culture medium is 60-110 ng/mL, preferably 70-90 ng/mL;

Optionally, the concentration of the insulin in the culture medium is 10-50 μg/mL, preferably 10-20 μg/mL;

Optionally, the concentration of the IGF in the medium is 10-50 μg/mL, preferably 10-20 μg/mL.
The medium according to claim 9 or 10, wherein the basic medium is a serum-free basic medium;

Optionally, the basal medium includes any one of DMEM/F12, α-MEM or DMEM high glucose.
A method for inducing stem cells to differentiate into chondrogenesis, which comprises culturing mesenchymal stem cells with the medium according to any one of claims 9-11.
The method according to claim 12, wherein the culture time does not exceed 3 days;

Optionally, the passage number of the mesenchymal stem cells is 4 to 8 generations;

Optionally, before culturing the mesenchymal stem cells, the method further includes the step of obtaining the mesenchymal stem cells from umbilical cord Huatong glue and/or umbilical cord amniotic membrane.
A pharmaceutical composition comprising chondrogenic cells prepared by inducing and culturing mesenchymal stem cells with the medium according to any one of claims 9-11;

Optionally, the pharmaceutical composition further includes any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.
A medicine for treating cartilage damage, which comprises the pharmaceutical composition according to claim 14.