Compositions and methods for cell reprogramming using circular RNA and microRNA

Abstract

The present invention provides a composition and method for cell reprogramming using circular RNA and microRNA. A composition and method for cell reprogramming using circular RNA and microRNA. The present invention combines a specific miRNA combination with circular RNA for transfection, which can simultaneously achieve transduction of reprogramming factors and regulation of target genes, improve cell reprogramming efficiency, and achieve extremely high reprogramming efficiency at low cell density.

Description

Technical Field

[0001] The present invention relates to the technical field of cell reprogramming, and in particular to a composition and method for cell reprogramming using circular RNA and microRNA. Background Art

[0002] 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] Researchers have reported reprogramming human fibroblast cell lines using circular RNA reprogramming factors, including Oct4, Sox2, Klf4, c-myc, Nanog, and Lin28 (OSKMLN), in combination with two other miRNAs, namely miR302 and miR367. These four circular RNAs of OSKMLN were synthesized in vitro using the permuted intron-exon (PIE) type I intron self-splicing system derived from the thymidylate synthase gene of T4 phage or pretRNALeu of Anabaena. The RNA circularization process after in vitro transcription requires Mg. 2+ and GTP cofactors, and the RNA products were subsequently treated with RNase-R and HPLC to remove linear mRNA and nicked circRNA. In addition, miR302 and miR367 were chemically synthesized and provided by Sangon.

[0004] Due to the poor stability of linear mRNA, multiple rounds of transfection are usually required to improve the success rate of reprogramming. For example, laboratory-made mRNA (reference: High-efficiency RNA-based reprogramming of human Primary filblasts) and mRNA reprogramming kits from ReproCELL (cat.00-0076) and Miltenyi (cat.130-132-990) usually require 6, 4-8, and 5-8 transfections. Compared with linear mRNA, circular RNA exhibits superior stability both in vivo and in vitro, so the number of transfections and the amount of RNA used can be reduced without affecting the reprogramming efficiency.

[0005] However, the current problem of induction efficiency of reprogramming induced by the combination of circular RNA and miRNA is low. Effectively improving the induction rate of induced pluripotent stem cells is the main technical obstacle faced by the industry. Summary of the invention

[0006] In view of the technical problems existing in the prior art, the present invention provides a composition and method for cell reprogramming using circular RNA and microRNA. The present invention combines a specific miRNA combination with circular RNA for co-transfection, which can simultaneously achieve transduction of reprogramming factors and regulation of target genes, improve cell reprogramming efficiency, and achieve extremely high reprogramming efficiency at low cell density.

[0007] One of the purposes of the present invention is a composition for reprogramming fibroblasts into induced pluripotent stem cells, characterized in that the composition comprises a circular RNA mixture and a miRNA mixture.

[0008] Preferably, the circularRNA mixture includes exogenous circular RNA molecules of OCT4, SOX2, KLF4, c-MYC, NANOG and LIN28A.

[0009] Preferably, the miRNA mixture includes a combination of hsa-miR-17-5p / hsa-miR-23a-3p, hsa-miR-17-5p / hsa-miR-144-3p, and hsa-miR-17-5p / hsa-miR-340-3p.

[0010] Preferably, the circular RNA mixture is prepared by preparing the circular RNA mixture at 1000 ng / μl in RNase-free ddH2O, wherein the molar stoichiometry of OCT4 and the other five factors is 3:1:1:1:1:1.

[0011] Preferably, the miRNA mixture is prepared by dissolving the lyophilized miRNA in RNase-free ddH2O to a final concentration of 5 μM. The stock solution is frozen at -80°C until further use. The individual microRNA stock solutions are mixed in a 1:1 ratio to prepare a 5 μM miRNA transfection mixture.

[0012] Further preferably, the composition comprises a circular RNA mixture and an hsa-miR-17-5p / hsa-miR-23a-3p mixture.

[0013] Further preferably, the composition comprises a circular RNA mixture and an hsa-miR-17-5p / hsa-miR-144-3p mixture.

[0014] Further preferably, the composition comprises a circular RNA mixture and an hsa-miR-17-5p / hsa-miR-340-3p mixture.

[0015] Another object of the present invention is to provide a method for preparing induced pluripotent stem cells (iPSCs) using the above composition, the method comprising the following steps:

[0016] 1) contacting the above composition with target cells;

[0017] 2) Cultivating target cells under conditions that can induce cell reprogramming, including appropriate culture medium, temperature, gas, hormones, growth factors, cytokines, compounds, etc.;

[0018] 3) Isolation and characterization of induced pluripotent stem cells (iPSCs) from cultured target cells.

[0019] Preferably, the target cells are primary somatic cells, such as fibroblasts, blood cells, skin cells, hepatocytes, pancreatic islet cells, kidney cells, lung cells, muscle cells, fat cells, neurons, glial cells, retinal cells, cardiomyocytes, bone cells, chondrocytes, endothelial cells, smooth muscle cells, epithelial cells, immune cells, etc.

[0020] Preferably, the conditions include appropriate culture medium, temperature, gas, hormones, growth factors, cytokines, compounds, etc.

[0021] Preferably, the identification includes detecting the morphology, phenotype, gene expression, epigenetic inheritance, karyotype, self-renewal ability, multidirectional differentiation ability, etc. of iPSCs.

[0022] The advantages of the present invention are as follows: the present invention screens and optimizes the miRNA combination, and further obtains the miRNA combination disclosed in the present invention. When it is co-transfected with circular RNA, the transduction of reprogramming factors and the regulation of target genes can be achieved simultaneously, thereby improving the reprogramming efficiency of cells and achieving extremely high reprogramming efficiency at low cell density. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 . Cell morphology analysis, wherein ae corresponds to Examples 1-3 and Comparative Examples 1-2, respectively;

[0024] Figure 2 . Statistical analysis of the number of positive iPSCs colonies;

[0025] Figure 3 .Analysis of iPSCs pluripotency indicators. DETAILED DESCRIPTION

[0026] 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.

[0027] 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.

[0028] 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.

[0029] Example 1

[0030] A composition for reprogramming fibroblasts into induced pluripotent stem cells, characterized in that the composition comprises a circular RNA mixture and an hsa-miR-17-5p / hsa-miR-23a-3p mixture.

[0031] The circularRNA mixture is prepared by preparing the transcripts of six reprogramming factors (OCT4, SOX2, KLF4, c-MYC, NANOG and LIN28A (abbreviated as "OSKMNL") into circularRNA. The specific preparation method of circularRNA can refer to the steps of the construction method of exogenous circular RNA molecules disclosed in CN201410604749.7.

[0032] The circular RNA mixture is prepared by preparing the circular RNA mixture at 1000 ng / μl in RNase-free ddH2O, wherein the molar stoichiometry of OCT4 and the other five factors is 3:1:1:1:1:1.

[0033] The hsa-miR-17-5p / hsa-miR-23a-3p mixture was prepared by dissolving the lyophilized microRNA in RNase free ddH2O to a final concentration of 5 μM. The stock solution was frozen at -80°C until further use. The individual microRNA stock solutions were mixed in a 1:1 ratio to prepare a 5 μM miRNA transfection mixture.

[0034] Example 2

[0035] A composition for reprogramming fibroblasts into induced pluripotent stem cells, characterized in that the composition comprises a circular RNA mixture and an hsa-miR-17-5p / hsa-miR-144-3p mixture.

[0036] The circularRNA mixture is prepared by preparing the transcripts of six reprogramming factors (OCT4, SOX2, KLF4, c-MYC, NANOG and LIN28A (abbreviated as "OSKMNL") into circularRNA. The specific preparation method of circularRNA can refer to the steps of the construction method of exogenous circular RNA molecules disclosed in CN201410604749.7.

[0037] The circular RNA mixture is prepared by preparing the circular RNA mixture at 1000 ng / μl in RNase-free ddH2O, wherein the molar stoichiometry of OCT4 and the other five factors is 3:1:1:1:1:1.

[0038] The hsa-miR-17-5p / hsa-miR-144-3p mixture was prepared by dissolving the lyophilized microRNA in RNase-free ddH2O to a final concentration of 5 μM. The stock solution was frozen at -80°C until further use. The individual microRNA stock solutions were mixed in a 1:1 ratio to prepare a 5 μM miRNA transfection mixture.

[0039] Example 3

[0040] A composition for reprogramming fibroblasts into induced pluripotent stem cells, characterized in that the composition comprises a circular RNA mixture and an hsa-miR-17-5p / hsa-miR-340-3p mixture.

[0041] The circularRNA mixture is prepared by preparing the transcripts of six reprogramming factors (OCT4, SOX2, KLF4, c-MYC, NANOG and LIN28A (abbreviated as "OSKMNL") into circularRNA. The specific preparation method of circularRNA can refer to the steps of the construction method of exogenous circular RNA molecules disclosed in CN201410604749.7.

[0042] The circular RNA mixture is prepared by preparing the circular RNA mixture at 1000 ng / μl in RNase-free ddH2O, wherein the molar stoichiometry of OCT4 and the other five factors is 3:1:1:1:1:1.

[0043] The hsa-miR-17-5p / hsa-miR-144-3p mixture was prepared by dissolving the lyophilized microRNA in RNase-free ddH2O to a final concentration of 5 μM. The stock solution was frozen at -80°C until further use. The individual microRNA stock solutions were mixed in a 1:1 ratio to prepare a 5 μM miRNA transfection mixture.

[0044] Comparative Example 1

[0045] A composition for reprogramming fibroblasts into induced pluripotent stem cells, characterized in that the composition comprises a circularRNA mixture and a has-miR-367-3p / has-miR-302a-3p mixture.

[0046] The circularRNA mixture is prepared by preparing the transcripts of six reprogramming factors (OCT4, SOX2, KLF4, c-MYC, NANOG and LIN28A (abbreviated as "OSKMNL") into circularRNA. The specific preparation method of circularRNA can refer to the steps of the construction method of exogenous circular RNA molecules disclosed in CN201410604749.7.

[0047] The circular RNA mixture is prepared by preparing the circular RNA mixture at 1000 ng / μl in RNase-free ddH2O, wherein the molar stoichiometry of OCT4 and the other five factors is 3:1:1:1:1:1.

[0048] The preparation method of the hsa-miR-367-3p / has-miR-302a-3p mixture is to dissolve the lyophilized microRNA in RNase-free ddH2O to a final concentration of 5 μM. The stock solution is frozen at -80°C until further use. The respective microRNA stock solutions are mixed in a 1:1 ratio to prepare a 5 μM miRNA transfection mixture.

[0049] Comparative Example 2

[0050] Compared with Comparative Example 1, several compositions only include the circular RNA mixture.

[0051] Verification Example

[0052] 1.1 Fibroblast treatment

[0053] 1) 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;

[0054] 2) Primary human neonatal fibroblasts were plated at a density of 200 to 100,000 cells per well in a 6-well cell culture dish. On coated culture dishes;

[0055] 3) Add culture medium to a 6-well cell culture dish, wherein the culture medium contains DMEM / F12, 10% heat-inactivated FBS, 0.4 mM GLUTAMAX TM Supplements;

[0056] 4) Incubate the plated cells in a 5% CO2 tissue culture incubator overnight.

[0057] 1.2 Transfection solution preparation

[0058] 1.2.1CircularRNA mixture transfection solution preparation

[0059] 1) Use the commercially available CALNP liposome transfection reagent to prepare the transfection complex, take 1.2 μg of the circular RNA mixture, add 10 μL of CALNP A solution and mix well;

[0060] 2) Add 2 μL of CALNP B solution, mix well, and incubate at room temperature for 10 min;

[0061] 3) Then, 50 μL of reproTSER complete culture medium was added thereto for dispersion, thereby completing the preparation of the circular RNA complex for transfection in one well of a six-well plate.

[0062] 1.2.1 miRNA mixture transfection solution configuration

[0063] 1) Take 20 pmol of miRNA mixture, then add 17 μL of CALNP A solution and mix well;

[0064] 2) Then add 3 μL of CALNP B solution, mix well and incubate at room temperature for 10 min;

[0065] 3) Then, 50 μL of reproTSER complete culture medium was added thereto for dispersion, thereby completing the configuration of the miRNA complex for transfection in one well of a six-well plate.

[0066] 1.3 Transfection

[0067] Transfection was performed every 48 hours. The medium was replaced with ordinary DMEM+10% FBS used for fibroblast culture 24 hours after each transfection in the first 8 days, and then replaced with reproTeSR complete medium after transfection.

[0068] After completing the four transfection series, the culture medium (reproTESR medium supplemented with 200 ng / mL B18R) was changed every day, and the cells were grown until day 12, when the morphological changes of neonatal fibroblasts were detected and the number of positive iPSCs colonies was counted.

[0069] The results are as follows Figure 1 As shown, under the action of Examples 1-3 of the present invention, the neonatal fibroblasts showed obvious morphological changes compared with Comparative Examples 1-2, and the cells showed a clustering phenomenon and a spherical appearance. The aggregation phenomenon of the cells in Comparative Example 1-2 was not obvious, and they showed a slender appearance. Further, the number of positive iPSCs colonies was statistically found to be significantly higher than that in Comparative Examples 1-2, thereby confirming that the composition of the present invention can efficiently achieve fibroblast reprogramming of induced pluripotent stem cells. In addition, by analyzing the iPSCs pluripotency indicators OCT4 and NANOG obtained in Example 1, it was found that they were consistent with the performance of positive commercially available iPSCs cell lines, which was much higher than the starting cell Fibroblast. It was once again confirmed that the combination of circularRNA and miRNA of the present invention can synergistically improve the efficiency of primary human fibroblasts in generating iPSCs.

[0070] 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 circularRNA mixture and a miRNA mixture; wherein the circularRNA mixture is exogenous circular RNA molecules of OCT4, SOX2, KLF4, c-MYC, NANOG and LIN28A; and the miRNA mixture is a combination of hsa-miR-17-5p / hsa-miR-23a-3p, a combination of hsa-miR-17-5p / hsa-miR-144-3p or a combination of hsa-miR-17-5p / hsa-miR-340-3p.

2. The composition according to claim 1, characterized in that The circular RNA mixture is prepared in RNase-free ddH2O at a final concentration of 1000 ng / μl, wherein the molar stoichiometry of OCT4 and the other five factors is 3:1:1:1:1:

1.

3. The composition according to claim 1, characterized in that The miRNA mixture is prepared by dissolving freeze-dried miRNA in RNase free ddH2O to prepare a stock solution with a final concentration of 5 μM; freezing the stock solution at -80°C until further use; and mixing the respective miRNA stock solutions at a ratio of 1:1 to prepare a 5 μM miRNA transfection mixture.

4. The composition according to claim 1, characterized in that The composition includes a circular RNA mixture and an hsa-miR-17-5p / hsa-miR-23a-3p mixture, wherein the circular RNA mixture is exogenous circular RNA molecules of OCT4, SOX2, KLF4, c-MYC, NANOG and LIN28A.

5. The composition according to claim 1, characterized in that The composition includes a circular RNA mixture and an hsa-miR-17-5p / hsa-miR-144-3p mixture, wherein the circular RNA mixture is exogenous circular RNA molecules of OCT4, SOX2, KLF4, c-MYC, NANOG and LIN28A.

6. The composition according to claim 1, characterized in that The composition includes a circular RNA mixture and an hsa-miR-17-5p / hsa-miR-340-3p mixture, wherein the circular RNA mixture is exogenous circular RNA molecules of OCT4, SOX2, KLF4, c-MYC, NANOG and LIN28A.

7. A method for preparing induced pluripotent stem cells (iPSCs) using the composition according to any one of claims 1 to 6, the method comprising the following steps: 1) contacting the above composition with target cells, wherein the target cells are fibroblasts; 2) culturing target cells under conditions capable of inducing cell reprogramming, including appropriate culture medium, temperature, gas, hormones, growth factors, cytokines, and compounds; 3) Isolation and characterization of induced pluripotent stem cells (iPSCs) from cultured target cells.

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