Composition, culture medium and culture method for inducing dental pulp cell reprogramming

Abstract

The invention discloses a composition for inducing dental pulp cell reprogramming, the composition comprises a TGF-beta pathway inhibitor, an MEK inhibitor, a GSK3 inhibitor, coptisine, astragaloside and artemisinin, and the components have a synergistic effect to realize the dental pulp cell reprogramming; the invention further discloses a reprogramming culture medium composed of the composition and an alpha-MEM basic culture medium. The invention also discloses a reprogramming method for reprogramming the dental pulp cells by using the reprogramming culture medium, the method comprises the steps of dental pulp cell culture, cell inoculation, reprogramming culture and the like, and the induced pluripotent stem cells can be obtained by culturing the dental pulp cells in the reprogramming culture medium for 14 days. The composition disclosed by the invention is definite in component, the reprogramming efficiency of the dental pulp cells can be remarkably improved, and the reprogrammed induced pluripotent stem cells are good in dryness and high in hereditary stability, and can be widely applied to the cell reprogramming fields such as regenerative medicine, oral disease treatment and drug development.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell reprogramming, and particularly relates to a composition for inducing dental pulp cell reprogramming, a culture medium and a culture method. BACKGROUND

[0002] Dental pulp disease is a common oral disease. According to statistics, the incidence of dental pulp disease is increasing year by year worldwide, among which dental pulpitis and dental pulp necrosis are particularly common. At present, root canal treatment is the main means for clinical treatment of dental pulp disease, which can relieve symptoms to a certain extent, but cannot restore the key functions of dental pulp such as nutritional supply, sensory nerve and induction of dentin formation. Therefore, functional regeneration of dental pulp tissue is a major problem to be solved in the field of oral medicine, and has extremely important significance for promoting the innovation of clinical treatment technology and the development of basic research in oral medicine. Pluripotent stem cells (PSCs) have the unique ability of self-renewal and multi-directional differentiation, and can differentiate into various cells in dental pulp tissue, realizing the regeneration and functional reconstruction of dental pulp tissue, and bringing new hope for dental pulp regeneration.

[0003] Cell reprogramming technology, as a new way to obtain pluripotent stem cells, has attracted widespread attention in recent years. At present, the mainstream method for obtaining induced pluripotent stem cells (iPSCs) through cell reprogramming includes introducing exogenous transcription factors (such as Shinya Yamanaka factors) by viral vectors or inducing by chemical small molecules (such as CN201010296987B, CN108884436B). However, the viral vector method has the risk of random integration of exogenous genes, and the operation is complex and costly. Although the traditional chemical reprogramming method can avoid the risk of gene introduction, it also has problems such as low reprogramming efficiency, difficulty in maintaining stem cell stemness, and poor genetic stability. The cell reprogramming technology induced by chemical small molecules alone effectively avoids the risk of gene manipulation, and has become a hot research direction. However, the current research is still in its infancy and faces many challenges. Therefore, it is of great practical significance to develop an efficient, safe, stable and component-specific chemical small molecule induction system and reprogramming method for promoting the clinical translation of dental pulp regeneration technology.

[0004] Among the many sources of stem cells, dental pulp cells have become the ideal seed cells for dental pulp regeneration due to their advantages of abundant source, relatively simple acquisition and minimal damage to patients. Dental pulp cells can be obtained non-invasively from orthodontic extracted teeth, wisdom teeth or deciduous teeth, avoiding the risk of invasive operation of traditional stem cell sources such as bone marrow and fat. More importantly, dental pulp cells have unique biological properties, strong proliferation capacity and multi-directional differentiation potential. In addition, dental pulp cells are derived from oral tissue, and their gene expression profile is naturally compatible with the dental pulp microenvironment, which has higher tissue compatibility and differentiation efficiency in dental pulp regeneration than heterologous stem cells such as bone marrow mesenchymal stem cells.

[0005] In summary, the current dental pulp cell reprogramming technology still faces many challenges in terms of reprogramming efficiency, stemness maintenance stability and medium optimization. Developing a chemical small molecule induction system targeting dental pulp cell characteristics has important practical significance for promoting the development of dental pulp cell reprogramming technology and its wide application in the fields of dental pulp disease treatment and regenerative medicine. SUMMARY

[0006] To solve the above problems, the present application provides a composition for inducing dental pulp cell reprogramming, a culture medium and a culture method.

[0007] In the first aspect of the present application, a composition for inducing dental pulp cell reprogramming is provided, characterized in that it comprises the following components: TGF-β pathway inhibitor, MEK inhibitor, GSK3 inhibitor, berberine, astragaloside and artemisinin. The composition of the present application is composed of chemical small molecules, wherein the chemical small molecule refers to an organic compound with a molecular weight of ≤1000 daltons, including artificially synthesized and naturally derived compounds.

[0008] The present inventors have found through extensive experimental research that each component of the composition alone, or the combination of the inhibitor components (TGF-β pathway inhibitor, MEK inhibitor, GSK3 inhibitor), or the combination of the active components of traditional Chinese medicine (berberine, astragaloside and artemisinin) does not have the function of inducing dental pulp cell reprogramming into induced pluripotent stem cells, but the combination of the TGF-β pathway inhibitor, MEK inhibitor, GSK3 inhibitor, berberine, astragaloside and artemisinin into a chemical small molecule composition has the function of inducing dental pulp cell reprogramming into induced pluripotent stem cells.

[0009] In the present application, for the inhibitor components, although the TGF-β pathway inhibitor alone can have some effect on the TGF-β signaling pathway, it cannot promote dental pulp cells to start the reprogramming process; the MEK inhibitor alone can only block MEK-related signal transduction and cannot guide dental pulp cells to transform into induced pluripotent stem cells; the GSK3 inhibitor alone can regulate the Wnt pathway, but also cannot make dental pulp cells reprogram. For the active components of traditional Chinese medicine, berberine alone can stabilize the ATF4 promoter G-quadruplex structure and inhibit the expression of stress-related genes, but cannot promote dental pulp cells to complete reprogramming; astragaloside alone can activate the AMPK signaling pathway and enhance cell energy metabolism, but also cannot induce dental pulp cell reprogramming; artemisinin alone cannot make dental pulp cells reprogram into induced pluripotent stem cells through the role of GABA receptor in regulating epigenetics.

[0010] However, when the TGF-β pathway inhibitor, the MEK inhibitor, the GSK3 inhibitor, berberine, astragaloside and artemisinin are combined according to the present application, they can interact with each other and produce a synergistic effect. The TGF-β pathway inhibitor and the MEK inhibitor synergize to effectively block the signal of cell differentiation in a specific direction, creating a suitable basic environment for cell reprogramming; the GSK3 inhibitor enhances the activity of the Wnt pathway to maintain the undifferentiated state of the cell, and cooperates with the former two to promote the process of cell reprogramming. At the same time, berberine inhibits cell apoptosis to ensure the survival of cells during the process of reprogramming; astragaloside provides sufficient energy to support the process of cell reprogramming; artemisinin regulates epigenetics to promote the activation of pluripotency genes. The three active components of traditional Chinese medicine and the three inhibitors cooperate with each other to optimize the intracellular environment from different angles, and together promote the change of the gene expression profile of dental pulp cells to realize the reprogramming and conversion of dental pulp cells to induced pluripotent stem cells. This small molecule composition after combination exhibits a whole new function and has the characteristics of reprogramming dental pulp cells to induced pluripotent stem cells, bringing new breakthroughs and hopes to the field of dental pulp regeneration.

[0011] In the formula, the TGF-β pathway inhibitor can be selected from SB431542, LY2157299 or RepSox, the MEK inhibitor can be selected from PD0325901, U0126 or Selumetinib, and the GSK3 inhibitor can be selected from CHIR99021, BIO or SB216763.

[0012] Preferably, the above-mentioned composition, in terms of final concentration, has the following component concentrations: 2-5 μM of TGF-β pathway inhibitor, 0.5-1 μM of MEK inhibitor, 2-3 μM of GSK3 inhibitor, 10-20 μM of berberine, 50-100 μM of astragaloside, and 5-10 μM of artemisinin. The 2-5 μM TGF-β pathway inhibitor can effectively inhibit the TGF-β signaling pathway and prevent the differentiation of dental pulp cells in a specific direction, thus creating favorable conditions for cell reprogramming. The 0.5-1 μM MEK inhibitor can synergize with the TGF-β pathway inhibitor to block cell differentiation signals and activate pluripotency gene networks. The 2-3 μM GSK3 inhibitor can enhance Wnt pathway activity, maintain the undifferentiated state of cells, and promote stem cell self-renewal. The 10-20 μM berberine can inhibit the expression of stress-related genes by stabilizing the ATF4 promoter G-quadruplex structure and reduce cell apoptosis during the reprogramming process. The 50-100 μM astragaloside can activate the AMPK signaling pathway, improve mitochondrial function, and enhance the efficiency of cell energy metabolism, thus providing sufficient energy for reprogramming. The 5-10 μM artemisinin can regulate epigenetics through GABA receptors, promote the activation of pluripotency genes, and promote the conversion of dental pulp cells into induced pluripotent stem cells. In the entire text of the present application, the final concentration refers to the ratio of the amount of substance of each component to the total volume of the solution when the composition is dissolved and configured into a solution for use. The solution for use refers to a solution that can be directly used for inducing the reprogramming of dental pulp cells, including a reprogramming medium.

[0013] Preferably, the above-mentioned composition has the following final concentrations of components: 5 μM of TGF-β pathway inhibitor, 1 μM of MEK inhibitor, 3 μM of GSK3 inhibitor, 20 μM of berberine, 100 μM of astragaloside, and 10 μM of artemisinin.

[0014] In the second aspect of the present application, a reprogramming medium for inducing the reprogramming of dental pulp cells into induced pluripotent stem cells is provided, characterized by comprising the composition proposed in the first aspect of the present application and a basic medium.

[0015] Preferably, the basic medium is an α-MEM medium. The α-MEM medium is rich in various nutrients such as amino acids, vitamins, and inorganic salts, and can meet the basic needs of dental pulp cell growth and reprogramming. Moreover, the composition is clear and beneficial to the repeatability and stability of experiments.

[0016] In the third aspect of the present application, a culture method for reprogramming dental pulp cells into induced pluripotent stem cells is provided, characterized by using the above-mentioned reprogramming medium for reprogramming culture of dental pulp cells, comprising the following steps: Step one, dental pulp cell culture: obtain dental pulp tissue, wash with PBS, cut into small pieces, add 0.25% trypsin-0.02% EDTA digestion solution, digest at 37°C for 30-60 min, add α-MEM medium to terminate digestion after digestion, centrifuge and discard the supernatant, resuspend the cell pellet with fresh α-MEM medium, inoculate into a culture bottle, and incubate in a 37°C, 5% CO2, saturated humidity incubator, replace the α-MEM medium every 3 days, when the cells grow to 80% confluence, obtain confluent dental pulp cells.

[0017] Step two, cell inoculation: the confluent dental pulp cells obtained in step one are digested with trypsin and counted to obtain digested cells, and then the digested cells are inoculated into Matrigel coated culture dishes at a density of 3×10 4 / cm 2 The Matrigel is rich in collagen, laminin and other extracellular matrix components, simulates the in vivo microenvironment, and promotes cell adhesion and growth.

[0018] Step three, reprogramming culture: add the reprogramming medium to the inoculated cell system obtained in step two, and culture in a 37°C, 5% CO2, saturated humidity incubator, replace the reprogramming medium with fresh reprogramming medium every 2 days during the culture process, and culture for 14 days to obtain induced pluripotent stem cells.

[0019] In addition, the present application also provides the application of the composition for reprogramming dental pulp cells into induced pluripotent stem cells, the reprogramming medium and the culture method in the field of cell reprogramming. The dental pulp cells cultured by using the technical solution of the present application have significantly improved reprogramming efficiency, and the induced pluripotent stem cells after reprogramming have good stemness, genetic stability and safety, and can be widely used in the fields of regenerative medicine, oral disease treatment, drug development, etc.

[0020] In summary, the beneficial effects of the present application are: (1) The composition and reprogramming medium of the present application significantly improve the efficiency of reprogramming dental pulp cells into induced pluripotent stem cells through the synergistic effect of adding multiple components. The Oct4 positive rate is >50% at the 14th day of culture, which is significantly higher than that of the traditional technology, and the induced pluripotent stem cells after reprogramming can stably express stemness genes such as Oct4 and Sox2, effectively solving the problems of low reprogramming efficiency and unstable stemness of induced pluripotent stem cells in chemical reprogramming technology in the prior art.

[0021] (2) The present application uses the unique mechanism of action of active ingredients of traditional Chinese medicine to effectively protect the genomic stability. The karyotype normal rate of the induced pluripotent stem cells after reprogramming is still ≥95% after being subcultured for the 80th time, solving the problems of genetic instability and easy mutation of cells in traditional reprogramming methods.

[0022] (3) The reprogramming medium of the present application adopts a formula with clear components, and does not rely on animal-derived components, thereby solving the problems of high contamination risk and large batch difference of animal-derived components. Meanwhile, the medium has clear components, facilitating quality control and research on reprogramming mechanisms.

[0023] (4) In the reprogramming culture process of the present application, Matrigel is used to coat and simulate the in-vivo microenvironment, combined with methods such as regular full replacement of the medium and strict control of the inoculation density, to ensure that the cells synchronously enter the reprogramming process, thereby avoiding excessive competition or insufficient signal transduction of the cells, and solving the problem of influence of culture conditions on reprogramming efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 are morphological diagrams of the dental pulp tissue and dental pulp cells after reprogramming, A is the dental pulp tissue, and B-I are morphological diagrams of the dental pulp cells after reprogramming, wherein B-D correspond to morphological diagrams of the dental pulp cells after reprogramming in the medium 1-3, respectively, and E-I correspond to morphological diagrams of the dental pulp cells after reprogramming in the comparative medium 1-5, respectively. DETAILED DESCRIPTION

[0025] In order to make the objectives, technical solutions and advantages of the present application clearer and more comprehensible, the present application will be further described in detail below in combination with specific examples. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as generally understood by those of ordinary skill in the art to which the present application belongs.

[0026] In the present application, the components and reagents involved are all conventional commercially available products, or can be obtained through conventional technical means in the art. Unless otherwise specified, the materials, methods and examples of the present application are only exemplary, and are not limiting.

[0027] Example 1: Preparation of the experimental group reprogramming medium In order to verify the effect of the composition of the present application, this example provides a variety of experimental group composition systems including the composition of the present application. The α-MEM medium is used as the basic medium, and the composition of the present application is added to prepare the experimental group reprogramming medium. The specific formula of the composition is shown in Table 1, and the obtained medium is respectively recorded as medium 1, medium 2 and medium 3. The specific operation of preparation is as follows: (1) Each component is accurately weighed and dissolved in an appropriate amount of dimethyl sulfoxide (DMSO) in sequence to prepare a high-concentration mother liquor for standby use.

[0028] (2) An appropriate amount of the mother liquor prepared in (1) is added to the α-MEM medium in a sterile container according to the required final concentration of each component, and the mixture is obtained by thoroughly stirring. (3) The mixed reprogramming medium is filtered through a 0.22 μm sterile filter to remove bacteria, and the reprogramming medium is obtained. The prepared reprogramming medium is stored at 4°C for standby.

[0029] Table 1 Composition of the reprogramming medium Comparative Example 1: Preparation of a comparative reprogramming medium To demonstrate the necessity of the composition combination of the present application, the comparative composition system of different missing or replaced components is designed based on the components of medium 1, and the comparative reprogramming medium is prepared. The specific formula of the comparative composition is shown in Table 2, and the obtained medium is denoted as comparative medium 1, comparative medium 2, comparative medium 3, comparative medium 4 and comparative medium 5, respectively. The specific operation of preparation is the same as that of Example 1.

[0030] Table 2 Composition of the comparative reprogramming medium Example 2: Acquisition and reprogramming culture of dental pulp cells In this example, dental pulp cells are reprogrammed using medium 1-3 and comparative medium 1-5, and the specific operation is as follows: Step one, acquisition and culture of dental pulp cells: dental pulp tissue is obtained from healthy third molars removed by orthodontics, and the dental pulp tissue is placed in a sterile culture dish containing PBS buffer under strict sterile operation conditions, and washed 3 times to remove bloodstains and impurities. The dental pulp tissue is cut into small pieces of about 1 mm 3 using an ophthalmic scissors, and an appropriate amount of digestion solution containing 0.25% trypsin-0.02% EDTA is added. The culture dish is shaken gently every 10 min to make the digestion more complete. After most of the tissue pieces are dispersed, the digestion is terminated by adding α-MEM medium, and the cell suspension is transferred to a centrifuge tube and centrifuged at 1000 rpm for 5 min. The supernatant is discarded. The dental pulp cells are resuspended with fresh α-MEM medium, and the cells are inoculated into culture bottles and cultured in a 37°C, 5% CO2, saturated humidity incubator. The α-MEM medium is replaced every 3 days, and the confluent dental pulp cells are obtained when the cells grow to 80% confluence.

[0031] Step two, inoculation of dental pulp cells: the confluent dental pulp cells obtained in step one are digested with 0.25% trypsin-0.02% EDTA digestion solution, and the number of cells after digestion is counted. The cells after digestion are inoculated at a density of 3×10 4 2 ​The cells were seeded at a density of 100 μg / mL in a Matrigel-coated culture dish to obtain a post-seeding cell system. Matrigel was melted at 4°C in advance and pipetted with a pre-cooled pipette tip before use. The cells were evenly spread on the bottom of the culture dish and incubated in a 37°C incubator for 30 minutes to fully coat the culture dish surface.

[0032] Step 3: Dental Pulp Cell Reprogramming Culture: Add experimental culture media 1-3 and control culture media 1-5 to the seeded cell system obtained in Step 2. Place the culture dish in an incubator at 37°C, 5% CO2, and saturated humidity for culture. Replace the culture medium with the corresponding new medium every two days for 14 days to obtain induced pluripotent stem cells. When changing the medium, carefully aspirate and discard the old medium. Gently wash the cells twice with PBS before adding fresh medium.

[0033] Example 3: Identification of induced pluripotent stem cells This part identifies the induced pluripotent stem cells obtained by reprogramming the above-mentioned dental pulp cells.

[0034] 1. Morphological identification On the 14th day of reprogramming culture, the cells in the culture dish were observed using an inverted microscope, and characteristics such as cell morphology, size, boundary clarity, and cell arrangement were recorded.

[0035] The culture results are as follows Figure 1 As shown, Figure 1 A is the original dental pulp tissue, which is a red solid and is the starting material for subsequent culture. Figure 1 B~D are the culture results of culture medium 1~3, respectively. The cells are dense and form obvious colonies with relatively clear colony boundaries. The cells grow well within the colonies, showing an appearance similar to that of pluripotent stem cell colonies, and gradually show the morphological characteristics of induced pluripotent stem cells, indicating that the culture medium of the experimental group can effectively induce reprogramming. Figure 1 E~I are the culture results of comparative culture medium 1~5, respectively. The overall cells are dispersed and sparse, and most retain the morphology of dental pulp cells. The colony formation is poor, indicating that the gene components of the comparative culture are missing or have problems with the combination, resulting in poor reprogramming induction effect, highlighting the key role of the synergistic effect of the components of the composition of the present invention. Figure 1 In E, the cells grew more dispersedly without forming obvious colony structures. The cell morphology was not much different from that of the original dental pulp cells, indicating that the lack of coptisine significantly hindered reprogramming and made it difficult to induce the cells to form morphological characteristics related to pluripotent stem cells. Figure 1 The cells in F are relatively sparsely distributed, and their morphology still retains many characteristics of dental pulp cells, with no obvious colonization trend, indicating that the lack of astragaloside IV is not conducive to the reprogramming of dental pulp cells; Figure 1G The cell morphology was irregular, the growth state was poor, and the colony formation was not obvious, indicating that the lack of artemisinin affected the reprogramming process, and the cells were difficult to present the morphological characteristics of pluripotent stem cells; Figure 1 H Although the cells in H had certain growth changes, the colony formation was not tight and typical, and compared with the experimental group, the degree of cell morphology transformation to pluripotent stem cells was weak, indicating that only the inhibitor component, the inhibitor combination alone was not enough to efficiently induce dental pulp cell reprogramming; Figure 1 I The cell morphology change was not significant, and the typical pluripotent stem cell colony structure was not formed, and the cell growth was loose, indicating that only the active ingredient combination of traditional Chinese medicine alone could not effectively induce dental pulp cell reprogramming to pluripotent stem cells.

[0036] II. Detection of stemness gene expression The detection was carried out according to the following steps: (1) Cell preparation: On the 14th day of reprogramming culture, the cells obtained by using different culture media (culture media 1-3, comparative culture media 1-5) were digested with 0.25% trypsin-0.02% EDTA digestion solution to make the cells separate from the culture dish surface. The cell suspension was transferred to a centrifuge tube and centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were washed with PBS twice, and after centrifugation, the supernatant was discarded to obtain clean cell pellets.

[0037] (2) Fixation and permeation: Add appropriate amount of 4% paraformaldehyde solution to the cell pellets, fix at room temperature for 15-20 min, and fix the cell morphology and antigen structure. After fixation, centrifuge at 1000 rpm for 5 min, discard the paraformaldehyde solution, and wash the cells with PBS twice. Then add PBS solution containing 0.1% Triton X-100, permeate at room temperature for 10-15 min, so that the antibody enters the cell to bind to the target antigen. After permeation, wash the cells with PBS twice.

[0038] (3) Antibody incubation: Resuspend the permeated cells in PBS solution containing Oct4 primary antibody, Sox2 primary antibody and Nanog primary antibody, and incubate at 4°C overnight in the dark. The next day, centrifuge at 1000 rpm for 5 min, discard the primary antibody solution, and wash the cells with PBS 3 times, 5 min each time. Then add the corresponding Alexa Fluor 488 fluorescently labeled secondary antibody to the primary antibody, and incubate at room temperature in the dark for 1-2 h. After incubation, wash the cells with PBS 3 times to remove unbound secondary antibodies.

[0039] (4) Flow cytometry detection: Resuspend the washed cells in appropriate amount of PBS, adjust the cell concentration to 1×10 6 ~1×10 7The cells were transferred to sample tubes dedicated for flow cytometry. The detection was performed using a flow cytometer, with an excitation wavelength of 488 nm and an emission wavelength of 519 nm, and the fluorescence signal of 510-530 nm wavelength was collected. The cells were analyzed one by one by the flow cytometer, and the fluorescence intensity of each cell was recorded. Unstained cells were set as negative controls to determine the background fluorescence level. The number of positive cells and the total number of detected cells were counted by the flow cytometer software, and the positive cell rate was calculated according to the following formula: Oct4 positive rate (%) = Oct4 gene expression positive cell number / total number of detected cells x 100% Sox2 positive rate (%) = Sox2 gene expression positive cell number / total number of detected cells x 100% Nanog positive rate (%) = Nanog gene expression positive cell number / total number of detected cells x 100% Table 3: Expression positive rate of stemness genes of reprogrammed cells The experimental results are shown in Table 3. Compared with the comparative medium 1-5, the expression positive rates of stemness genes Oct4, Sox2 and Nanog of induced pluripotent stem cells obtained by the medium 1-3 are significantly higher, wherein the Oct4 positive rate of the medium 1 is 58%, the Sox2 positive rate is 55%, and the Nanog positive rate is 52%. This shows that the composition and medium comprising the TGF-β pathway inhibitor, the MEK inhibitor, the GSK3 inhibitor, berberine, astragaloside and artemisinin in the present application can efficiently induce dental pulp cells to reprogram into induced pluripotent stem cells, and exhibit excellent performance in maintaining cell stemness, which effectively guarantees the high expression level of stemness genes. From the results of the comparative medium 1-5, it can be seen that each component in the medium of the present application is crucial for the reprogramming of dental pulp cells. With the synergistic effect of each component, a suitable environment is created for the reprogramming of dental pulp cells, which greatly promotes the expression of stemness genes such as Oct4, Sox2 and Nanog, and effectively maintains the stemness of induced pluripotent stem cells.

[0040] III. Karyotype analysis The detection was performed according to the following steps: (1) Cell culture and treatment: the induced pluripotent stem cells obtained by using different media were subcultured, and when the cells were subcultured to the 80th generation, colchicine was added to the medium when the cells were cultured to the logarithmic growth phase, with a final concentration of 0.05 μg / mL, and the cells were cultured for another 4 h to inhibit the formation of cell spindles and make the cells arrest at the metaphase.

[0041] (2) Hypotonic treatment: the cells were collected and subjected to hypotonic treatment with 0.075 M KCl solution at 37°C for 20-30 min to make the cells swell and the chromosomes disperse.

[0042] (3) Fixation and slide preparation: the cells after hypotonic treatment were fixed with methanol-glacial acetic acid (3:1) fixing solution for 3 times, 15-20 min each time. The fixed cells were dropped on pre-cooled glass slides and slide preparation was performed by air drying method.

[0043] (4) G-banding staining: the prepared slides were appropriately treated with trypsin solution, then stained with Giemsa staining solution for 10-15 min. After staining, the slides were washed with water and naturally dried.

[0044] (5) Microscopic detection: the morphology and number of chromosomes were observed under a microscope. 100 metaphase cells were selected for karyotype analysis. The chromosomes were paired, grouped and numbered according to the morphology, size and kinetochore position of the chromosomes, and whether the karyotype of the cells was normal was determined. The karyotype normality rate was calculated according to the following formula: Karyotype normality rate (%) = number of karyotype normal cells / total number of cells x 100% Table 4 Karyotype normality rate of reprogrammed cells The detection results are shown in Table 4. The karyotype normality rate of the induced pluripotent stem cells cultured in the experimental group medium 1-3 of the application reached 95% or above, indicating that the composition and reprogramming medium of the application performed well in maintaining the genetic stability of the induced pluripotent stem cells. The karyotype normality rate of the comparative medium 1-5 was significantly lower than that of the experimental group, being between 82% and 88%. The comparative medium 1 lacked berberine, and the karyotype normality rate was 82%; the comparative medium 2 lacked astragaloside, and the karyotype normality rate was 85%; the comparative medium 3 lacked artemisinin, and the karyotype normality rate was 86%, indicating that berberine, astragaloside and artemisinin played an important role in maintaining the genetic stability of the cells, and they cooperated with the inhibitor components to jointly ensure the stability of the genome during the cell reprogramming process. The comparative medium 4 only contained inhibitor components, and the karyotype normality rate was 88%; the comparative medium 5 only contained traditional Chinese medicine active ingredients, and the karyotype normality rate was 84%, further proving that the synergistic combination of the inhibitor and the traditional Chinese medicine active ingredients in the application was more conducive to maintaining the karyotype normality of the induced pluripotent stem cells and ensuring the genetic stability of the cells compared with using the inhibitor or the traditional Chinese medicine active ingredients alone.

[0045] The synergistic effect between the components of the composition of the application has a key impact on the genetic stability of the induced pluripotent stem cells. Through precise component combination and synergistic effect, a stable microenvironment is provided for the reprogramming of dental pulp cells, effectively reducing the chromosomal abnormalities of the cells during the reprogramming process, and providing a solid genetic stability foundation for the application of reprogramming technology in dental pulp regeneration and related fields.

[0046] It can be known from the above examples that the composition and the reprogramming medium have remarkable effects in inducing the reprogramming of dental pulp cells into induced pluripotent stem cells, and can effectively maintain the cell stemness and genetic stability, thereby providing a new effective method for the dental pulp regeneration field.

[0047] In conclusion, the composition and the reprogramming medium of the present application have the effects of synergistic cooperation, mutual promotion, strict and reasonable use method, remarkable effects in the efficient conversion of dental pulp cells into induced pluripotent stem cells, stemness maintenance, and genetic stability guarantee, thereby providing a new effective method for the dental pulp regeneration field.

[0048] Meanwhile, the present application also provides scientific ideas and methods for reprogramming dental pulp cells into induced pluripotent stem cells by using the composition and the reprogramming medium, which covers the whole process from the design of chemical small molecule composition to the preparation of reprogramming medium and cell culture. Any improvement, optimization and innovative application based on the principle of the present application should be considered to fall within the protection scope of the present application.

Claims

1. A composition for inducing reprogramming of dental pulp cells, characterized in that: Includes the following ingredients: TGF-β pathway inhibitor, MEK inhibitor, GSK3 inhibitor, coptisine, astragaloside IV and artemisinin; The TGF-β pathway inhibitor is selected from SB431542, LY2157299 or RepSox; The MEK inhibitor is selected from PD0325901, U0126 or Selumetinib; The GSK3 inhibitor is selected from CHIR99021, BIO or SB216763.

2. The composition according to claim 1, characterized in that In terms of final concentration, the concentrations of each component are: TGF-β pathway inhibitor 2~5μM, MEK inhibitor 0.5~1μM, GSK3 inhibitor 2~3μM, coptisine 10~20μM, astragaloside IV 50~100μM, and artemisinin 5~10μM.

3. The composition according to claim 2, characterized in that The final concentrations of each component were as follows: TGF-β pathway inhibitor 5 μM, MEK inhibitor 1 μM, GSK3 inhibitor 3 μM, coptisine 20 μM, astragaloside IV 100 μM, and artemisinin 10 μM.

4. A reprogramming medium for inducing reprogramming of dental pulp cells into induced pluripotent stem cells, characterized in that: Comprising the composition according to any one of claims 1 to 3 and a basal culture medium.

5. The reprogramming medium according to claim 4, characterized in that The basic culture medium is α-MEM culture medium.

6. A method for culturing dental pulp cells to reprogram induced pluripotent stem cells, characterized in that: Reprogramming and culturing dental pulp cells using the reprogramming medium according to claim 4 or 5 comprises the following steps: Step 1: Dental pulp cell culture: obtain dental pulp tissue, wash it with PBS, cut it into small pieces, add 0.25% trypsin-0.02% EDTA digestion solution, and digest it at 37°C for 30-60 minutes. After digestion, add α-MEM medium to terminate digestion, centrifuge and discard the supernatant, resuspend the cell pellet with fresh α-MEM medium, inoculate it into a culture flask, and culture it in an incubator at 37°C, 5% CO2, and saturated humidity. Change the α-MEM medium every 3 days. When the cells grow to 80% confluence, obtain confluent dental pulp cells. Step 2: Cell inoculation: The confluent dental pulp cells obtained in step 1 were digested with trypsin and counted to obtain digested cells. The digested cells were then plated at 3×10 4 pieces / cm 2 The cells were seeded at a density of 100 μg / mL in a Matrigel-coated culture dish to obtain a seeded cell system; Step 3: Reprogramming culture: Add the reprogramming medium to the inoculated cell system obtained in step 2, and culture in an incubator at 37°C, 5% CO2, and saturated humidity. During the culture process, replace the reprogramming medium with new one every 2 days. Culture for 14 days to obtain induced pluripotent stem cells.

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