CN117431258A - Method for inducing reprogramming of human cells using reprogramming factor containing Tet1 gene - Google Patents
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Abstract
The invention provides a method for inducing reprogramming of human cells by using reprogramming factors containing Tet1 genes, which comprises the steps of preparing circular RNA containing the Tet1 gene reprogramming factors and introducing the circular RNA into the human cells by using an electrotransfection method. According to the reprogramming factor method containing the Tet1 gene, the annular RNA is used as a delivery carrier of the reprogramming factor, so that the breakthrough of reprogramming iPSC in human peripheral blood mononuclear cells is successfully realized, the quality and the size of a gene transfection carrier are effectively reduced, and the reprogramming of the Tet1 for human peripheral blood mononuclear cells is successfully realized by optimizing electrotransformation parameters and factor combinations, so that human induced pluripotent stem cells can be efficiently prepared; the use of circular RNA instead of plasmid or mRNA for reprogramming avoids the drawbacks of genomic integration and cytotoxicity, and thus the present invention can prepare human induced pluripotent stem cells more efficiently and safely.
Description
Technical Field
The invention belongs to the field of molecular biology and cell technology, and mainly relates to a method for inducing reprogramming of human cells by using reprogramming factors containing Tet1 genes.
Background
Induced pluripotent stem cells (ipscs) were originally obtained by the mountain stretch team of university of kyoto, japan by introducing exogenous 4 genes (Oct 4, sox2, klf4, c-Myc) into the fibroblasts of mice, a pluripotent stem cell with properties similar to embryonic stem cells was successfully obtained, the most classical reprogramming factors being the 4 genes described above, also known as OSKM factors. After that, a large number of biologists began to study and improve reprogramming factors to increase the induction efficiency of reprogramming, and in one study on the effect of epigenetic inheritance on reprogramming efficiency, experimental staff found that expression of DNA hydroxymethylase 1 (Tet 1) protein in mouse embryo fibroblasts could increase reprogramming efficiency and could replace part of the OSKM factor to establish a mouse iPSC cell line.
The traditional iPSC induction method adopts virus-mediated reprogramming, so that foreign genes in human genome are inserted, cancer is easy to be induced, and the application of iPSC is not facilitated. Currently, the induction mode of human iPSC has gradually changed from the original retrovirus or lentivirus induction method to the non-virus-mediated non-integrated induction mode without foreign gene insertion, however, most of the induction methods have various defects, such as difficult transduction, low reprogramming induction efficiency, genome integration and the like of episomal vectors, and mRNA-based reprogramming has problems of easy degradation and cytotoxicity.
The methylation and the demethylation of the DNA play a very important role in the totipotency of embryonic stem cells, and the invention utilizes the activity of DNA hydroxymethylase Tet1 on the DNA to accelerate the demethylation process of a multipotent gene regulatory region and activate the expression of multipotent genes, thereby promoting the establishment of the multipotency of cells and improving the speed and the efficiency of induced reprogramming. The report that the Tet1 gene is used for replacing Oct4 by a slow virus mode and a reprogramming system is successfully established in Mouse Embryo Fibroblasts (MEFs) together with Sox2, klf4 and c-Myc is provided, compared with the traditional four factors, the reprogramming efficiency is greatly improved, the whole methylation level of the MEF cell genome is lower, and the reprogramming system is also a cell line which is accepted in the industry and is easy to reprogram. However, many problems need to be solved in order to reproduce the same experimental results in human somatic cells.
Disclosure of Invention
In order to find a reprogramming method which can replace OSKM factor and has the same or better induction efficiency, and overcome the defects of the traditional induction method, the invention provides the following technical scheme:
a method for inducing reprogramming of human cells by using a reprogramming factor containing a Tet1 gene, characterized in that a circular RNA containing a Tet1 gene reprogramming factor is prepared and introduced into human cells by using an electrotransfection method.
A method of inducing reprogramming of human cells using a reprogramming factor comprising a Tet1 gene as described above, wherein the reprogramming factor comprises a Tet1 gene and other genes selected from one or a combination of several of Oct4, sox2, klf4, c-Myc genes.
A method of inducing reprogramming of human cells using a reprogramming factor comprising a Tet1 gene as described above, wherein the circular RNA comprises the sequence as set forth in SEQ ID NO:7 and a nucleic acid sequence as set forth in SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 13. SEQ ID NO:15, or a sequence at least 90% or at least 95% identical to the above sequence.
A method of inducing reprogramming of human cells using a reprogramming factor comprising a Tet1 gene as described above, wherein the precursor RNA sequence for preparing the circular RNA comprises the sequence set forth in SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO: 14. SEQ ID NO:16, or a sequence at least 90% or at least 95% identical to the above sequence.
The invention has the beneficial effects that: according to the invention, the annular RNA is used as a delivery carrier of the reprogramming factors, so that the breakthrough of reprogramming iPSC in human peripheral blood mononuclear cells is successfully realized, the quality and the size of the gene transfection carrier are effectively reduced, and the reprogramming of the human peripheral blood mononuclear cells by using Tet1 is successfully realized by optimizing the electrotransformation parameters and the factor combination. The induction method using reprogramming factors containing the Tet1 gene of the present invention can reprogram ipscs with higher efficiency.
Drawings
FIG. 1 is a graph showing comparison of protein expression levels of different transfection modes of Tet1 gene;
FIG. 2 is a graph of circular RNA yield;
FIG. 3 is a photograph of a cell clone obtained by reprogramming human cells to iPSC induced by OSKM factor and OT, TSKM factor;
FIG. 4 is a graph comparing the expression levels of endogenous cellular pluripotency markers in iPSCs induced by OSKM and OT factors;
FIG. 5 is a flow chart of markers of iPSC pluripotency induced by OSKM and TSKM factors;
FIG. 6 is a graph showing the comparison of the efficiency of OSKM factor and OT and TSKM factor in inducing human cell reprogramming.
Detailed Description
The human cell genome used in the invention has higher methylation degree, and compared with MEF cells, the reprogramming difficulty is high and the efficiency is low. Tet1 is required to have an effect of improving efficiency in reprogramming human cells, and it is required that Tet1 can be stably and highly expressed in human cells, and the expression level is required to be higher than that in MEF.
The present invention has made numerous attempts to increase Tet1 expression in human cells.
Firstly, the invention uses slow virus to transfect, and although cell strains stably expressing Tet1 protein are obtained, the stable transformation efficiency is lower, and the expressed Tet1 is insufficient to realize reprogramming of human cells.
Second, since the human Tet1 protein shares 2136 amino acid residues, transfection by adeno-associated virus or sendai virus is not possible.
Again, the plasmid constructed was larger by the usual plasmid transfection method, also because of the longer sequence of Tet1 itself. Although the plasmid structure is designed optimally to reduce unnecessary sequences, the expression level after transfection can not effectively improve the success rate of reprogramming.
Fourth, although the expression level of Tet1 can be increased by using the transfection of messenger RNA (mRNA) technique, the high level expression cannot be stably and continuously maintained due to the rapid degradation of mRNA, and the aim of improving the reprogramming efficiency cannot be achieved.
Finally, the present invention utilizes synthetic or in vitro transcribed circular RNAs. The circular RNA is extremely stable in a living cell because of its special closed structure, is not affected by an exonuclease, and is therefore very suitable as a vector for introducing a reprogramming factor into a somatic cell. Because of the non-integration, degradability and low immunogenicity of the circular RNA, the method can obtain the 'footprint-free' iPSC which is safer than the method using an integrated viral vector, and overcomes the major defects of foreign gene insertion, cancer induction and the like in the human genome in the prior art. However, the loop forming efficiency of the Tet1 gene sequence is low because the Tet1 gene sequence is long, so the invention predicts the loop forming efficiency of the Tet1 sequence by using the SIRI2 algorithm, and optimizes the available circular RNA sequence after testing the predicted sequence.
Example 1: protein expression quantity comparison of different transfection modes of Tet1 gene
The lentivirus, episomal plasmid, messenger RNA and circular RNA protocols of the Tet1 gene were designed separately. The lentiviral vector skeleton selects pLVX plasmid obtained from Addgene, and the CDS region sequence of Tet1 gene is inserted into the pLVX plasmid expression cassette by a molecular cloning method, and the corresponding vector sequence information is shown in SEQ ID NO:1, then co-transfecting HEK293T cells with packaging plasmid and envelope plasmid, culturing for two days, collecting viruses and measuring titer; the eukaryotic expression vector skeleton selects pCDNA plasmid obtained from Addgene, and the CDS region sequence of Tet1 gene is inserted into the pCDNA plasmid expression cassette by a molecular cloning method, and the corresponding vector sequence information is shown in SEQ ID NO:2 is shown in the figure; messenger RNA is obtained by using a T7 promoter on a eukaryotic expression vector and purifying after transcription by using an in vitro transcription kit; the circular RNA is required to prepare a precursor vector, a substitution ribozyme sequence is firstly added at two sides of the CDS region of the eukaryotic expression vector, and the corresponding precursor vector sequence information is shown as SEQ ID NO:3, then in vitro transcription, RNA cyclization and RNA purification.
According to the conventional experimental operation, the lentivirus, the episomal plasmid, the messenger RNA and the circular RNA are respectively transfected into HEK293T cells with the same cell quantity, and the cells are continuously cultured, and part of the cells are respectively collected on 4 th day, 8 th day, 12 th day, 16 th day and 20 th day, and the Tet1 protein expression quantity in each group of cells is detected by a WB method, so that the transfected circular RNA group can maintain stable and high-level protein expression quantity for a long time as shown in figure 1.
Example 2: optimization of circular RNA sequences
In comparison of protein expression levels of different transfection methods of Tet1 gene in example 1, transfection with messenger RNA was performed, and although relatively stable high level expression could be maintained for a long period of time, loop formation efficiency of loop RNA was low, and a large amount of reaction was required to obtain loop RNA required to satisfy the experiment, and therefore, optimization of loop RNA structure was required.
Based on the degenerate codon principle, SIRI2 algorithm is used to predict the secondary structure of circular RNA, the circular RNA sequence favorable to cyclization reaction is screened, three groups of circular RNA precursor vectors are redesigned, and the sequence information of the corresponding precursor vectors is shown as SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO:6, then using the vector with the same plasmid quantity as a starting template, preparing precursor RNA in vitro, cyclizing and purifying in vitro to obtain circular RNA, measuring the concentration of the circular RNA, comparing the cyclizing success rate of each group, and obtaining the sequence shown in figure 2 as SEQ ID NO: the cyclization yield of the 4 groups of annular RNA sequences is higher, so that the subsequent reprogramming experiment adopts SEQ ID NO: 4.
Example 3: isolation and expansion of human peripheral blood mononuclear cells
Isolation of peripheral blood mononuclear cells from a blood sample using a density gradient medium, followed by a 5X 10 protocol 6 The density of cells/well is inoculated in six-well plates, and the culture is carried out in an incubator at 37 ℃ for 7 days, and the culture medium is changed every two days, wherein 20% of KSR, 50 ug/mL of Ascorbic Acid, 100 ng/mL of SCF, 10 ng/mL of IL-3, 2U/mL of EPO, 40 ng/mL of IGF, 1 uM Dexamethasone and 100 nM Hydrocortisone are added to the basic culture medium.
Example 4: preparation of reprogramming factor circular RNA
The main elements of the vector for transcription of circular RNA are composed of T7 promoter, 3 'substituted ribozyme sequence (composed of 3' intron, 3 'exon fragment and flanking sequence), target factor CDS region sequence, 5' substituted ribozyme sequence (composed of 5 'intron, 5' exon fragment and flanking sequence). Preparing precursor RNA or directly synthesizing the precursor RNA by the carrier according to the steps in an in vitro transcription kit, wherein the sequence information of the precursor RNA is shown as SEQ ID NO in a sequence table: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO: 14. SEQ ID NO:16 The nucleic acid sequence (CDS region sequences of Tet1, oct4, sox2, klf4 and c-Myc genes respectively) is shown, incubated for 2 to 3 hours at 37 ℃, then DNase is added to the reaction (DNase is added in a proportion of 4 units of DNase per μg of template DNA), mixed, and incubated for 30 minutes at 37 ℃ to obtain precursor RNA, and finally, after in vitro cyclization and purification, circular RNA is obtained, namely, the circular RNA can be used for reprogramming human cells, and the circular RNA sequence information is shown as SEQ ID NO in a sequence table: 7. SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 13. SEQ ID NO:15 The nucleic acid sequences (CDS region sequences of Tet1, oct4, sox2, klf4, c-Myc genes, respectively) are shown.
Example 5: human cell reprogramming by electrotransfection of circular RNA
Experiments were performed to compare the efficiency of using conventional reprogramming factors with OT, TSKM reprogramming factors. A first group, a circular RNA carrying a traditional reprogramming factor; a second group, a circular RNA carrying an OT reprogramming factor; third group, cyclic RNAs carrying TSKM reprogramming factors.
On the day of electrotransfer, 500. Mu.L of diluted basement membrane matrix (matrigel) was added to the 12-well plate and placed in a carbon dioxide incubator for coating. The cells after the expansion in example 3 were removed, washed and counted, and the counted peripheral blood mononuclear cells were counted at 1X 10 6 centrifuging the density of cell/well, removing supernatant, adding buffer solution according to operation steps of electrotransformation instrument, mixing with corresponding annular RNA, electrotransformation, immediately adding peripheral blood amplification culture medium, inoculating in coated six-hole plate, standing in carbon dioxide incubator at 37deg.C for 48 hr, observing cell attachment condition, changing human pluripotent stem cell induction culture medium, culturing for 10-14 days, changing primary liquid for two days, observing cloning growth condition, and continuing until induced pluripotent stem cells are completely mature,about day 14 or 15 after electrotransformation, clones were then picked for amplification.
Example 6: characterization of iPSC colonies
A number of cell-like clones resembling human embryonic stem cells were observed on the fifteenth day of culture, and the clonal masses were photographed and counted under a microscope. FIG. 3 is a photograph of a cell clone obtained by reprogramming human cells to iPSC induced by OSKM factor and OT, TSKM factor, and a brighter and better morphology of a cell clone resembling human embryonic stem cells was observed on the fifteenth day of culture, as shown in FIG. 3.
The mRNA expression level of the endogenous cell pluripotency marker in the iPSC induced by OSKM and OT factors was detected by qPCR, and the result is shown in FIG. 4, wherein the mRNA level of the endogenous cell pluripotency marker in the OT-iPSC is similar to that in the iPSC induced by OSKM factors.
The results of immunofluorescent staining and flow cytometry analysis of intracellular multipotential markers OCT4, SOX2 are shown in fig. 5, demonstrating that the obtained cells have the characteristics of multipotential stem cells.
Comparing the efficiency of inducing human cells to reprogram by different factors, the results are shown in FIG. 6, wherein the OT and TSKM groups containing Tet1 genes have significantly higher efficiency of inducing human cells to reprogram than the conventional OSKM group. The invention successfully realizes the breakthrough of reprogramming iPSC in human peripheral blood mononuclear cells by taking the annular RNA as a delivery system of reprogramming factors, and lays a foundation for the induction reprogramming system containing the Tet1 gene to be used for industrial transformation. Firstly, because mouse embryo fibroblasts are easy to reprogram, the mouse embryo fibroblasts are usually used as the most common mode cells for basic research in industry, and in the field of stem cell therapeutic drug development, the most common reprogramming initiating cells are peripheral blood mononuclear cells, but compared with fibroblasts, the reprogramming efficiency and success rate of the cells are lower, and whether the efficient reprogramming of the peripheral blood mononuclear cells can be realized is a key for industrialization of the technology. According to the invention, the quality and the size of the gene transfection vector are effectively reduced by using the annular RNA as a delivery vector of the reprogramming factor, and the reprogramming of the human peripheral blood mononuclear cells by using the Tet1 is successfully realized by optimizing the electrotransformation parameters and the factor combination.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Claims (4)
1. A method for inducing reprogramming of human cells by using a reprogramming factor containing a Tet1 gene, characterized in that a circular RNA containing a Tet1 gene reprogramming factor is prepared and introduced into human cells by using an electrotransfection method.
2. The method of claim 1, wherein the reprogramming factors comprise Tet1 gene and other genes selected from one or more of Oct4, sox2, klf4, c-Myc genes.
3. The method of claim 1, wherein the circular RNA comprises the sequence set forth in SEQ ID NO:7 and a nucleic acid sequence as set forth in SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 13. SEQ ID NO:15, or a sequence at least 90% or at least 95% identical to the above sequence.
4. A method of inducing reprogramming of human cells using a reprogramming factor comprising Tet1 gene according to claim 1, wherein the precursor RNA sequence for preparing the circular RNA comprises the sequence set forth in SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO: 14. SEQ ID NO:16, or a sequence at least 90% or at least 95% identical to the above sequence.
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