Totipotent stem cell culture solution containing maternal factors

Abstract

The invention relates to the field of biomedicine, in particular to a totipotent stem cell culture solution containing maternal factors. The maternal factor and the pomegranate seed active peptide extract are jointly used for preparing the totipotent stem cell culture solution for the first time, the maternal factor in the culture solution is selected from a combination of TPRX1, TPRXL, miR-302 and IL1alpha, verification analysis shows that the combination has a synergistic effect, expression of an Oct3 / 4 gene and the cell colony number in the totipotent stem cell can be effectively improved, and the total cell colony number of the totipotent stem cell can be effectively increased. According to the present invention, with the application of the totipotent stem cell culture solution, the induction ability of the somatic cells to the totipotent stem cells can be further improved so as to induce the generation of the totipotent stem cells on a large scale, and the totipotent stem cell culture solution can be adopted as the fibroblast proliferation promoting culture solution so as to effectively promote the proliferation of the fibroblasts, such that the application in the wound healing aspect is easily achieved.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a pluripotent stem cell culture medium containing maternal factors. Background Technology

[0002] During animal fertilization, sperm mainly release the male pronucleus into the egg. In the zygote formed, the female and male pronuclei fuse to form the zygote nucleus. However, the genome of the fertilized egg is not transcribed during the first few mitotic divisions. A reasonable logical assumption is that its early development depends entirely on the RNA and proteins stored in the egg plasma, i.e., maternal factors.

[0003] Recent research has used a four-factor expression cassette (Hsf1, Zar1, Padi6, and Npm2) to directly convert mouse or human somatic cells into 2C-like pluripotent stem cells (MFi2CLTSCs) in vitro, expressing 2C markers such as MERVL and Zscan4, and enabling them to form blastocysts and chimeric embryos. This strategy avoids the drawbacks of iPSCs (tumor risk), SCNTs (oocyte source limitation), and EPSCs (insufficient small molecule specificity), providing a highly efficient and controllable new approach for regenerative medicine, endangered species conservation, and the preparation of oocyte-free pluripotent cells. However, how to effectively improve the induction efficiency of pluripotent stem cells using maternal factors remains an urgent problem to be solved. Summary of the Invention

[0004] To address the technical problems existing in the prior art, this invention provides a pluripotent stem cell culture medium containing maternal factors. This invention is the first to combine maternal factors with pomegranate seed active peptide extract to prepare a pluripotent stem cell culture medium. The maternal factors in the culture medium are selected from a combination of TPRX1, TPRXL, miR-302, and IL1α. Verification analysis shows that this combination has a synergistic effect, effectively increasing the expression of the Oct3 / 4 gene and the number of cell colonies in pluripotent stem cells, further enhancing the ability of somatic cells to induce pluripotent stem cells, thereby enabling the large-scale induction of pluripotent stem cell production. Simultaneously, this invention utilizes the pluripotent stem cell culture medium as a fibroblast proliferation-promoting culture medium, effectively promoting fibroblast proliferation, which is beneficial for applications in wound healing.

[0005] Specifically, this invention first provides a pluripotent stem cell culture medium containing maternal factors, comprising the following components: 10% (v / v) FBS, 1-3 mM TPRX1, 1-3 mM TPRXL, 0.5-1.5 mM miR-302, 1-2 mM IL1α, 0.05 mM β-mercaptoethanol, 0.1-3 mM pomegranate seed active peptide extract, 100 u / L penicillin, 100 u / L streptomycin, and the balance being RPMI-1640 culture medium. The above substances are prepared into a pluripotent stem cell culture medium according to conventional preparation methods. Before use, the culture medium should be preheated to 37°C.

[0006] Preferably, the pomegranate seed active peptide extract is the active polypeptide shown in SEQ ID NO.1.

[0007] Preferably, the separation method of the pomegranate seed active peptide extract includes the following steps: after physically crushing the pomegranate seeds, distilled water is added at a mass ratio of 1:(10-20), and the mixture is heated at 50-60℃ for 3-5 hours. Then, bromelain is added at a mass ratio of 100:(5-9) for enzymatic hydrolysis for 6-8 hours. Subsequently, an equal amount of papain is added to the hydrolysate for a second enzymatic hydrolysis treatment for 12-18 hours. After the enzymatic hydrolysis is completed, ultrafiltration is performed, and the active peptides in the hydrolysate are separated by HPLC-MS and identified.

[0008] Preferably, the pluripotent stem cell culture medium containing maternal factors comprises the following components: 10% (v / v) FBS, 1 mM TPRX1, 1 mM TPRXL, 0.5 mM miR-302, 1 mM IL1α, 0.05 mM β-mercaptoethanol, 0.1 mM pomegranate seed active peptide extract, 100 u / L penicillin, 100 u / L streptomycin, and the balance being RPMI-1640 culture medium. The above substances are prepared into a pluripotent stem cell culture medium according to conventional preparation methods. Before use, the culture medium should be preheated to 37°C.

[0009] Preferably, the pluripotent stem cell culture medium containing maternal factors comprises the following components: 10% (v / v) FBS, 2mM TPRX1, 2mM TPRXL, 1.0mM miR-302, 1.5mM IL1α, 0.1mM β-mercaptoethanol, 0.2mM pomegranate seed active peptide extract, 100u / L penicillin, 100u / L streptomycin, and the balance being RPMI-1640 culture medium. The above substances are prepared into a pluripotent stem cell culture medium according to conventional preparation methods. Before use, the culture medium should be preheated to 37°C.

[0010] Preferably, the pluripotent stem cell culture medium containing maternal factors comprises the following components: 10% (v / v) FBS, 3mM TPRX1, 3mM TPRXL, 1.5mM miR-302, 2.0mM IL1α, 0.15mM β-mercaptoethanol, 0.3mM pomegranate seed active peptide extract, 100u / L penicillin, 100u / L streptomycin, and the balance being RPMI-1640 culture medium. The above substances are prepared into a pluripotent stem cell culture medium according to conventional preparation methods. Before use, the culture medium should be preheated to 37°C.

[0011] Furthermore, the present invention also provides the use of maternal factors in the preparation of pluripotent stem cell culture medium, wherein the maternal factors are selected from one or more of TPRX1, TPRXL, miR-302 and IL1α.

[0012] Furthermore, the present invention also provides the use of pomegranate seed active peptide extract in the preparation of pluripotent stem cell culture medium, wherein the pomegranate seed active peptide extract is the active polypeptide shown in SEQ ID NO.1.

[0013] Furthermore, the present invention also provides the use of maternal factors combined with pomegranate seed active peptide extract in the preparation of pluripotent stem cell culture medium, wherein the maternal factors are selected from any one or more of TPRX1, TPRXL, miR-302 and IL1α; and the pomegranate seed active peptide extract is the active polypeptide shown in SEQ ID NO.1.

[0014] The advantages of this invention are as follows: This invention is the first to use maternal factors in combination with pomegranate seed active peptide extract to prepare pluripotent stem cell culture medium. The maternal factors in the culture medium are selected from a combination of TPRX1, TPRXL, miR-302 and IL1α. Verification analysis shows that the above combination has a synergistic effect, which can effectively improve the expression of Oct3 / 4 genes and the number of cell colonies in pluripotent stem cells, further enhancing the ability of somatic cells to induce pluripotent stem cells, thereby enabling the large-scale induction of pluripotent stem cells. At the same time, this invention utilizes the pluripotent stem cell culture medium as a fibroblast proliferation-promoting culture medium, which can effectively promote fibroblast proliferation and is beneficial for applications in wound healing. Detailed Implementation

[0015] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0016] The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the specific embodiments of the invention without inventive effort are within the protection scope of the invention.

[0017] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well known to those skilled in the art.

[0018] All components of the inducing agent of the present invention are commercially available products: RPMI-1640, FBS, β-mercaptoethanol, TPRX1, TPRXL, miR-302 and IL1α, penicillin and streptomycin are all commercially available. Example 1

[0019] A pluripotent stem cell culture medium containing maternal factors comprises the following components: 10% (v / v) FBS, 1 mM TPRX1, 1 mM TPRXL, 0.5 mM miR-302, 1 mM IL1α, 0.05 mM β-mercaptoethanol, 0.1 mM pomegranate seed active peptide extract, 100 u / L penicillin, 100 u / L streptomycin, and the balance being RPMI-1640 culture medium. The above substances are prepared into a pluripotent stem cell culture medium according to conventional preparation methods. Before use, the culture medium should be preheated to 37°C.

[0020] The pomegranate seed active peptide extract is the active polypeptide shown in SEQ ID NO.1. The separation method of this pomegranate seed active peptide extract includes the following steps: After physically pulverizing pomegranate seeds, distilled water is added at a mass ratio of pulverized material to distilled water of 1:10. The mixture is then heated at 50°C for 3 hours. Bromelain (enzyme parameter 10,000 u / g) is added at a mass ratio of hydrolysate to bromelain of 100:5 for enzymatic hydrolysis for 6 hours. Subsequently, papain (enzyme parameter 10,000 u / g) is added to the hydrolysate in an equal amount to bromelain for a second enzymatic hydrolysis treatment of 12 hours. After hydrolysis, ultrafiltration is performed, and the active peptides in the hydrolysate are separated using HPLC-MS. The pomegranate seed active peptide extract shown in SEQ ID NO.1 is obtained and used for future reference. Example 2

[0021] A pluripotent stem cell culture medium containing maternal factors comprises the following components: 10% (v / v) FBS, 2 mM TPRX1, 2 mM TPRXL, 1.0 mM miR-302, 1.5 mM IL1α, 0.1 mM β-mercaptoethanol, 0.2 mM pomegranate seed active peptide extract, 100 u / L penicillin, 100 u / L streptomycin, and the balance being RPMI-1640 culture medium. The above substances are prepared into a pluripotent stem cell culture medium according to conventional preparation methods. Before use, the culture medium should be preheated to 37°C. The pomegranate seed active peptide extract is the pomegranate seed active peptide extract prepared in Example 1 as shown in SEQ ID NO. 1. Example 3

[0022] A pluripotent stem cell culture medium containing maternal factors comprises the following components: 10% (v / v) FBS, 3mM TPRX1, 3mM TPRXL, 1.5mM miR-302, 2.0mM IL1α, 0.15mM β-mercaptoethanol, 0.3mM pomegranate seed active peptide extract, 100u / L penicillin, 100u / L streptomycin, and the balance being RPMI-1640 culture medium. The above substances are prepared into a pluripotent stem cell culture medium according to conventional preparation methods. Before use, the culture medium should be preheated to 37°C. The pomegranate seed active peptide extract is the pomegranate seed active peptide extract prepared in Example 1 as shown in SEQ ID NO. 1. Comparative Example 1

[0023] A pluripotent stem cell culture medium comprises the following components: 10% (v / v) FBS, 3.5 mM TPRX1, 0.05 mM β-mercaptoethanol, 100 u / L penicillin, 100 u / L streptomycin, and the balance being RPMI-1640 culture medium. The above substances are prepared into a pluripotent stem cell culture medium according to conventional preparation methods. Before use, the culture medium should be preheated to 37°C. Comparative Example 2

[0024] Compared to Comparative Example 1, 3.5mM TPRXL was used instead of 3.5mM TPRX1. Comparative Example 3

[0025] Compared to Comparative Example 1, 3.5mM miR-302 was used instead of 3.5mM TPRX1. Comparative Example 4

[0026] Compared to Comparative Example 1, 3.5 mM IL1α was used instead of 3.5 mM TPRX1. Comparative Example 5

[0027] Compared to Comparative Example 1, it also contains 0.1 mM pomegranate seed active peptide extract. Example 4

[0028] A method for culturing pluripotent stem cells, comprising the following steps: 1) Take 1×10 4 Cell human neonatal dermal fibroblasts (Catalog #2310, purchased from Zhongqiao Xinzhou Biotechnology); 2) The above-mentioned human neonatal dermal fibroblasts were seeded into the pluripotent stem cell culture medium of the above-mentioned different embodiments and comparative examples, and cultured continuously for 7 days, with fresh culture medium replaced every 1 day. 3) Centrifuge to collect cells cultured in step 2), and use immunohistochemistry to detect the Oct3 / 4 positive rate in the cells, and use alkaline phosphatase staining to count the number of cell colonies.

[0029] The blank control group used RPMI-1640 culture medium. The statistical results are shown in Table 1 below: Table 1: Test Results Blank group Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Positive rate (%) on October 3 / 4 0 12 15 20 4 4 5 2 8 Cell colony count 0 120 136 152 42 45 56 25 98 The results above confirm that the maternal factors TPRX1, TPRXL, miR-302 and IL1α can effectively increase the expression of Oct3 / 4 genes and the number of cell colonies in stem cells. Furthermore, when combined with pomegranate seed active peptide extract, they can further enhance the ability of somatic cells to be pluripotent stem cells, thereby inducing the generation of pluripotent stem cells. Example 5

[0030] Effects of pluripotent stem cell culture medium containing maternal factors on fibroblast proliferation: The effect of pluripotent stem cell culture medium containing maternal factors on the proliferation of HFF-1 fibroblasts was detected using the MTT cell proliferation assay kit. Specific assay procedures can be routinely adjusted according to the kit instructions.

[0031] The blank group consisted of RPMI-1640 culture medium, experimental groups 1-3 each contained the pluripotent stem cell culture medium containing maternal factors prepared in Examples 1-3, and control groups 1-5 each contained the pluripotent stem cell culture medium described in Comparative Examples 1-5. The cell volume in both experimental and control groups was 1×10⁻⁶. 3 indivual.

[0032] Table 2. Analysis of fibroblast proliferation Group OD value Blank group 0.175±0.012 Experimental group 1 0.543±0.025 Experimental group 2 0.596±0.014 Experimental group 3 0.658±0.031 Control group 1 0.198±0.012 Control group 2 0.209±0.032 Control group 3 0.212±0.021 Control group 4 0.246±0.014 Control group 5 0.319±0.011 The test results are shown in Table 2: The pluripotent stem cell culture medium containing maternal factors obtained using any one of the experimental groups 1-3 of this invention efficiently promoted the proliferation of fibroblasts, and its proliferation-promoting effect was significantly better than that of the control groups 1-5 (P<0.05). This further confirms that the combination of maternal factors and pomegranate seed active peptide extract in this invention can serve as a fibroblast proliferation-promoting culture medium, effectively promoting wound healing.

[0033] It should be noted that the above embodiments are only for further elaboration and explanation of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention. The method of the present invention is only a preferred embodiment and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A pluripotent stem cell culture medium containing maternal factors, characterized in that, The composition includes the following components: 10% (v / v) FBS, 1-3mM TPRX1, 1-3mM TPRXL, 0.5-1.5mM miR-302, 1-2mM IL1α, 0.05mM β-mercaptoethanol, 0.1-3mM pomegranate seed active peptide extract, 100u / L penicillin, 100u / L streptomycin, and the balance of RPMI-1640 culture medium.

2. The pluripotent stem cell culture medium as described in claim 1, characterized in that, The pomegranate seed active peptide extract is the active polypeptide shown in SEQ ID NO.

1.

3. The pluripotent stem cell culture medium as described in claim 2, characterized in that, The separation method of the pomegranate seed active peptide extract includes the following steps: after physically crushing the pomegranate seeds, distilled water is added at a mass ratio of 1:(10-20), and the mixture is heated at 50-60℃ for 3-5 hours. Then, bromelain is added at a mass ratio of 100:(5-9) for enzymatic hydrolysis for 6-8 hours. Subsequently, an equal amount of papain is added to the hydrolysate for a second enzymatic hydrolysis treatment for 12-18 hours. After the enzymatic hydrolysis is completed, ultrafiltration is performed, and the active peptides in the hydrolysate are separated by HPLC-MS and identified.

4. The pluripotent stem cell culture medium according to any one of claims 1-3, characterized in that, The pluripotent stem cell culture medium containing maternal factors comprises the following components: 10% (v / v) FBS, 1mM TPRX1, 1mM TPRXL, 0.5mM miR-302, 1mM IL1α, 0.05mM β-mercaptoethanol. 0.1 mM pomegranate seed active peptide extract, 100 u / L penicillin, 100 u / L streptomycin, and the remainder of RPMI-1640 culture medium.

5. The pluripotent stem cell culture medium according to any one of claims 1-3, characterized in that, The pluripotent stem cell culture medium containing maternal factors comprises the following components: 10% (v / v) FBS, 2mM TPRX1, 2mM TPRXL, 1.0mM miR-302, 1.5mM IL1α, 0.1mM β-mercaptoethanol, 0.2mM pomegranate seed active peptide extract, 100u / L penicillin, 100u / L streptomycin, and the remainder of RPMI-1640 culture medium.

6. The pluripotent stem cell culture medium according to any one of claims 1-3, characterized in that, The pluripotent stem cell culture medium containing maternal factors comprises the following components: 10% (v / v) FBS, 3mM TPRX1, 3mM TPRXL, 1.5mM miR-302, 2.0mM IL1α, 0.15mM β-mercaptoethanol, 0.3mM pomegranate seed active peptide extract, 100u / L penicillin, 100u / L streptomycin, and the remainder of RPMI-1640 culture medium.

7. The use of maternal factors in the preparation of pluripotent stem cell culture medium, characterized in that, The maternal factor is selected from one or more of TPRX1, TPRXL, miR-302 and IL1α.

8. The use as described in claim 7, characterized in that, The maternal factors selected are a combination of TPRX1, TPRXL, miR-302 and IL1α.

9. The use of pomegranate seed active peptide extract in the preparation of pluripotent stem cell culture medium, characterized in that, The pomegranate seed active peptide extract is the active polypeptide shown in SEQ ID NO.

1.

10. The use of maternal factors combined with pomegranate seed active peptide extract in the preparation of pluripotent stem cell culture medium, characterized in that, The maternal source factor is a combination of TPRX1, TPRXL, miR-302 and IL1α; the pomegranate seed active peptide extract is the active polypeptide shown in SEQ ID NO.1.