Pharmaceutical composition and culture medium for in-vitro culture of odontogenic mesenchymal stem cells

Abstract

The invention provides a pharmaceutical composition and a culture medium for in-vitro culture of odontogenic mesenchymal stem cells, and belongs to the technical field of in-vitro culture of cells, the pharmaceutical composition comprises an epidermal growth factor (EGF), a basic fibroblast growth factor (bFGF) and a smooth receptor agonist (SAG); or epidermal growth factor (EGF) + smooth receptor agonist SAG + CHIR99021; or the epidermal growth factor (EGF), the basic fibroblast growth factor (bFGF), the fibroblast growth factor (FGF) and the CHIR99021. The pharmaceutical composition can significantly prolong the in-vitro culture time of the odontogenic mesenchymal stem cells, enhance cell proliferation, maintain the in-vitro tooth formation potential of the mesenchymal stem cells, and establish a more stable dental organ in-vitro culture system.

Description

Technical Field

[0001] The invention belongs to the technical field of in vitro cell culture, and in particular relates to a pharmaceutical composition and a culture medium for in vitro culture of dental mesenchymal stem cells. Background Art

[0002] The development of teeth depends on the interaction between the oral epithelium and the surrounding mesenchyme, and is also regulated by various signaling pathways. Tooth germ mesenchymal stem cells at a specific embryonic period (cap stage) have the ability to form teeth and can form structures such as enamel and dentin. Therefore, an effective strategy for the in vitro culture of such dental mesenchymal stem cells is crucial for tooth regeneration. However, prior art studies have shown that E14.5 mouse tooth germ mesenchymal stem cells are prone to lose their odontogenic potential during in vitro culture. Therefore, it is a huge challenge to culture dental mesenchymal stem cells in vitro and maintain their odontogenic potential.

[0003] Small molecule drugs refer to synthetic compounds with a molecular weight of less than 1000 Daltons and biological functions. Unlike cytokines and proteins, these compounds can directly penetrate the cell membrane into the cell and then exert their corresponding functions. At present, the effects of many small molecule drugs on the in vitro culture of dental mesenchymal stem cells are unclear. Finding drugs that can maintain the odontogenic potential of dental mesenchymal stem cells in vitro is of great significance for the in vitro culture of dental mesenchymal stem cells and tooth regeneration. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a pharmaceutical composition and culture medium for in vitro culture of dental mesenchymal stem cells; the pharmaceutical composition can significantly prolong the in vitro culture time of dental mesenchymal stem cells, enhance cell proliferation while maintaining the in vitro odontogenic potential of mesenchymal stem cells, and establish a more stable dental organ culture system.

[0005] The present invention provides a pharmaceutical composition for in vitro culture of dental mesenchymal stem cells, characterized in that it comprises epidermal growth factor EGF+basic fibroblast growth factor bFGF+smoothened receptor agonist SAG;

[0006] Or epidermal growth factor EGF + smoothened receptor agonist SAG + CHIR99021;

[0007] Or epidermal growth factor EGF + basic fibroblast growth factor bFGF + fibroblast growth factor FGF3 + CHIR99021.

[0008] In the present invention, CHIR99021 is a GSK-3α / β inhibitor.

[0009] Preferably, when the pharmaceutical composition includes epidermal growth factor EGF+basic fibroblast growth factor bFGF+smoothened receptor agonist SAG; the concentration of epidermal growth factor EGF is 15-25 ng / ml, the concentration of basic fibroblast growth factor bFGF is 15-25 ng / ml, and the concentration of smoothened receptor agonist SAG is 0.4-0.6 ng / ml.

[0010] Preferably, when the pharmaceutical composition includes epidermal growth factor EGF+smoothened receptor agonist SAG+CHIR99021, the concentration of epidermal growth factor EGF is 15-25 ng / ml, the concentration of smoothened receptor agonist SAG is 0.4-0.6 ng / ml, and the concentration of CHIR99021 is 2.5-3.5 ng / ml.

[0011] Preferably, when the pharmaceutical composition includes epidermal growth factor EGF+basic fibroblast growth factor bFGF+fibroblast growth factor FGF3+CHIR99021, the concentration of epidermal growth factor EGF is 15-25 ng / ml, the concentration of basic fibroblast growth factor bFGF is 15-25 ng / ml, the concentration of fibroblast growth factor FGF3 is 15-25 ng / ml, and the concentration of CHIR99021 is 2.5-3.5 ng / ml.

[0012] The invention provides a culture medium for in vitro culture of dental mesenchymal stem cells, comprising the pharmaceutical composition and a basic culture medium.

[0013] Preferably, the basic culture medium is DMEM / F12+10% KSR.

[0014] The present invention provides a method for culturing tooth germ organoids in vitro, comprising the following steps:

[0015] 1) isolating tooth germ mesenchymal stem cells, and culturing the tooth germ mesenchymal stem cells in the culture medium for 48 to 72 hours;

[0016] 2) After the cultured tooth germ mesenchymal stem cells are recombined with the dental epithelial tissue, they are further cultured to obtain tooth germ organoids.

[0017] Preferably, the time for continuing culturing in step 2) is 1 to 6 days.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] During the in vitro culture process, the odontogenic potential of the cultured induced dental mesenchymal stem cells was impaired, and the odontogenic potential was lost after 48 hours of in vitro culture. The present invention focuses on the odontogenic potential of dental mesenchymal stem cells, and uses high-throughput sequencing screening technology to screen out a small molecule drug composition that maintains the odontogenic potential of dental mesenchymal stem cells in vitro. The drug composition provided by the present invention can significantly prolong the in vitro culture time of dental mesenchymal stem cells. Currently, the in vitro culture time can be up to 72 hours, and the cell proliferation is nearly 3 times. At the same time, the odontogenic potential of mesenchymal stem cells in vitro can be maintained. The drug composition can establish a more stable tooth organ culture system. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the separation process of the mandibular arch and tongue of an E14.5 fetal mouse, and the position of the first molar tooth germ in the mandibular arch. The morphology of the E14.5 first molar tooth germ was observed under a stereomicroscope; the scale is 1 mm;

[0021] Figure 2 The cell morphology after digestion, where A represents the separation of epithelium and mesenchyme after digestion of the E14.5 first molar tooth germ, and B represents the morphology of epithelial tissue separated after digestion of the tooth germ (the red dotted line represents the enamel organ epithelium in the cap stage, and the gray dotted line represents the oral epithelium)

[0022] C represents the morphology of mesenchymal tissue separated after tooth germ digestion. The scale bar is 1 mm;

[0023] Figure 3 Negative control group: DMSO stimulated tooth germ mesenchymal stem cells and then reconstituted. After 6 days of in vitro culture, no organoid formation was observed; Stereo: 100μm; H&E: 50μm (40x) 20μm (100x);

[0024] Figure 4 The results of CCK-8 experiment show the proliferation growth curve of E14.5 dental mesenchymal stem cells treated with different single drugs; the scale bar is 100 μm;

[0025] Figure 5 The CCK-8 experimental results show the proliferation growth curve of E14.5 dental mesenchymal stem cells treated with different small molecule drug compositions;

[0026] Figure 6 The results of paraffin sectioning and H&E staining of composition A after 6 days of in vitro culture; Stereo: 100 μm; H&E: 50 μm (40x) 20 μm (100x);

[0027] Figure 7 The results of paraffin sectioning and H&E staining of composition B after 6 days of in vitro culture; Stereo: 100 μm; H&E: 50 μm (40x) 20 μm (100x);

[0028] Figure 8 The results of paraffin sectioning and H&E staining of C composition after 6 days of in vitro culture, stereo: 100 μm; H&E: 50 μm (40x) 20 μm (100x);

[0029] Fig. 9 The tooth germ mesenchymal stem cells of composition B were cultured in vitro and then recombined with epithelial cells. The recombinants were transplanted into the subrenal capsule of mice and cultured for 8 weeks. The growth of hard tissue with tooth-like morphology was observed under a stereo microscope; stereo: 100 μm; Micro CT: 5 mm. DETAILED DESCRIPTION

[0030] The present invention provides a pharmaceutical composition for in vitro culture of dental mesenchymal stem cells, comprising epidermal growth factor EGF+basic fibroblast growth factor bFGF+smoothened receptor agonist SAG;

[0031] Or epidermal growth factor EGF + smoothened receptor agonist SAG + CHIR99021;

[0032] Or epidermal growth factor EGF + basic fibroblast growth factor bFGF + fibroblast growth factor FGF3 + CHIR99021.

[0033] In the present invention, when the pharmaceutical composition includes epidermal growth factor EGF+basic fibroblast growth factor bFGF+smoothened receptor agonist SAG; the concentration of epidermal growth factor EGF is preferably 15-25ng / ml, more preferably 18-22ng / ml, and further preferably 20ng / ml; the concentration of basic fibroblast growth factor bFGF is preferably 15-25ng / ml, more preferably 18-22ng / ml, and further preferably 20ng / ml; the concentration of smoothened receptor agonist SAG is preferably 0.4-0.6ng / ml, more preferably 0.45-0.55ng / ml, and further preferably 0.5ng / ml.

[0034] In the present invention, when the pharmaceutical composition includes epidermal growth factor EGF+smoothened receptor agonist SAG+CHIR99021, the concentration of epidermal growth factor EGF is preferably 15-25 ng / ml, more preferably 18-22 ng / ml, and further preferably 20 ng / ml; the concentration of smoothened receptor agonist SAG is preferably 0.4-0.6 ng / ml, more preferably 0.45-0.55 ng / ml, and further preferably 0.5 ng / ml; the concentration of CHIR99021 is preferably 2.5-3.5 ng / ml, more preferably 2.7-3.3 ng / ml, and further preferably 3.0 ng / ml.

[0035] In the present invention, when the pharmaceutical composition includes epidermal growth factor EGF+basic fibroblast growth factor bFGF+fibroblast growth factor FGF3+CHIR99021, the concentration of epidermal growth factor EGF is preferably 15-25 ng / ml, more preferably 18-22 ng / ml, and further preferably 20 ng / ml; the concentration of basic fibroblast growth factor bFGF is preferably 15-25 ng / ml, more preferably 18-22 ng / ml, and further preferably 20 ng / ml; the concentration of fibroblast growth factor FGF3 is preferably 15-25 ng / ml, more preferably 18-22 ng / ml, and further preferably 20 ng / ml; the concentration of CHIR99021 is preferably 2.5-3.5 ng / ml, more preferably 2.7-3.3 ng / ml, and further preferably 3.0 ng / ml.

[0036] The invention provides a culture medium for in vitro culture of dental mesenchymal stem cells, comprising the pharmaceutical composition and a basic culture medium.

[0037] In the present invention, the basal culture medium is preferably DMEM / F12+10% KSR; the concentration of each component in the pharmaceutical composition is the final concentration in the culture medium.

[0038] The present invention also provides a method for culturing tooth germ organoids in vitro, comprising the following steps: 1) isolating tooth germ mesenchymal stem cells, and culturing the tooth germ mesenchymal stem cells in the culture medium for 45 to 75 hours; 2) recombining the cultured tooth germ mesenchymal stem cells, and continuing to culture to obtain tooth germ organoids.

[0039] In the present invention, tooth germ mesenchymal stem cells are isolated, and the tooth germ mesenchymal stem cells are isolated from ICR pregnant mice. After the tooth germ of the first molar of the ICR pregnant mice is isolated, the tooth germ is digested, preferably treated with dispase, the tooth germ epithelium is separated from the mesenchymal tissue, the separated mesenchymal tissue is collected, and then the mesenchymal tissue is digested with a mixed digestive enzyme of dispase, collagenase and pancreatin at 37°, and filtered through a 40μm cell sieve to obtain single cells. Then the tooth germ mesenchymal stem cells are cultured in the culture medium for 48 to 72 hours to obtain proliferated tooth germ mesenchymal stem cells that maintain odontogenic potential in vitro.

[0040] In the present invention, the cultured tooth germ mesenchymal stem cells are recombined with dental epithelial cells and then further cultured to obtain tooth germ organoids. In the present invention, the time for further culture is preferably 1 to 6 days.

[0041] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0042] Example 1

[0043] Materials and Methods

[0044] Experimental materials: 14.5-day-old ICR pregnant mice

[0045] Experimental methods:

[0046] ICR mice at 14.5 days of gestation were dissected with surgical scissors, the embryos were taken out, the head and neck of the embryos were separated, the mandibular arch was separated from the head of the fetal mouse, the first molar tooth germ was separated with a fine needle, and the tooth germ was treated with 10 mg / ml dispase at room temperature for 12 minutes to separate the tooth germ epithelium and mesenchymal tissue, and the separated mesenchymal tissue was collected into ep tubes, and the mesenchymal tissue was digested with a digestive enzyme mixed with dispase (4 mg / ml), collagenase (3 mg / ml) and pancreatin (0.05%) at 37° for 3 minutes, and filtered through a 40 μm cell sieve to obtain single cells.

[0047] Using different growth factors and small molecule drugs to culture dental mesenchymal stem cells in vitro, the culture time can be extended to 72 hours. Then, freshly isolated epithelial tissue and stimulated mesenchymal stem cells cultured in vitro were reconstituted and cultured in vitro, divided into a negative control group (DMSO) and a drug-induced group, and verified in vitro by morphological analysis and HE staining, proving the in vitro odontogenic potential of mesenchymal stem cells under the regulation of drug combination.

[0048] Experimental results and analysis

[0049] 1. Morphological characteristics of E14.5 fetal mice and tooth germs

[0050] The tooth germ of E14.5 fetal mouse under stereo microscope is translucent and 3D long spindle-shaped. Figure 1 As shown in the figure, the first molar tooth germ of a pregnant mouse at day E14.5. After the tooth germ is digested, the epithelium and mesenchyme are naturally separated. The epithelium is a translucent cap-shaped structure, and the mesenchyme is a semicircular tissue below, which is light pink and contains blood-like tissue. The volume of the mesenchyme is larger than that of the epithelium. Figure 2 shown.

[0051] 2. After 48 hours of in vitro culture, tooth germ mesenchymal stem cells were reconstituted without further development.

[0052] The isolated tooth germ mesenchymal stem cells were stimulated with negative control DMSO, cultured in DMEM / F-12+10% KSR containing 0.1% DMSO for 48 hours, and then recombined with epithelial cells. Figure 3As shown, after 48 hours of in vitro culture, tooth germ mesenchymal stem cells were reorganized and no further development was observed. It can be seen that after 48 hours of in vitro culture, tooth germ mesenchymal stem cells were reorganized and organoids were cultured in vitro for 6 days without organoid development.

[0053] 3. Changes in cell morphology and proliferation after drug stimulation

[0054] Single drug treatment: Five small molecule drugs, EGF, bFGF, FGF3, SAG, and CHIR99021, were used to act on tooth germ mesenchymal stem cells and cultured in vitro for 24h, 48h, and 72h. The results showed that the number of cells gradually increased with the extension of culture time, and the cell density reached almost 100% at 72h.

[0055] The culture medium was DMEM / F-12 medium containing 10% KSR;

[0056] When treated with single drugs, the concentrations of EGF, bFGF, FGF3, SAG and CHIR99021 in the culture medium were 20 ng / ml, 20 ng / ml, 20 ng / ml, 0.5 ng / ml and 3 ng / ml, respectively.

[0057] The results are as follows Figure 4 As shown, small molecule drugs had no significant effect on cell proliferation within 24 hours. As the culture time prolonged, the number of cells gradually increased, reaching a maximum OD of 0.4 at 48 hours, and began to decline after 48 hours.

[0058] The small molecule drug composition treatment groups were divided into group A, group B and group C. The culture medium was DMEM / F-12 culture medium containing 10% KSR. The concentration of the drug in the culture medium was as follows:

[0059] Group A: EGF 20ng / ml+bFGF 20ng / ml+SAG 0.5ng / ml;

[0060] Group B: EGF 20ng / ml+SAG 0.5ng / ml+CHIR990213ng / ml;

[0061] Group C: EGF 20ng / ml+bFGF 20ng / ml+FGF320ng / ml+CHIR990213ng / ml

[0062] Every 10 4 Add 100 μl culture medium per cell;

[0063] Cell proliferation ability test was performed, and the results were as follows Figure 5As shown, small molecule drugs had no significant effect on cell proliferation within 24 hours. As the culture time prolonged, the cell number gradually increased, doubling in 72 hours, with the highest OD reaching about 0.6. Moreover, at 72 hours, the proliferation effect of small molecule drug group C was more significant than that of groups A and B, reaching a peak.

[0064] 4. Characteristics and histological morphology of tooth germ organoids cultured in vitro

[0065] After small molecule drugs were used to treat tooth germ mesenchymal cells, they were recombined with dental epithelial tissue (10 4 The organoids were cultured in an in vitro incubator for 5-7 days. It was observed that the surrounding mesenchymal stem cells continued to gather in the inner enamel epithelium of the epithelial tissue, the mesenchymal stem cells and the epithelial tissue continued to develop and differentiate, and the inner and outer enamel epithelium of the epithelial tissue continued to extend downward to form a cap-like structure.

[0066] 4.1 Morphological changes of tooth germ organoids formed by the action of small molecule drugs on mesenchymal stem cells in vitro for 72 hours

[0067] like Figure 6 As shown, mesenchymal stem cells were cultured in group A culture medium containing EGF, bFGF, and SAG for 72 hours, and then recombined with E14.5 mouse dental epithelium. After 6 days of in vitro culture, paraffin sections and H&E staining were performed. The results showed that they developed into bioengineered teeth with tooth morphology and structure in vitro. This result indicates that group A small molecule drugs can maintain the in vitro odontogenic potential of E14.5 mesenchymal stem cells for up to 72 hours in vitro, and promote the in vitro development of epithelial and mesenchymal stem cell recombinants into bioengineered teeth.

[0068] like Figure 7 As shown, tooth germ mesenchymal stem cells were cultured in group B culture medium containing EGF, SAG, and CHIR99021 for 72 hours, and then recombined with E14.5 mouse dental epithelium. After 6 days of in vitro culture, paraffin sections and H&E staining were performed. The results showed that they developed into bioengineered teeth with tooth morphology and structure in vitro. The results showed that group B small molecule drugs can maintain the in vitro odontogenic potential of E14.5 mesenchymal stem cells for up to 72 hours in vitro, and promote the in vitro development of epithelial and mesenchymal stem cell recombinants into bioengineered teeth.

[0069] like Figure 8As shown, mesenchymal stem cells were cultured in a medium containing four components, EGF, bFGF, FGF3, and CHIR99021, for 72 hours, and then recombined with E14.5 mouse dental epithelium. After 6 days of in vitro culture, paraffin sections and H&E staining were performed. The results showed that they developed into bioengineered teeth with tooth morphology and structure in vitro. The results showed that the small molecule drugs in group C can maintain the odontogenic potential of E14.5 mesenchymal stem cells in vitro for up to 72 hours, and promote the development of epithelial and mesenchymal stem cell recombinants into bioengineered teeth in vitro

[0070] 4.2 Validation of in vitro organoid culture system by in vivo transplantation

[0071] Organoid morphology formed by in vitro reconstitution and in vivo transplantation of mouse molar mesenchymal stem cells under the action of small molecule drugs

[0072] like Fig. 9 As shown, tooth germ mesenchymal stem cells were cultured in type B culture medium containing EGF, SAG, and CHIR99021 for 48 hours and then recombined with E14.5 mouse dental epithelium. After 6 days of in vitro recombinant culture, the recombinant was surgically transplanted under the mouse renal capsule and cultured for 8 weeks. The growth of hard tissue with tooth-like morphology was observed under a stereomicroscope, and micro-ct analysis and HE staining results confirmed the formation of tooth structure, confirming that the small molecule drug combination EGF, SAG, and CHIR99021 can not only help prolong the in vitro culture time of tooth germ mesenchymal stem cells, but also maintain the in vitro odontogenic potential of tooth germ mesenchymal stem cells.

[0073] It can be seen from the above examples that the small molecule drug composition provided by the present invention can maintain and promote the in vitro odontogenic potential of tooth germ mesenchymal stem cells and establish a more stable in vitro tooth organoid culture system.

[0074] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A pharmaceutical composition for in vitro culture of dental mesenchymal stem cells, characterized in that: Including epidermal growth factor EGF + basic fibroblast growth factor bFGF + smooth receptor agonist SAG; Or epidermal growth factor EGF + smoothened receptor agonist SAG + CHIR99021; Or epidermal growth factor EGF + basic fibroblast growth factor bFGF + fibroblast growth factor FGF3 + CHIR99021.

2. The pharmaceutical composition according to claim 1, characterized in that When the pharmaceutical composition includes epidermal growth factor EGF+basic fibroblast growth factor bFGF+smoothened receptor agonist SAG; the concentration of epidermal growth factor EGF is 15-25 ng / ml, the concentration of basic fibroblast growth factor bFGF is 15-25 ng / ml, and the concentration of smoothened receptor agonist SAG is 0.4-0.6 ng / ml.

3. The pharmaceutical composition according to claim 1, characterized in that When the pharmaceutical composition includes epidermal growth factor EGF+smoothened receptor agonist SAG+CHIR99021, the concentration of epidermal growth factor EGF is 15-25 ng / ml, the concentration of smoothened receptor agonist SAG is 0.4-0.6 ng / ml, and the concentration of CHIR99021 is 2.5-3.5 ng / ml.

4. The pharmaceutical composition according to claim 1, characterized in that When the pharmaceutical composition includes epidermal growth factor EGF+basic fibroblast growth factor bFGF+fibroblast growth factor FGF3+CHIR99021, the concentration of epidermal growth factor EGF is 15-25 ng / ml, the concentration of basic fibroblast growth factor bFGF is 15-25 ng / ml, the concentration of fibroblast growth factor FGF3 is 15-25 ng / ml, and the concentration of CHIR99021 is 2.5-3.5 ng / ml.

5. A culture medium for in vitro culture of dental mesenchymal stem cells, characterized in that: The invention comprises the pharmaceutical composition according to any one of claims 1 to 4 and a basic culture medium.

6. The culture medium according to claim 5, characterized in that The basic culture medium is DMEM / F12+10% KSR.

7. A method for culturing tooth germ organoids in vitro, characterized in that: The following steps are involved: 1) isolating tooth germ mesenchymal stem cells, and culturing the tooth germ mesenchymal stem cells in the culture medium of claim 5 or 6 for 48 to 72 hours; 2) After the cultured tooth germ mesenchymal stem cells are recombined with the dental epithelial tissue, they are further cultured to obtain tooth germ organoids.

8. The method according to claim 7, characterized in that The time of continuing cultivation in step 2) is 1 to 6 days.

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