Composition and method for cell reprogramming using circular RNA and microrna

Abstract

Provided are a composition and method for cell reprogramming using circular RNA and microRNA. The composition for cell reprogramming using circular RNA and microRNA comprises a circular RNA mixture and a miRNA mixture. Under co-transfection with the specific miRNA combination and the circular RNA, the transduction of reprogramming factors and the regulation and control of target genes can be achieved simultaneously, thereby improving the reprogramming efficiency of cells and achieving extremely high reprogramming efficiency at low cell density.

Description

Compositions and methods for cell reprogramming using circular RNA and microRNA Technical Field

[0001] This invention relates to the field of cell reprogramming technology, and in particular to compositions and methods for cell reprogramming using circular RNA and microRNA. Background Technology

[0002] Reprogramming refers to the process of altering cell fate through epigenetic modifications such as DNA methylation without changing the gene sequence. Originally, it referred to the process of eliminating epigenetic markers carried by the parents during the development of mammalian germ cells. However, it has been proven that in vitro manipulations of embryos, such as nuclear transfer and cell fusion, can also alter their original epigenetic characteristics. Currently, reprogramming mainly refers to two processes: first, the process of reversing differentiated cells back to a totipotent state; and second, the process of transforming one type of differentiated cell into another.

[0003] Researchers have previously reported reprogramming human fibroblast cell lines using circular RNA reprogramming factors, including Oct4, Sox2, Klf4, c-myc, Nanog, and Lin28 (OSKMLN), combined with two other miRNAs (miR302 and miR367). These four OSKMLN circular RNAs were synthesized in vitro using a permuted intron-exon (PIE) type I intron self-splicing system derived from the thymidylate synthase gene of T4 phage or the pretRNA Leu from Anabaena. The in vitro RNA circularization process requires Mg... 2+ Along with GTP cofactor, the RNA product was subsequently treated with RNase-R and HPLC to remove linear mRNA and nicked circRNA. Additionally, miR302 and miR367 were chemically synthesized and supplied by Sangon Biotech.

[0004] Because linear mRNAs are relatively unstable, multiple transfections are usually required to improve reprogramming success rates. For example, lab-made mRNA reprogramming kits (see: High-efficiency RNA-based reprogramming of human primary filblasts) and ReproCELL (cat.00-0076) and Miltenyi (cat.130-132-990) typically require 6, 4-8, or 5-8 transfections, respectively. Compared to linear mRNAs, circular RNAs exhibit superior stability both in vivo and in vitro, thus allowing for a reduction in the number of transfections and the amount of RNA used without compromising reprogramming efficiency.

[0005] However, current methods for inducing reprogramming by combining circular RNA and miRNA suffer from low induction efficiency, and effectively improving the induction rate of induced pluripotent stem cells is a major technical obstacle facing the industry.

[0006] Summary of the Invention

[0007] To address the technical problems existing in the prior art, this invention provides a composition and method for cell reprogramming using circular RNA and microRNA. By co-transfecting specific miRNA combinations with circular RNA, this invention can simultaneously achieve the transduction of reprogramming factors and the regulation of target genes, improving cell reprogramming efficiency and achieving extremely high reprogramming efficiency even at low cell densities.

[0008] One of the objectives of this invention is to provide a composition for fibroblast reprogramming induced pluripotent stem cells, characterized in that the composition comprises a mixture of circular RNA and a mixture of miRNA.

[0009] Preferably, the circular RNA mixture comprises exogenous circular RNA molecules of OCT4, SOX2, KLF4, c-MYC, NANOG, and LIN28A.

[0010] Preferably, the miRNA mixture comprises 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.

[0011] Preferably, the circular RNA mixture is prepared in RNase-free ddH2O at a concentration of 1000 ng / μl, wherein the molar stoichiometry of OCT4 with the other five factors is 3:1:1:1:1:1.

[0012] Preferably, the miRNA mixture is prepared by dissolving lyophilized miRNA in RNase-free ddH2O to a final concentration of 5 μM. The stock solution is then 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.

[0013] More preferably, the composition comprises a mixture of circular RNA and a mixture of hsa-miR-17-5p / hsa-miR-23a-3p.

[0014] More preferably, the composition comprises a mixture of circular RNA and a mixture of hsa-miR-17-5p / hsa-miR-144-3p.

[0015] More preferably, the composition comprises a mixture of circular RNA and a mixture of hsa-miR-17-5p / hsa-miR-340-3p.

[0016] 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:

[0017] 1) Contact the above composition with the target cells;

[0018] 2) Cultivate target cells under conditions that can induce cell reprogramming, including appropriate culture medium, temperature, gas, hormones, growth factors, cytokines, compounds, etc.

[0019] 3) Isolate and identify induced pluripotent stem cells (iPSCs) from cultured target cells.

[0020] Preferably, the target cell is a primary somatic cell, such as fibroblasts, blood cells, skin cells, hepatocytes, pancreatic islet cells, kidney cells, lung cells, muscle cells, adipocytes, neurons, glial cells, retinal cells, cardiomyocytes, bone cells, chondrocytes, endothelial cells, smooth muscle cells, epithelial cells, immune cells, etc.

[0021] Preferably, these conditions include appropriate culture medium, temperature, gas, hormones, growth factors, cytokines, compounds, etc.

[0022] Preferably, the identification includes detecting the morphology, phenotype, gene expression, epigenetics, karyotype, self-renewal capacity, and multi-lineage differentiation capacity of iPSCs.

[0023] The advantages of this invention are as follows: This invention screens and optimizes miRNA combinations, and further obtains the miRNA combinations disclosed in this invention. When these combinations are 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 densities. Attached Figure Description

[0024] Figure 1. Cell morphology analysis, where ae correspond to Examples 1-3 and Comparative Examples 1-2, respectively;

[0025] Figure 2. Statistical analysis of the number of positive iPSCs colonies;

[0026] Figure 3. Analysis of the multipotency index of iPSCs. Detailed Implementation

[0027] 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.

[0028] 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.

[0029] 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.

[0030] Example 1

[0031] A composition for fibroblast reprogramming induced pluripotent stem cells, characterized in that the composition comprises a mixture of circular RNA and a mixture of hsa-miR-17-5p / hsa-miR-23a-3p.

[0032] The circularRNA mixture is prepared from transcripts of six reprogramming factors (OCT4, SOX2, KLF4, c-MYC, NANOG, and LIN28A (abbreviated as "OSKMNL")) into circularRNA. For specific methods of preparing circularRNA, please refer to the construction method of exogenous circular RNA molecules disclosed in CN201410604749.7.

[0033] The circular RNA mixture is prepared by using RNase-free ddH2O at a concentration of 1000 ng / μl, wherein the molar stoichiometry of OCT4 with the other five factors is 3:1:1:1:1:1.

[0034] The hsa-miR-17-5p / hsa-miR-23a-3p mixture was prepared by dissolving 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.

[0035] Example 2

[0036] A composition for fibroblast reprogramming induced pluripotent stem cells, characterized in that the composition comprises a mixture of circular RNA and a mixture of hsa-miR-17-5p / hsa-miR-144-3p.

[0037] The circularRNA mixture is prepared by converting transcripts of six reprogramming factors (OCT4, SOX2, KLF4, c-MYC, NANOG, and LIN28A (abbreviated as "OSKMNL")) into circularRNA. For specific methods of preparing circularRNA, please refer to the construction method of exogenous circular RNA molecules disclosed in CN201410604749.7.

[0038] The circular RNA mixture is prepared by using RNase-free ddH2O at a concentration of 1000 ng / μl, wherein the molar stoichiometry of OCT4 with the other five factors is 3:1:1:1:1:1.

[0039] The hsa-miR-17-5p / hsa-miR-144-3p mixture was prepared by dissolving 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.

[0040] Example 3

[0041] A composition for fibroblast reprogramming induced pluripotent stem cells, characterized in that the composition comprises a mixture of circular RNA and a mixture of hsa-miR-17-5p / hsa-miR-340-3p.

[0042] The circularRNA mixture is prepared from transcripts of six reprogramming factors (OCT4, SOX2, KLF4, c-MYC, NANOG, and LIN28A (abbreviated as "OSKMNL")) into circularRNA. For specific methods of preparing circularRNA, please refer to the construction method of exogenous circular RNA molecules disclosed in CN201410604749.7.

[0043] The circular RNA mixture is prepared by using RNase-free ddH2O at a concentration of 1000 ng / μl, wherein the molar stoichiometry of OCT4 with the other five factors is 3:1:1:1:1:1.

[0044] The hsa-miR-17-5p / hsa-miR-144-3p mixture was prepared by dissolving 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.

[0045] Comparative Example 1

[0046] A composition for fibroblast reprogramming induced pluripotent stem cells, characterized in that the composition comprises a mixture of circular RNA and a mixture of has-miR-367-3p / has-miR-302a-3p.

[0047] The circularRNA mixture is prepared from transcripts of six reprogramming factors (OCT4, SOX2, KLF4, c-MYC, NANOG, and LIN28A (abbreviated as "OSKMNL")) into circularRNA. For specific methods of preparing circularRNA, please refer to the construction method of exogenous circular RNA molecules disclosed in CN201410604749.7.

[0048] The circular RNA mixture is prepared by using RNase-free ddH2O at a concentration of 1000 ng / μl, wherein the molar stoichiometry of OCT4 with the other five factors is 3:1:1:1:1:1.

[0049] The has-miR-367-3p / has-miR-302a-3p mixture was prepared by dissolving 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.

[0050] Comparative Example 2

[0051] Compared to Comparative Example 1, the operand composition consisted only of a mixture of circular RNA.

[0052] Verification Example

[0053] 1.1 Fibroblast treatment

[0054] 1) Add Matrigel 354277 (Corning Inc., NY) diluted 100-fold with DMEM / F12 to a 6-well cell culture dish and incubate at 37°C for 1 hour in a 5% CO2 tissue culture incubator;

[0055] 2) Primary human neonatal fibroblasts were seeded in 6-well cell culture dishes at a density of 200 to 100,000 cells per well. On the coated petri dish;

[0056] 3) Add culture medium to a 6-well cell culture dish, wherein the culture medium contains DMEM / F12, 10% heat-inactivated FBS, and 0.4 mM GLUTAMAX. TM Supplements;

[0057] 4) Incubate the plated cells overnight in a 5% CO2 tissue culture incubator.

[0058] 1.2 Preparation of transfection solution

[0059] 1.2.1 Preparation of circular RNA mixture transfection solution

[0060] 1) Prepare the transfection complex using commercially available CALNP liposome transfection reagent. Take 1.2 μg of the above circular RNA mixture and add 10 μL of CALNP A solution to it and mix well.

[0061] 2) Add 2 μL of CALNP B solution, mix well, and incubate at room temperature for 10 min;

[0062] 3) Then add 50 μL of reproTSER complete medium to disperse, thus preparing one well of the six-well plate for transfection of the circular RNA complex.

[0063] 1.2.1 Preparation of miRNA mixture transfection solution

[0064] 1) Take 20 pmol of miRNA mixture and then add 17 μL of CALNP A solution and mix well;

[0065] 2) Then add 3 μL of CALNP B solution, mix well, and incubate at room temperature for 10 min;

[0066] 3) Then add 50 μL of reproTSER complete medium to disperse, thus preparing the miRNA complex for transfection in one well of a six-well plate.

[0067] 1.3 Transfection

[0068] Transfection was performed every 48 hours. For the first 8 days, the medium was changed to the original DMEM + 10% FBS medium from the fibroblast culture institute 24 hours after each transfection. After that, the medium was changed to reproTeSR complete medium after transfection.

[0069] After completing four transfection series, the culture medium was changed daily (reproTESR medium supplemented with 200 ng / mL B18R). On day 12, the morphological changes of neonatal fibroblasts were detected, and the number of positive iPSC colonies was counted.

[0070] As shown in Figure 1, under the influence of Examples 1-3 of this invention, neonatal fibroblasts exhibited significant morphological changes compared to Comparative Examples 1-2, showing cell aggregation and a spherical appearance. In contrast, the aggregation of cells in Comparative Examples 1-2 was not obvious, and they presented a slender, elongated shape. Furthermore, statistical analysis of the number of positive iPSC colonies revealed that the number of positive iPSC colonies in Examples 1-3 was significantly higher than that in Comparative Examples 1-2, thus confirming that the composition of this invention can efficiently reprogram fibroblasts into induced pluripotent stem cells. Additionally, analysis of the iPSC pluripotency indices OCT4 and NANOG obtained in Example 1 showed that they were consistent with the performance of commercially available positive iPSC cell lines and far exceeded those of the starting cell Fibroblast. This further confirms that the combination of circular RNA and miRNA in this invention can synergistically improve the efficiency of primary human fibroblasts in generating iPSCs.

[0071] The above embodiments are intended to illustrate the essential content of the present invention, but are not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of protection of the present invention.

Claims

1. A composition for fibroblast reprogramming induced pluripotent stem cells, characterized in that, The composition comprises a mixture of circular RNA and a mixture of miRNA.

2. The composition according to claim 1, characterized in that, The circular RNA mixture includes exogenous circular RNA molecules such as OCT4, SOX2, KLF4, c-MYC, NANOG, and LIN28A.

3. The composition according to claim 1, characterized in that, The miRNA mixture includes combinations 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.

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

1.

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

6. The composition according to claim 1, characterized in that, The composition comprises a mixture of circular RNA and a mixture of hsa-miR-17-5p / hsa-miR-23a-3p.

7. The composition according to claim 1, characterized in that, The composition comprises a mixture of circular RNA and a mixture of hsa-miR-17-5p / hsa-miR-144-3p.

8. The composition according to claim 1, characterized in that, The composition comprises a mixture of circular RNA and a mixture of hsa-miR-17-5p / hsa-miR-340-3p.

9. A method for preparing induced pluripotent stem cells (iPSCs) using the above composition, the method comprising the following steps: 1) Contact the above composition with the target cells; 2) Cultivate target cells under conditions that can induce cell reprogramming, including appropriate culture medium, temperature, gas, hormones, growth factors, cytokines, compounds, etc. 3) Isolate and identify induced pluripotent stem cells (iPSCs) from cultured target cells.

10. The method as described in claim 9, characterized in that, The target cells are primary somatic cells, such as fibroblasts, blood cells, skin cells, hepatocytes, pancreatic islet cells, kidney cells, lung cells, muscle cells, adipocytes, neurons, glial cells, retinal cells, cardiomyocytes, bone cells, chondrocytes, endothelial cells, smooth muscle cells, epithelial cells, and immune cells.

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