A cell reprogramming composition and a method and application thereof for inducing embryonic fibroblasts to reprogram into osteoblast-like cells

Abstract

The present invention belongs to the field of regenerative medicine, and specifically relates to a cell reprogramming composition and a method and application thereof for inducing embryonic fibroblasts to be reprogrammed into osteoblast-like cells. The composition comprises amiloride pyrazine amidine methylsulfonic acid, forskolin and psoralen, and the concentrations used are 1-10µmol / L, 1-10µmol / L and 1-100µmol / L, respectively. Psoralen can significantly improve the efficiency of FP-induced MEFs reprogramming to osteoblasts on the basis of amiloride pyrazine amidine methylsulfonic acid and forskolin, and the osteoblasts have a high content of calcium nodules and highly express Runx2, ALP, BMP2 and OCN.

Description

Technical Field

[0001] The present invention belongs to the field of regenerative medicine, and specifically relates to a cell reprogramming composition and a method and application thereof for inducing embryonic fibroblasts to reprogram into osteoblast-like cells. Background Art

[0002] Osteoblasts are the main functional cells of bone formation, responsible for the synthesis, secretion and mineralization of bone matrix. Human and animal bone tissue is constantly being rebuilt, and the bone reconstruction process includes bone decomposition and absorption and the formation of new bone. Osteoclasts are responsible for bone decomposition and absorption, while osteoblasts are responsible for new bone formation. Osteoclasts attach to the old bone area, secrete acidic substances to dissolve minerals, secrete proteases to digest bone matrix, and form bone absorption pits; then, osteoblasts migrate to the absorbed area, secrete bone matrix, and the bone matrix mineralizes to form new bone. The balance between osteoclasts and osteoblasts is the key to maintaining normal bone mass.

[0003] Due to the key role of osteoblasts in bone formation, they are widely used in the field of bone tissue engineering and regenerative medicine. Osteoblasts are expanded by in vitro culture and applied to the repair and regeneration of bone defects, achieving significant therapeutic effects. Patent WO2024053957A1 provides a culture medium composition for direct differentiation of osteoblasts and a method for direct differentiation of osteoblasts, which induce osteoblasts from somatic cells, the composition comprising chemical substances as active ingredients, namely TGF-β receptor inhibitors (RepSox), cAMP signal activators (Forskolin) and epithelial Na+ channel inhibitors (Phenamil). It has been confirmed that the composition containing osteoblasts prepared by the method is effective in the treatment or prevention of osteogenesis imperfecta and Proteus syndrome, which are prototype bone diseases, and can therefore be effectively used as a composition for preventing or treating bone diseases.

[0004] Although reprogramming into osteoblast-like cells has great value in biomedical research and clinical applications, it still faces some technical challenges. For example, issues such as reprogramming efficiency, cell quality, safety, and large-scale production still need to be further studied and resolved.

[0005] The importance of reprogramming osteoblast-like cells in biomedical research and clinical applications is self-evident. It not only provides a powerful tool for disease model construction, drug screening and development, gene function research, etc., but also brings new hope to the fields of bone tissue repair and regeneration, treatment of bone diseases such as osteoporosis, and personalized medicine. However, improving the efficiency of reprogramming to osteoblast-like cells is of great significance for promoting the development of biomedical research and clinical applications. It can not only accelerate the research process, reduce costs, and improve cell quality, but also promote clinical transformation, promote the development of personalized medicine, and promote the advancement of cell therapy technology. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a composition for inducing cell reprogramming, which can significantly improve the efficiency of inducing embryonic fibroblasts to reprogram into osteoblast-like cells and improve cell quality.

[0007] In one aspect, the present invention provides a composition for inducing cell reprogramming, comprising amiloride, forskolin and psoralen.

[0008] Specifically, the concentrations of amiloride pyrazine amidine methylsulfonic acid, forskolin and psoralen can be 1-10µmol / L, 1-10µmol / L and 1-100µmol / L, respectively.

[0009] In some embodiments, the concentrations of amiloride, forskolin, and psoralen can be 10 µmol / L, 10 µmol / L, and 25 µmol / L, respectively.

[0010] In other embodiments, the concentrations of amiloride, forskolin and psoralen can be 10 µmol / L, 10 µmol / L and 1 µmol / L, respectively.

[0011] In some other embodiments, the concentrations of amiloride, forskolin and psoralen can be 1 µmol / L, 1 µmol / L and 100 µmol / L, respectively.

[0012] Specifically, the composition is added to a basal culture medium; the basal culture medium includes, but is not limited to, at least one of DMEM / F12, IMDM, M-199, RPMI-1640, MEM or DMEM.

[0013] Preferably, the basal culture medium may be DMEM / F12.

[0014] Specifically, the basic culture medium further includes one or more of fetal bovine serum, penicillin-streptomycin, ascorbic acid, dexamethasone and sodium β-glycerophosphate.

[0015] More specifically, the concentrations of fetal bovine serum, penicillin-streptomycin, ascorbic acid, dexamethasone and β-sodium glycerophosphate can be: 5%-15% v / v, 0.5%-2% v / v, 30-70 mg / L, 0.05-0.2 μM, 5-15 mM, respectively.

[0016] Preferably, the concentrations of fetal bovine serum, penicillin-streptomycin, ascorbic acid, dexamethasone and β-glycerophosphate sodium can be: 10%-15% v / v, 1%-2% v / v, 30-50 mg / L, 0.1-0.2 μM, 5-10 mM, respectively.

[0017] Further preferably, the concentrations of fetal bovine serum, penicillin-streptomycin, ascorbic acid, dexamethasone and β-glycerophosphate sodium can be: 10% v / v, 1% v / v, 50 mg / L, 0.1 μM, 10 mM, respectively.

[0018] In yet another aspect, the present invention provides a method for inducing fibroblasts to reprogram into osteoblast-like cells, wherein the method utilizes the aforementioned composition to culture fibroblasts.

[0019] Specifically, the fibroblasts are derived from mouse embryos.

[0020] More specifically, the fibroblasts are derived from 10-15 day old mouse embryos.

[0021] Preferably, the fibroblasts are derived from 12-14 day old mouse embryos.

[0022] Further preferably, the fibroblasts are derived from 13.5-day-old mouse embryos.

[0023] In yet another aspect, the present invention provides an osteoblast-like cell, wherein the osteoblast-like cell is obtained by inducing fibroblast reprogramming using the aforementioned composition or method.

[0024] Specifically, the fibroblasts are derived from mouse embryos.

[0025] More specifically, the fibroblasts are derived from 10-15 day old mouse embryos.

[0026] Preferably, the fibroblasts are derived from 12-14 day old mouse embryos.

[0027] Further preferably, the fibroblasts are derived from 13.5-day-old mouse embryos.

[0028] In yet another aspect, the present invention provides a kit comprising the aforementioned composition.

[0029] In yet another aspect, the present invention provides use of the aforementioned composition, method or kit in preparing a method for inducing fibroblasts to reprogram into osteoblast-like cells.

[0030] Specifically, the fibroblasts are derived from mouse embryos.

[0031] More specifically, the fibroblasts are derived from 10-15 day old mouse embryos.

[0032] Preferably, the fibroblasts are derived from 12-14 day old mouse embryos.

[0033] Further preferably, the fibroblasts are derived from 13.5-day-old mouse embryos.

[0034] The technical effects achieved by the present invention are:

[0035] (1) The combined use of psoralen and small molecule compounds forskolin and amiloride pyrazine methanesulfonic acid can induce more alkaline phosphatase-positive cells within 4 days.

[0036] (2) The combined use of psoralen with small molecule compounds forskolin and amiloride pyrazine methanesulfonic acid can induce more Runx2-positive cells.

[0037] (3) The combined use of psoralen with small molecule compounds forskolin and amiloride pyrazine methanesulfonic acid can induce more calcium nodules.

[0038] (4) The combined use of psoralen and small molecule compounds forskolin and amiloride pyrazine methanesulfonic acid can induce the relative expression levels of Runx2, ALP, BMP2, and OCN mRNA in cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 These are the cell morphology pictures of the OM group, F+Ph group and P+F+Ph group in Example 1 after 6 days of induction.

[0040] Figure 2 This is the expression of RUNX2 in MEFs in the OM group, F+Ph group and P+F+Ph group after 4 days of induction in Example 1.

[0041] Figure 3 This is the average fluorescence expression intensity of the osteogenic-related protein RUNX2 in MEFs in the OM group, F+Ph group and P+F+Ph group after 4 days of induction in Example 1.

[0042] Figure 4 The alkaline phosphatase activity of MEFs in the OM group, F+Ph group and P+F+Ph group after 6 days of induction and the mineralization of MEF matrix after 21 days of induction in Example 1.

[0043] Figure 5 The expression of osteogenic-related genes in MEFs in the OM group, F+Ph group and P+F+Ph group after 6 days of induction in Example 1.

[0044] Figure 6 The expression of OCN mRNA in the OM group, F+Ph group and P+F+Ph group after 21 days of induction in Example 1

[0045] Figure 7 This is the expression of osteogenesis-related genes RUNX2, ALP, and BMP2 mRNA in MEFs in the OM group, F+Ph group, and P+F+Ph group after 6 days of induction, and the expression of OCN mRNA after 21 days of induction in Example 2.

[0046] Figure 8 This is the expression of osteogenesis-related genes RUNX2, ALP, and BMP2 mRNA in MEFs in the OM group, F+Ph group, and P+F+Ph group after 6 days of induction, and the expression of OCN mRNA after 21 days of induction in Example 3.

[0047] Fig. 9 The expression changes of alkaline phosphatase activity in the P+F+Ph group in Example 1 and Comparative Examples 1, 2 and 3 at 6 days after induction, and the deposition of extracellular calcium matrix in each group after 21 days of induction.

[0048] Fig.10 The expression of osteogenic genes RUNX2 and ALP mRNA in the P+F+Ph group in Example 1 and Comparative Examples 1, 2 and 3 at 6 days of induction, and the expression of osteogenic gene OCN mRNA in each group after 21 days of induction. DETAILED DESCRIPTION

[0049] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0050] In the present invention, amiloride pyrazine amidine methylsulfonic acid is Phenamil (Ph); forskolin is Forskolin (F); and psoralen is Psoralen (P).

[0051] Example 1

[0052] 1.1 Materials

[0053] DMEM / F12 (1:1) basic (1X) (gibco, 6123073), fetal bovine serum (Punosai, 164210-50), L-ascorbic acid (Sigma, SLCF5765), penicillin-streptomycin mixture (double antibody, 100x) (Servicebio, G4003-100ML), β-glycerophosphate sodium disodium salt hydrate (Biogene, BN35422-10g), RUNX2 Polyclonal antibody (RUNX family transcription factor 2 polyclonal antibody, Protientech, 20700-1-AP), Alexa Fluor 488-labeled goat anti-rabbit IgG (Servicebio, GB25303), DAPI staining reagent (Servicebio, G1012), pluripotent stem cell alkaline phosphatase colorimetric kit (Biyuntian, C3250S), OriCell® Alizarin Red Staining Solution (Saiye Bio, ALIR-10001), primers synthesized by Shanghai Shenggong Biotechnology, Forskolin (Forskolin, GLPBIO, GC11920), Psoralen (Psoralen, Beijing Betterkang Biotechnology Co., Ltd., 23010802), Phenamil (Amilopyrazine Methanesulfonic Acid, GLPBIO, GC13994).

[0054] 1.2 Methods

[0055] 1.2.1 Isolation and primary culture of mouse embryonic fibroblasts (MEFs)

[0056] C57BL / 6 pregnant mice at 13.5 days of gestation were killed by cervical dislocation and then immersed in 75% ethanol for 5 minutes. The skin of the lower abdomen was cut open with scissors and forceps, and the skin was pulled to both sides to fix it. After exposing and separating the uterus, the uterus was transferred to a sterile dish and washed 2-3 times. The uterus was opened, the fetal membranes were removed, the fetuses were taken out and counted, and the fetuses were transferred to a culture dish containing PBS. The head, limbs and internal organs of the fetus were removed, and the trunk was retained. It was washed 2-3 times with PBS until it was bloodless. The clean mouse embryo trunk was transferred to a small dish and cut into about 1mm 3 The following fragments were mixed with PBS, centrifuged at 800rpm for 1min to remove the supernatant, and complete culture medium (DMEM / F12+10%v / v fetal bovine serum+1%v / v double antibody) was added to the precipitate, and inoculated into a T25 culture flask. The first medium change was performed after 24h, and the medium was changed every 2 days thereafter. When fibroblasts crawled out of the tissue block to 50%, the tissue block was gradually removed, and the fibroblasts were passaged when they grew and merged to 90%. P4 was used for the experiment.

[0057] 1.2.2 Observation of cell morphology using an inverted microscope

[0058] P4 MEFs were cultured at a rate of 1×10 5 100 μmol / L, 10 μmol / L Phytopathogens were plated in 12-well plates. When the cells grew to about 80%, the complete medium was replaced with osteogenic induction medium (OM) (DMEM / F12 + 10% v / v fetal bovine serum + 1% v / v penicillin-streptomycin + 50 mg / L ascorbic acid + 0.1 μM dexamethasone + 10 mM sodium β-glycerophosphate). The cells were divided into OM group (osteogenic induction medium), F+Ph group (osteogenic induction medium + F+Ph), and P+F+Ph group (osteogenic induction medium + P+F+Ph), where the concentration of F was 10 μmol / L, the concentration of Ph was 10 μmol / L, and the concentration of P was 25 μmol / L. There were 6 wells in each group, and the cells were cultured for 6 days. The cell morphology was observed under an inverted microscope.

[0059] 1.2.3 Alkaline phosphatase staining (ALP staining) to detect osteogenic differentiation ability

[0060] Cell culture and grouping refer to 1.2.2. After 4 days of culture, the culture medium was discarded, and the cells were washed with PBS three times. 4% paraformaldehyde tissue fixative was added and fixed for 10 minutes. After discarding the fixative, the cells were washed again with PBS, and the prepared ALP staining solution was added. The cells were kept at room temperature in the dark for 30 minutes, the staining solution was discarded, and the cells were washed with PBS. Blue-purple osteoblastic differentiated cells could be seen under the microscope.

[0061] 1.2.4 Real-time fluorescence quantitative PCR detection of osteogenesis-related gene expression

[0062] Cell culture and grouping refer to 1.2.2. Three parallel wells were set up in each group. The medium was changed every 2-3 days. The cells induced for 6 days were collected, and total RNA was extracted using TRIzol. 500 ng of total RNA was synthesized into first-strand cDNA using the Bio-rad CFX Opus 96 real-time fluorescence quantitative PCR instrument. The qPCR reaction was performed using the following conditions: 95 ℃ pre-denaturation for 30 s; 95 ℃ 10 s, 56 ℃ 30 s, 72 ℃ 30 s, and 40 cycles; the melting curve reaction conditions were 95 ℃ 15 s, 60 ℃ 60 s, and 95 ℃ 15 s. The primer sequences are shown in Table 2. GAPDH was used as the internal reference. The comparative cycle threshold (CT) method was used. The results were analyzed by 2 -ΔΔCT Methods The relative expression levels of RUNX2, ALP, and OCN mRNA were calculated. The specific primer designs are shown in Table 1.

[0063] Table 1 Primer sequence list

[0064]

[0065] 1.2.5 Immunofluorescence detection of the expression level of RUNX2, a typical osteoblast marker, during psoralen induction

[0066] Grouping was as described in 1.2.2. Immunofluorescence staining was performed on the cells at 4 days. The cells were washed with PBS 3 times and fixed in 4% paraformaldehyde fixative for 30 minutes. The cells were washed with PBS 3 times, each time for 5 minutes. The cells were permeabilized with permeabilization solution for 10 minutes. After washing, the cells were blocked in 5% BSA solution at room temperature for 30 minutes. The primary antibody RUNX2 (1:75) was added and incubated at 4°C overnight. After washing, Alexa Fluor 488-labeled goat anti-rabbit IgG was added and incubated at 37°C for 1 hour. The cells were stained with DAPI for nuclei, washed, sealed, and observed under a fluorescence microscope.

[0067] 1.2.6 Alizarin red staining to detect calcium nodules

[0068] Cell culture was performed as described in 1.2.2. The medium was changed every 3 days. After induction for 21 days, the medium was discarded, the cells were washed 3 times with PBS, and fixed with 95% ethanol at room temperature for 10 min. After discarding the ethanol, the cells were washed 3 times with pure water, and then stained with Alizarin red staining solution for 30 min. The staining solution was discarded, and the cells were washed 3 times with pure water. Orange-red calcium deposits were observed under the microscope.

[0069] 1.3 Statistical methods

[0070] SPSS 26.0 software was used for statistical analysis. Data were expressed as mean ± standard deviation (x¯±s). One-way analysis of variance was used for comparison among groups. LSD test was used for multiple comparisons when variances were equal, and Tambane's T2 test was used when variances were unequal. p The difference was statistically significant when the value was <0.05.

[0071] 1.4 Results and Analysis

[0072] 1.4.1 Cell morphology changes after 6 days of induction

[0073] Under the microscope, P4 generation MEFs were spindle-shaped or polygonal, with small nuclei; cells in the OM group were spindle-shaped or polygonal, with unclear edges, low refractive index, small nuclei, and unclear nucleoli; under the microscope, a small number of cells in the F+Ph group were round, cubic, or short columnar, with clear edges, high refractive index, large nuclei, and obvious nucleoli; the number of round, cubic, or short columnar cells in the P+F+Ph group was significantly higher than that in the F+Ph group. Figure 1 .

[0074] 1.4.2 Changes in fluorescence expression intensity of osteogenic marker protein RUNX2 after 4 days of induction

[0075] After 4 days of culture, the results of immunofluorescence showed that compared with the OM group, the fluorescence expression intensity of RUNX2 in the F+Ph group showed an upward trend but no significant difference, while the fluorescence expression intensity of RUNX2 in the P+F+Ph group increased significantly ( p <0.01); compared with the F+Ph group, the fluorescence expression intensity of RUNX2 in the P+F+Ph group increased significantly ( p <0.01). Figure 2-Figure 3 .

[0076] 1.4.3 Changes in alkaline phosphatase and calcium nodule staining 6 and 21 days after induction

[0077] The results of alkaline phosphatase staining showed that compared with the OM group, the alkaline phosphatase activity of the F+Ph group was slightly increased after 4 days of induction, and occasional blue-purple alkaline phosphatase-positive cells were observed under the microscope after staining; compared with the F+Ph group, the alkaline phosphatase activity of the P+F+Ph group was significantly increased, and more blue-purple alkaline phosphatase-positive cells were observed under the microscope. Alizarin red staining showed that after 21 days of induction, a small amount of calcium nodules were produced in the F+Ph group, and a large amount of calcium nodules were produced in the P+F+Ph group, and more red precipitates were observed under the microscope. Figure 4 .

[0078] 1.4.4 Comparison of RUNX2, ALP, BMP2, and OCN mRNA expression levels in each group after 6 and 21 days of induction

[0079] qPCR results showed that after 6 days of culture, the mRNA expressions of RUNX2, ALP, and BMP2 in the F+Ph group were significantly increased compared with those in the OM group ( p <0.001); compared with the F+Ph group, the mRNA expressions of RUNX2, ALP, and BMP2 in the P+F+Ph group were significantly increased ( p <0.001). After 21 days of culture, compared with the OM group, the OCN mRNA expression in the F+Ph group increased significantly ( p <0.001), compared with the F+Ph group, the OCN mRNA expression in the P+F+Ph group was significantly increased ( p <0.001). See Table 2, Figure 5-Figure 6 .

[0080] Table 2

[0081]

[0082] The above results suggest that psoralen can improve the efficiency of F+Ph-induced MEFs reprogramming into osteoblasts.

[0083] Example 2

[0084] Example 2 was set up with reference to Example 1. The difference between Example 2 and Example 1 is that it was divided into OM group (osteoblast induction medium), F+Ph group (osteoblast induction medium + F+Ph), and P+F+Ph group (osteoblast induction medium + P+F+Ph), wherein the concentration range of F was 10µmol / L, the concentration range of Ph was 10µmol / L, and the concentration range of P was 1µmol / L. Real-time fluorescence quantitative PCR was performed to detect the expression of osteogenesis-related genes with reference to 1.2.4 in Example 1.

[0085] The results are shown in Table 3. Figure 7 .

[0086] Table 3

[0087]

[0088] The results of PCR experiments after 6 days of culture are shown in Table 3. Figure 7 As shown, compared with the OM group, the mRNA expressions of RUNX2, ALP, and BMP2 in the F+Ph group were significantly increased ( p <0.001); compared with the F+Ph group, the mRNA expressions of RUNX2, ALP, and BMP2 in the P+F+Ph group were significantly increased ( p <0.001). After 21 days of culture, compared with the OM group, the OCN mRNA expression in the F+Ph group increased significantly ( p <0.001); compared with the F+Ph group, the OCN mRNA expression in the P+F+Ph group was significantly increased ( p <0.001)

[0089] The above results suggest that F concentration of 10µmol / L, Ph concentration of 10µmol / L, and P concentration of 1µmol / L can induce embryonic fibroblasts to reprogram into osteoblast-like cells, and psoralen can significantly enhance the effect of F+Ph in inducing the reprogramming of embryonic fibroblasts into osteoblast-like cells.

[0090] Example 3

[0091] Divided into OM group (osteoblast induction medium), F+Ph group (osteoblast induction medium + F+Ph), P+F+Ph group (osteoblast induction medium + P+F+Ph), where the F concentration range is 1µmol / L, the Ph concentration range is 1µmol / L, and the P concentration range is 100µmol / L. Real-time fluorescence quantitative PCR was performed to detect the expression of osteogenesis-related genes according to 1.2.4 in Example 1.

[0092] The results are shown in Table 4. Figure 8

[0093] Table 4

[0094]

[0095] The results of PCR experiments after 6 days of culture are shown in Table 4. Figure 8 As shown, compared with the OM group, the BMP2 mRNA expression in the F+Ph group was significantly increased ( p <0.05), there was no significant difference in the expression of RUNX2 and ALP mRNA; compared with the F+Ph group, the expression of RUNX2, ALP, and BMP2 mRNA in the P+F+Ph group was significantly increased ( p <0.001). After 21 days of culture, compared with the OM group, the OCN mRNA expression in the F+Ph group increased significantly ( p <0.001); compared with the F+Ph group, the OCN mRNA expression in the P+F+Ph group was significantly increased ( p <0.001).

[0096] The above results suggest that F concentration of 1µmol / L, Ph concentration of 1µmol / L, and P concentration of 100µmol / L can induce embryonic fibroblasts to reprogram into osteoblast-like cells, and psoralen can significantly enhance the effect of F+Ph inducing embryonic fibroblasts to reprogram into osteoblast-like cells.

[0097] Comparative Example

[0098] Refer to the P+F+Ph group setting comparison example in Example 1, and the setting method is shown in Table 5:

[0099] Table 5

[0100]

[0101] The results of alkaline phosphatase staining showed that at 4 days of induction, the alkaline phosphatase activity induced by the P+F+Ph group in Example 1 was higher than that of the comparative example 1 group, the comparative example 2 group, and the comparative example 3 group. The results of alizarin red staining showed that at 21 days, the P+F+Ph group in Example 1 induced more calcium nodules than that of the comparative example 1 group, the comparative example 2 group, and the comparative example 3 group, and the positive area of ​​alizarin red staining was larger. Fig. 9 .

[0102] Table 6

[0103]

[0104] The qPCR results showed that at 6 days after induction, the mRNA expression of bone-related genes RUNX2 and ALP in the P+F+Ph group in Example 1 was significantly increased compared with that in the control group 1, control group 2, and control group 3 ( p <0.001). At 21 days after induction, the expression of the bone-related gene OCN in Example 1 group was significantly higher than that in Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group ( p<0.001). The above results suggest that the efficiency of direct reprogramming of MEFs into osteoblasts induced by P+F+Ph in Example 1 is higher than that of Comparative Example 1, Comparative Example 2, and Comparative Example 3. Comparative Example 1, Comparative Example 2, and Comparative Example 3 did not show obvious effects of direct reprogramming of MEFs into osteoblasts. See Table 6, Fig.10 .

Claims

1. A composition for inducing cell reprogramming, characterized in that The composition consists of amiloride pyrazine amidine methyl sulfonic acid, forskolin and psoralen; the concentrations of amiloride pyrazine amidine methyl sulfonic acid, forskolin and psoralen are 1-10µmol / L, 1-10µmol / L and 1-100µmol / L respectively.

2. The composition according to claim 1, characterized in that The composition is added to a basic culture medium; the basic culture medium comprises at least one of DMEM / F12, IMDM, M-199, RPMI-1640, MEM or DMEM.

3. The composition according to claim 2, characterized in that The basic culture medium further comprises one or more of fetal bovine serum, penicillin-streptomycin, ascorbic acid, dexamethasone and sodium β-glycerophosphate.

4. The composition according to claim 3, characterized in that The concentrations of the fetal bovine serum, penicillin-streptomycin, ascorbic acid, dexamethasone and sodium β-glycerophosphate are respectively: 5%-15% v / v, 0.5%-2% v / v, 30-70 mg / L, 0.05-0.2 μM, 5-150 mM.

5. A method for inducing fibroblasts to reprogram into osteoblast-like cells, characterized in that: Fibroblasts are cultured using the composition according to any one of claims 1 to 4.

6. The method according to claim 5, characterized in that The fibroblasts are derived from mouse embryos.

7. A kit, characterized in that: The kit comprises the composition according to any one of claims 1 to 4.

8. Use of the composition according to any one of claims 1 to 4, the method according to any one of claims 5 to 6, or the kit according to claim 7 in inducing reprogramming of fibroblasts into osteoblast-like cells.

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