Application of miRNA in regulation and control of cell reprogramming

Abstract

The invention relates to application of miRNA (micro Ribonucleic Acid) in regulation and control of cell reprogramming. It is found for the first time that a mixture of three miRNAs of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p can induce fibroblasts to be reprogrammed into induced pluripotent stem cells, the three miRNAs have a synergistic effect, the statistical number of positive iPSCs colonies under combined transfection is obviously increased, and the expression quantity of Oct4, Sox2, c-Myc and Klf4 transcription factors generated through induction is also obviously increased. The reprogramming effect of the mixture of the three miRNAs is further proved, and the mixture can be used for realizing reprogramming of somatic cells. Compared with compound-induced reprogramming, the method disclosed by the invention has the advantages that a large number of induced pluripotent stem cells can be rapidly and efficiently obtained through in-vitro induction, the reediting efficiency is far higher than that of a currently known reediting method in the prior art, and the method has a good commercial application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of cell reprogramming, and in particular to the application of miRNA in regulating cell reprogramming. Background Art

[0002] At present, reprogramming refers to the process of changing cell fate through epigenetic modifications such as DNA methylation without changing the gene sequence. It originally referred to the process of eliminating the epigenetic marks carried by its parents during the development of mammalian germ cells. Later, it was confirmed that in vitro operations of embryos such as nuclear transplantation and cell fusion can also change their original epigenetic characteristics. At present, reprogramming mainly refers to two processes: first, the process of reversing differentiated cells to restore to a omnipotent state; second, the process of transforming from one differentiated cell to another differentiated cell.

[0003] The in vitro induction of pluripotent stem cells usually uses a series of induction factors to be introduced into mature somatic cells and re-edited into a type of pluripotent stem cells with characteristics similar to embryonic stem cells. However, the induction process of this type of method is extremely slow and the success rate is extremely low, which is not conducive to industrial application. The technology of using small molecules to achieve re-editing has long been reported by Deng Hongkui's research group. Although its effective efficiency has been improved, the cell biosafety needs to be further improved. Therefore, it is necessary to focus on discovering biological molecules related to somatic cell dedifferentiation and achieve high-efficiency re-editing through fine-tuning of biological molecules. Summary of the invention

[0004] In view of the technical problems existing in the prior art, the present invention provides the application of miRNA in regulating cell reprogramming. The present invention first discovered that a mixture of three miRNAs, hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p, can induce fibroblasts to be reprogrammed into induced pluripotent stem cells, and there is a synergistic effect between the three miRNAs. The number of positive iPSCs colonies under combined transfection is significantly increased, and the expression levels of induced Oct4, Sox2, c-Myc and Klf4 transcription factors are also significantly increased. It is further confirmed that the reprogramming efficacy of the three miRNA mixtures of the present invention can be used to achieve the reprogramming of somatic cells.

[0005] One of the objects of the present invention is to provide a composition for reprogramming fibroblasts into induced pluripotent stem cells, characterized in that the composition comprises a combination of one or more of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p.

[0006] Preferably, the composition comprises a combination of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p.

[0007] Preferably, the mass ratio of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p in the composition is (1-3):(2-5):(1-9).

[0008] Further preferably, the mass ratio of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p in the composition is 1:2:1.

[0009] Further preferably, the mass ratio of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p in the composition is 2:3:4.

[0010] Further preferably, the mass ratio of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p in the composition is 3:5:9.

[0011] On the other hand, the present invention also provides a method for preparing a composition for reprogramming fibroblasts into induced pluripotent stem cells, characterized in that the method comprises the following steps: adding a miRNA mixture with a final concentration of 50-70 μg / mL to RNase-free ddH2O, wherein the composition contains hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p in a mass ratio of (1-3):(2-5):(1-9).

[0012] Preferably, the method comprises the following steps: adding a miRNA mixture with a final concentration of 50 μg / mL in RNase-free ddH2O, wherein the mass ratio of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p is 1:2:1.

[0013] Preferably, the method comprises the following steps: adding a miRNA mixture with a final concentration of 60 μg / mL in RNase-free ddH2O, wherein the mass ratio of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p is 2:3:4.

[0014] Preferably, the method comprises the following steps: adding a miRNA mixture with a final concentration of 70 μg / mL in RNase-free ddH2O, wherein the mass ratio of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p is 3:5:9.

[0015] On the other hand, the present invention also provides a method for rapidly obtaining induced pluripotent stem cells, comprising the following steps:

[0016] 1) Providing human adult cells;

[0017] 2) inoculating the human adult cells in the above-mentioned basal culture medium containing the above-mentioned miRNA mixture, culturing continuously for 7-14 days, and replacing fresh culture medium every other day;

[0018] 3) collecting the cells cultured in step 2) by centrifugation, and using flow cytometry and RT-PCR to sort and identify the gene expressions of Oct4, Sox2, c-Myc and Klf4 in the induced cells;

[0019] 4) The results of flow cytometry and RT-PCR were used to identify the induced pluripotent stem cells obtained.

[0020] Preferably, the sequences of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p are shown in SEQ ID No.1-3 respectively.

[0021] The advantages of the present invention are as follows: The present invention first discovered that a mixture of three miRNAs, hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p, can induce fibroblasts to be reprogrammed into induced pluripotent stem cells, and there is a synergistic effect between the three miRNAs. The statistical number of positive iPSCs colonies under combined transfection is significantly increased, and the expression levels of induced Oct4, Sox2, c-Myc and Klf4 transcription factors are also significantly increased. It is further confirmed that the reprogramming efficacy of the three miRNA mixtures of the present invention can be used to achieve the reprogramming of somatic cells. Compared with compound-induced reprogramming, the method of the present invention can quickly and efficiently induce a large number of induced pluripotent stem cells in vitro, and the re-editing efficiency is much higher than the re-editing methods currently known in the prior art, and has good commercial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 .Statistical number of positive iPSCs colonies induced by the miRNA mixture;

[0023] Figure 2 .Analysis of the expression of Oct4, Sox2, c-Myc and Klf4 transcription factors induced by the miRNA mixture. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0025] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0026] In the examples 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 examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0027] Example 1

[0028] A composition for reprogramming induced pluripotent stem cells from fibroblasts, characterized in that the composition comprises a mixture of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p. The preparation method of the mixture comprises the following steps: adding a miRNA mixture with a final concentration of 50 μg / mL to RNase-free ddH2O, wherein the mass ratio of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p is 1:2:1.

[0029] Example 2

[0030] A composition for reprogramming induced pluripotent stem cells from fibroblasts, characterized in that the composition comprises a mixture of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p. The preparation method of the mixture comprises the following steps: adding a miRNA mixture with a final concentration of 60 μg / mL to RNase-free ddH2O, wherein the mass ratio of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p is 2:3:4.

[0031] Example 3

[0032] A composition for reprogramming induced pluripotent stem cells from fibroblasts, characterized in that the composition comprises a mixture of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p. The preparation method of the mixture comprises the following steps: adding a miRNA mixture with a final concentration of 70 μg / mL to RNase-free ddH2O, wherein the mass ratio of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p is 3:5:9.

[0033] Comparative Example

[0034] Compared with Comparative Example 1, Comparative Examples 1-6 contain equal amounts of hsa-miR-647, hsa-miR-450b-5p or hsa-miR-875-5p as shown in the following table, and Comparative Example 7 is a combination containing equal amounts of miR302 and miR367. Among them, + represents the presence of the component, and - represents the absence of the component. The composition results of the comparative examples are shown in Table 1.

[0035] Table 1: Comparative Example Composition

[0036]

[0037] Verification Example

[0038] 1.1 Fibroblast treatment

[0039] Matrigel 354277 (Corning Inc., NY) diluted 100-fold using DMEM / F12 was added to a 6-well cell culture dish and cultured at 37°C in a 5% CO2 tissue culture incubator for 1 hour; primary human neonatal fibroblasts were plated at a density of 200 to 100,000 cells per well in a 6-well cell culture dish format. Coated culture dish; add culture medium to the 6-well cell culture dish, wherein the culture medium contains DMEM / F12, 10% heat-inactivated FBS, 0.4mM GLUTAMAX TM Supplements; incubate the plated cells overnight in a 5% CO2 tissue culture incubator.

[0040] 1.2 Transfection solution preparation

[0041] The commercially available CALNP liposome transfection reagent was used to prepare the transfection complex. 2.5 μg of the miRNA mixture in the above-mentioned examples and comparative examples was added with 15 μL of CALNP A solution and mixed; 4 μL of CALNP B solution was added, mixed, and incubated at room temperature for 15 min; and then 50 μL of reproTSER complete culture medium was added thereto for dispersion, thereby completing the configuration of the miRNA mixture for transfection in one well of a six-well plate.

[0042] 1.3 Transfection

[0043] Transfection was performed every 48 hours. The medium was replaced with ordinary DMEM + 10% FBS for fibroblast culture 24 hours after each transfection in the first 8 days, and then replaced with reproTeSR complete medium after transfection. After completing the four transfection series, the medium was replaced every day (reproTESR medium supplemented with 200ng / mL B18R). The cells were grown to day 12, the morphological changes of neonatal fibroblasts were detected, and the number of positive iPSCs colonies was counted. Statistical results are shown in Figure 1 content.

[0044] Statistical results show that under the action of Examples 1-3 of the present invention, neonatal fibroblasts can efficiently reprogram fibroblasts into induced pluripotent stem cells compared to Comparative Examples 1-7, and the number of positive iPSCs colonies is significantly higher than that of the Comparative Examples.

[0045] The expression levels of Oct4, Sox2, c-Myc and Klf4 were further detected by qPCR, and the results were statistically analyzed. Figure 2 The results showed that under the combination of the four miRNAs of the present invention, the expression levels of the four transcription factors in the transfected cells were significantly increased, suggesting that they have the potential to be reprogrammed into induced pluripotent stem cells.

[0046] The above embodiments are intended to illustrate the essential content of the present invention, but are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and protection scope of the technical solution of the present invention.

Claims

1. A composition for reprogramming fibroblasts into induced pluripotent stem cells, characterized in that: The composition includes a combination of one or more of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p.

2. The composition according to claim 1, characterized in that The composition includes a combination of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p.

3. The composition according to claim 2, characterized in that The mass ratio of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p in the composition is (1-3):(2-5):(1-9).

4. The composition according to claim 3, characterized in that The mass ratio of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p in the composition is 1:2:

1.

5. The composition according to claim 3, characterized in that The mass ratio of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p in the composition is 2:3:

4.

6. The composition according to claim 3, characterized in that The mass ratio of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p in the composition is 3:5:

9.

7. A method for preparing a composition for reprogramming fibroblasts into induced pluripotent stem cells, characterized in that: The method comprises the following steps: adding a miRNA mixture with a final concentration of 50-70 μg / mL to RNase-free ddH2O, wherein the composition contains hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p in a mass ratio of (1-3):(2-5):(1-9).

8. The method according to claim 7, characterized in that The method comprises the following steps: adding a miRNA mixture with a final concentration of 50 μg / mL into RNase-free ddH2O, wherein the mass ratio of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p in the composition is 1:2:

1.

9. The method according to claim 7, characterized in that The method comprises the following steps: adding a miRNA mixture with a final concentration of 60 μg / mL into RNase-free ddH2O, wherein the mass ratio of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p in the composition is 2:3:

4.

10. The method according to claim 7, characterized in that The method comprises the following steps: adding a miRNA mixture with a final concentration of 70 μg / mL into RNase-free ddH2O, wherein the mass ratio of hsa-miR-647, hsa-miR-450b-5p and hsa-miR-875-5p in the composition is 3:5:9.

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