CN118272378B - Method for maintaining stem property of pluripotent stem cells and removing residues thereof in differentiated cells - Google Patents
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Abstract
The invention discloses a method for maintaining the stem property of a pluripotent stem cell and removing the residue of the pluripotent stem cell in differentiated cells, which can maintain the stem property and differentiation potential of the pluripotent stem cell in a pluripotent stem cell passage stage, simultaneously eliminate the clinical risk of a derivative product caused by the residue of the pluripotent stem cell, create preconditions for clinical application of cell treatment products and even organoids, and have important scientific significance and clinical application value.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a method for maintaining the stem property of pluripotent stem cells and removing residues in differentiated cells.
Background
The core technology of regenerative medicine and cell therapy is to use pluripotent stem cells as a platform to induce and differentiate different cells, tissues and organs, thereby promoting the self-repair and regeneration of organisms or constructing new tissues and organs to improve or restore the functions of damaged tissues and organs. Among them, pluripotent stem cells are a type of pluripotent cells with self-renewal and self-replication capacity, and embryonic stem cells (Embryonic STEM CELLS, ESCs) were first discovered to be capable of infinitely self-renewing and differentiating into any tissue cells in the body, however, the use of ESCs in clinical therapies is greatly limited due to immune rejection, tumorigenicity and ethical issues. The pluripotent stem cells (Induced Pluripotent STEM CELLS, IPSC) and the potent expansile stem cells (Expanded Potential STEM CELLS, EPSC) are then induced into human vision, which are pluripotent stem cells obtained from the human body by dedifferentiation, which can proliferate indefinitely, store for a long period of time, and differentiate into any type of tissue cells of the body, and are widely used in a variety of fields of drug screening, disease modeling, disease treatment, and the like.
In order to realize the clinical application of therapeutic products derived from pluripotent stem cells, obtaining cells and organs with high differentiation efficiency and high safety is important. For this purpose, the stem nature of the pluripotent stem cells is first maintained, ensuring that they have the potential to differentiate towards the individual cells; secondly, since pluripotent stem cells have the ability to proliferate indefinitely, there is a risk of producing teratomas in vivo, and it is necessary to ensure that no pluripotent stem cells remain in the differentiated cells or organs.
The removal of residual pluripotent stem cells requires three basic requirements to be met: high efficiency, controllability and specificity. By "highly potent" is meant that the pluripotent stem cell remains in the end product of differentiation are cleared as completely as possible; by "controllable" is meant that the time for the removal of pluripotent stem cells is controllable, and cannot be removed during passaging and differentiation culture, only when needed, such as after completion of the differentiation stage; by "specific" is meant that the process of clearance is directed only to the pluripotent stem cells that are not normally differentiated, and does not cause damage to the normally differentiated target cell population.
The pluripotent stem cells have unlimited proliferation capability, living cell products containing pluripotent stem cell residues have teratogenic properties in vivo, and the method for removing the pluripotent stem residues by using flow separation has the problems of low specificity and efficiency, single judgment method, low detection precision and difficulty in eliminating false negatives. On the other hand, the platform of pluripotent stem cell induction differentiation technology requires that pluripotent stem cells maintain as high a pluripotent stem cell capacity as possible at the pre-differentiation stage.
Disclosure of Invention
In view of the above, the present invention aims to solve the above technical problems faced in the present art, and an object of the present invention is to provide a method for maintaining the stem cell viability and eliminating the residual in differentiated cells, which can maintain the stem cell viability and differentiation potential of pluripotent stem cells in the passaging stage of pluripotent stem cells, and eliminate the clinical risk of derived products due to the residual pluripotent stem cells, thereby creating a prerequisite for clinical application of cell therapy products and organoids.
The above object of the present invention is achieved by the following technical solutions:
In a first aspect the invention provides a pluripotent stem cell-specific promoter.
Further, the pluripotent stem cell specific promoter is selected from any one of mEtnAhC p, mEtnAHS4p and mANOGP;
The nucleotide sequence of mEtnAhC p is shown as SEQ ID NO. 1;
The nucleotide sequence of mEtnAhS p is shown as SEQ ID NO. 2;
the nucleotide sequence of mNANOGp is shown as SEQ ID NO. 3.
In some embodiments, the pluripotent stem cell-specific promoters mEtnAhC p, mEtnAHS4p, mANOGP of the invention are not limited to the nucleotide sequences described above, and a recombinant promoter corresponding to a nucleotide sequence having at least 90% homology to the nucleotide sequence described above, that is, a nucleotide sequence obtained by replacing a nucleotide at any one or more positions with any other nucleotide on the basis of the corresponding nucleotide sequence, is within the scope of the invention as long as the nucleotide sequence having at least 90% homology to the nucleotide sequence described above is within the scope of the invention.
In a second aspect, the invention provides a gene expression cassette for maintaining the stem properties of pluripotent stem cells in a culture system comprising an antibiotic.
Further, the gene expression cassette comprises the pluripotent stem cell-specific promoter and the resistance gene according to the first aspect of the invention;
preferably, the resistance gene comprises Puromycin、Zeocin、Blasticidin、Geneticin、Hygromycin B、Mycophenolic Acid、Neomycin、Bekanamycin、Tetracycline;
Preferably, the pluripotent stem cell-specific promoter comprises mEtnAhC p, mEtnAHS4p, mANOGP;
more preferably, the resistance gene is Puromycin;
More preferably, the pluripotent stem cell specific promoter is mEtnAhS p, a sequence obtained by inserting a 88bp human RNA polymerase II intron into the mEtnAhS p promoter, and the purpose of the intron addition is to improve the expression of a target gene;
more preferably, the pluripotent stem cell-specific promoter and resistance gene constitute a structure of mEtnAhS p-Puromycin;
most preferably, the sequence of mEtnAhS4p-Puromycin is shown in SEQ ID NO. 7;
Preferably, the pluripotent stem cells comprise ESC, iPSC, EPSC.
Further, mEtnAhS p and Puromycin of the mEtnAhS p-Puromycin structure are connected in series. Wherein the sequence of mEtnAhS p is shown as SEQ ID NO. 2, and the sequence of Puromycin is shown as SEQ ID NO. 6.
In the present invention, the resistance genes include, but are not limited to: puromycin (puromycin), zeocin (bleomycin), blasticidin (blasticidin), geneticin (Geneticin, G418), hygromycin B (Hygromycin B), mycophenolic Acid (mycophenolic acid), neomycin (Neomycin), bekanamycin (kanamycin), TETRACYCLINE (tetracyclomycin), any resistance selection gene known to those skilled in the art is within the scope of the present invention. In a specific embodiment of the invention, the resistance gene is Puromycin.
In the present invention, the pluripotent stem cells include, but are not limited to: ESCs (embryonic stem cells), iPSCs (induced pluripotent stem cells), EPSCs (expanded pluripotent stem cells), any stem cells known to those skilled in the art to have the potential for polyblastic differentiation are within the scope of the present invention. In a specific embodiment of the invention, the pluripotent stem cells are ipscs or EPSCs.
In some embodiments, the invention constructs a stable strain of mEtnAhS p and resistance gene Puromycin in tandem expression, which is capable of maintaining the stem cell in a culture system containing the corresponding antibiotic (Puromycin).
To ensure that the pluripotent stem cells used in the cytomedicine have one hundred percent of stem property, a stable transgenic strain in which a pluripotent stem promoter (for example, mEtnAhS p) and a resistance gene (for example, puromycin) are expressed in series can be constructed, and the pluripotent stem cells stem property is maintained in a culture system containing a corresponding antibiotic (for example, puromycin), so that the positive rate and the efficiency of differentiated cells can be improved.
In a third aspect, the present invention provides a gene expression cassette for clearing pluripotent stem cells remaining in cells obtained after differentiation of pluripotent stem cells.
Further, the gene expression cassette comprises the pluripotent stem cell-specific promoter and suicide gene according to the first aspect of the invention;
Preferably, the suicide gene comprises CD, HSV-TK, NTR, and/or mutants or truncations based thereon;
preferably, the pluripotent stem cell-specific promoter comprises mEtnAhC p, mEtnAHS4p, mANOGP;
Preferably, the prodrugs of suicide genes CD, HSV-TK, NTR are 5-FC, ganciclovir and Valacyclovir, CB1954 and PR-104A, respectively;
More preferably, the suicide gene is CD;
More preferably, the pluripotent stem cell-specific promoter is mEtnAhS p;
more preferably, the prodrug is 5-FC;
More preferably, the structure of the pluripotent stem cell-specific promoter and suicide gene is mEtnAhS p-CDm3;
Most preferably, CDm is a CD comprising three mutation sites;
Most preferably, the sequence of mEtnAhS4p-CDm3 is shown in SEQ ID NO. 5;
Preferably, the pluripotent stem cells comprise ESC, iPSC, EPSC;
Preferably, the cells obtained after differentiation of the pluripotent stem cells include neural cells, cardiomyocytes, endothelial cells, retinal pigment epithelial cells, keratocytes, ovum precursor cells, sperm precursor cells, bone precursor cells, blood cells, islet cells and/or hepatocytes.
Further, mEtnAhS p and CDm3 in the mEtnAhS p-CDm3 structure are connected in series. Wherein the sequence of mEtnAhS p is shown as SEQ ID NO.2, and the sequence of CDm3 is shown as SEQ ID NO. 4.
In the present invention, the cells obtained after differentiation of the pluripotent stem cells include neural cells, cardiac muscle cells, endothelial cells, retinal pigment epithelial cells, cornea cells, ovum precursor cells, sperm precursor cells, bone precursor cells, blood cells, islet cells and/or liver cells, and any type of cells which are known to those skilled in the art to be differentiated by the pluripotent stem cells are within the scope of the present invention.
In the present invention, the CD refers to cytosine deaminase (Cytosine Deaminase, CD) and the corresponding prodrug is 5-fluorocytosine (5-Fluorocytosine, 5-FC); the HSV-TK refers to herpes simplex virus thymidine kinase (Herpes Simplex Virus THYMIDINE KINASE, HSV-TK, TK), and corresponding prodrugs are Ganciclovir (Ganciclovir, ganc) and Valacyclovir (valacyclovir); the NTR refers to escherichia coli nitroreductase (Nitroreductase, NTR), and corresponding prodrugs are 5- (1-aziridine) -2, 4-dinitrobenzamide (CB 1954) and PR-104A. The CD, HSV-TK and NTR are suicide genes commonly used in tumor gene therapy research.
In some embodiments, the inventors of the present invention have verified through comparative experiments that, for the first time, it was unexpectedly found that the effect of the combination of 5-FC and CD employed in the present invention on the removal of pluripotent stem cells remaining in cells obtained after differentiation of pluripotent stem cells is significantly better than the effect of the combination of Ganciclovir and HSV-TK, which is far less than the effect of the combination of 5-FC and CD employed in the present invention, and that the 5-FC employed in the present invention has no significant toxic or side effect, is a preferred effect of killing the pluripotent stem cells remaining in cells obtained after differentiation of pluripotent stem cells, which result belongs to technical effects that would not be expected by a person skilled in the art based on the prior art.
In a fourth aspect, the present invention provides a gene expression cassette for simultaneously maintaining the stem properties of pluripotent stem cells in a culture system containing an antibiotic and removing pluripotent stem cells remaining in cells obtained after differentiation of the pluripotent stem cells.
Further, the gene expression cassette comprises the pluripotent stem cell-specific promoter according to the first aspect of the invention, the resistance gene according to the second aspect of the invention, and the suicide gene according to the third aspect of the invention.
In a fifth aspect the present invention provides a recombinant expression vector comprising a gene expression cassette according to the second aspect of the invention, a gene expression cassette according to the third aspect of the invention and/or a gene expression cassette according to the fourth aspect of the invention.
In some embodiments, the expression vector comprises a viral vector, a plasmid vector; the viral vectors include adeno-associated viral vectors, adenovirus vectors, lentiviral vectors, retrovirus vectors, alphavirus vectors, herpesvirus vectors, EB virus vectors, vaccinia virus vectors; the adeno-associated viral vector comprises AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV2/1、AAV2/2、AAV2/5、AAV2/6、AAV2/8、AAV2/9.
In some embodiments, the recombinant expression vector may further comprise one or more of the following elements: an origin of replication, a selectable marker and a multiple cloning site. The recombinant expression vector may be any vector (e.g., a plasmid or virus) in which recombinant DNA procedures can be conveniently performed and expression of the polynucleotide can be produced. The choice of vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.
In some embodiments, the expression vector may be an autonomously replicating vector, i.e., a vector, which replicates independently of chromosomal replication as an extrachromosomal entity, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The expression vector may contain any structure for ensuring self-replication. Alternatively, the expression vector may be integrated into the genome and replicated together with the chromosome(s) into which it has been integrated upon introduction into the host cell. Transposons may also be used.
In a sixth aspect the invention provides a host cell comprising a recombinant expression vector according to the fifth aspect of the invention.
Further, the host cell is a pluripotent stem cell;
Preferably, the pluripotent stem cells comprise ESC, iPSC, EPSC.
In the present invention, the host cell includes, in addition to the host cell itself, any progeny of a parent cell (the host cell) that are different from the parent cell due to mutations that occur during replication. In some embodiments, the host cell may be a unicellular microorganism, such as a prokaryote, or a non-unicellular microorganism, such as a eukaryote. In a preferred embodiment, the host cell is a eukaryotic cell, including mammalian, insect, plant or fungal cells. In particular embodiments of the invention, the host cell is a pluripotent stem cell, including but not limited to: ESC, iPSC, EPSC.
A seventh aspect of the invention provides the use of any one of the following:
(1) The application of the pluripotent stem cell specific promoter in the first aspect of the invention in preparing a reagent for promoting the specific expression of a target gene in pluripotent stem cells;
(2) Use of a pluripotent stem cell-specific promoter according to the first aspect of the invention in combination with a resistance gene according to the second aspect of the invention for the preparation of a reagent for maintaining the stem cell-like properties of pluripotent stem cells in a culture system comprising an antibiotic;
(3) Use of a pluripotent stem cell-specific promoter according to the first aspect of the invention in combination with a suicide gene according to the third aspect of the invention for the preparation of a reagent for clearing pluripotent stem cells remaining in cells obtained after differentiation of pluripotent stem cells;
(4) Use of a pluripotent stem cell-specific promoter according to the first aspect of the invention in combination with a resistance gene according to the second aspect of the invention, a suicide gene according to the third aspect of the invention, for the preparation of a reagent for simultaneously maintaining the stem cell-specific properties of pluripotent stem cells in a culture system containing an antibiotic and for removing residual pluripotent stem cells in cells obtained after differentiation of pluripotent stem cells;
(5) The use of a gene expression cassette according to the second aspect of the invention in the preparation of a reagent for maintaining the stem cell stem properties of pluripotent stem cells in a culture system comprising an antibiotic;
(6) The use of a gene expression cassette according to the third aspect of the present invention for the preparation of a reagent for clearing pluripotent stem cells remaining in cells obtained after differentiation of pluripotent stem cells.
In an eighth aspect, the invention provides a method of maintaining pluripotent stem cell stem in a culture system comprising an antibiotic.
Further, the method comprises: transfecting pluripotent stem cells with the recombinant expression vector comprising the gene expression cassette of the second aspect of the invention of the fifth aspect of the invention, and maintaining the stem cell in a culture system containing the corresponding antibiotic;
preferably, the resistance gene comprises Puromycin、Zeocin、Blasticidin、Geneticin、Hygromycin B、Mycophenolic Acid、Neomycin、Bekanamycin、Tetracycline;
Preferably, the pluripotent stem cell-specific promoter comprises mEtnAhC p, mEtnAHS4p, mANOGP;
more preferably, the resistance gene is Puromycin;
More preferably, the pluripotent stem cell-specific promoter is mEtnAhS p;
more preferably, the pluripotent stem cell-specific promoter and resistance gene constitute a structure of mEtnAhS p-Puromycin;
most preferably, the sequence of mEtnAhS4p-Puromycin is shown in SEQ ID NO. 7;
Preferably, the pluripotent stem cells comprise ESC, iPSC, EPSC;
preferably, the antibiotic is Puromycin;
Preferably, the Puromycin is used at a concentration of 0.5 μg/mL.
In a ninth aspect, the present invention provides a method for removing pluripotent stem cells remaining in cells obtained after differentiation of pluripotent stem cells.
Further, the method comprises: transfecting the recombinant expression vector comprising the gene expression cassette of the third aspect of the present invention into pluripotent stem cells, and adding corresponding prodrug into a culture system to remove residues in cells obtained after differentiation of pluripotent stem cells;
Preferably, the suicide gene comprises CD, HSV-TK, NTR, and/or mutants or truncations based thereon;
preferably, the pluripotent stem cell-specific promoter comprises mEtnAhC p, mEtnAHS4p, mANOGP;
Preferably, the prodrugs of suicide genes CD, HSV-TK, NTR are 5-FC, ganciclovir and Valacyclovir, CB1954 and PR-104A, respectively;
More preferably, the suicide gene is CD;
More preferably, the pluripotent stem cell-specific promoter is mEtnAhS p;
more preferably, the prodrug is 5-FC;
More preferably, the structure of the pluripotent stem cell-specific promoter and suicide gene is mEtnAhS p-CDm3;
Most preferably, CDm is a CD comprising three mutation sites;
Most preferably, the sequence of mEtnAhS4p-CDm3 is shown in SEQ ID NO. 5;
Preferably, the pluripotent stem cells comprise ESC, iPSC, EPSC;
preferably, the cells obtained after differentiation of the pluripotent stem cells include neural cells, cardiomyocytes, endothelial cells, retinal pigment epithelial cells, keratocytes, ovum precursor cells, sperm precursor cells, bone precursor cells, blood cells, islet cells and/or hepatocytes;
Preferably, the transfection means comprises lentiviral vector transfection, electroporation transfection, calcium phosphate transfection, liposome-mediated transfection, adenovirus vector transfection, retroviral vector transfection;
more preferably, the transfection means is lentiviral vector transfection;
More preferably, the 5-FC is used at a concentration of 0.5mM-2mM;
More preferably, the 5-FC treatment time is 48 hours.
In a tenth aspect, the present invention provides a method for simultaneously maintaining the stem property of pluripotent stem cells in a culture system containing an antibiotic, and removing pluripotent stem cells remaining in cells obtained after differentiation of pluripotent stem cells.
Further, the recombinant expression vector comprising the gene expression cassette according to the fourth aspect of the present invention in the fifth aspect of the present invention is transfected into a pluripotent stem cell, or the recombinant expression vector comprising the gene expression cassette according to the second aspect of the present invention in the fifth aspect of the present invention and the recombinant expression vector comprising the gene expression cassette according to the third aspect of the present invention in the fifth aspect of the present invention are transfected into a pluripotent stem cell, the pluripotent stem cell is maintained in a culture system containing the corresponding antibiotic, and the corresponding prodrug is added to the culture system to remove the residual in the cells obtained after differentiation of the pluripotent stem cell;
Preferably, the means for transfecting the recombinant expression vector comprising the gene expression cassette of the second aspect of the present invention in the fifth aspect of the present invention and the recombinant expression vector comprising the gene expression cassette of the third aspect of the present invention in the fifth aspect of the present invention comprises: simultaneously and successively carrying out transfection;
preferably, the resistance gene comprises Puromycin、Zeocin、Blasticidin、Geneticin、Hygromycin B、Mycophenolic Acid、Neomycin、Bekanamycin、Tetracycline;
Preferably, the pluripotent stem cell-specific promoter comprises mEtnAhC p, mEtnAHS4p, mANOGP;
Preferably, the suicide gene comprises CD, HSV-TK, NTR, and/or mutants or truncations based thereon;
Preferably, the prodrugs of suicide genes CD, HSV-TK, NTR are 5-FC, ganciclovir and Valacyclovir, CB1954 and PR-104A, respectively;
more preferably, the resistance gene is Puromycin;
More preferably, the pluripotent stem cell-specific promoter is mEtnAhS p;
More preferably, the suicide gene is CD;
more preferably, the prodrug is 5-FC;
more preferably, the pluripotent stem cell-specific promoter and resistance gene constitute a structure of mEtnAhS p-Puromycin;
most preferably, the sequence of mEtnAhS4p-Puromycin is shown in SEQ ID NO. 7;
More preferably, the structure of the pluripotent stem cell-specific promoter and suicide gene is mEtnAhS p-CDm3;
Most preferably, CDm is a CD comprising three mutation sites;
Most preferably, the sequence of mEtnAhS4p-CDm3 is shown in SEQ ID NO. 5;
Preferably, the pluripotent stem cells comprise ESC, iPSC, EPSC;
preferably, the antibiotic is Puromycin;
Preferably, the Puromycin is used at a concentration of 0.5 μg/mL;
preferably, the cells obtained after differentiation of the pluripotent stem cells include neural cells, cardiomyocytes, endothelial cells, retinal pigment epithelial cells, keratocytes, ovum precursor cells, sperm precursor cells, bone precursor cells, blood cells, islet cells and/or hepatocytes;
More preferably, the 5-FC is used at a concentration of 0.5mM-2mM;
More preferably, the 5-FC treatment time is 48 hours.
Drawings
Fig. 1: the mEtnAhS p promoter has multipotent stem cell specificity;
fig. 2: as a conditional suicide gene, the clearance effect of CD is better than TK, and the toxicity of prodrug 5-FC is lower than TK prodrug Ganc;
Fig. 3: the pluripotent stem cells overexpressing mEtnAhS p-CDm3 in the presence of 5-FC were completely cleared;
fig. 4: the pluripotent stem cell line stably transformed mEtnAhS p-CDm3 in the presence of 5-FC was completely cleared;
Fig. 5: the addition of 5-FC can completely remove iPSC and EPSC of stable rotation mEtnAhS p-CDm3, but has no obvious influence on the survival rate of mesenchymal stem cells iMSC, fibrochondrocyte AGN, osteoblast D6E8, monocyte MONO and hematopoietic stem cells HSC obtained by differentiation of iPSC and EPSC of stable rotation mEtnAhS p-CDm 3;
Fig. 6: stable rotation CDm does not affect proliferation and differentiation potential of the multifunctional stem cells;
fig. 7: CDm3 does not affect proliferation of pluripotent stem-derived differentiated cells;
fig. 8: mEtnAhS4p-PuroR can be used to maintain multipotent dryness.
Detailed Description
The invention is further illustrated below in conjunction with specific examples, which are provided solely to illustrate the invention and are not to be construed as limiting the invention. One of ordinary skill in the art can appreciate that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents.
The reagents and raw materials used in the present invention are readily available to those of ordinary skill in the art, and unless otherwise indicated, are commercially available, and the experimental methods of the present invention without specifying the specific conditions are generally carried out according to conventional conditions or according to conditions suggested by the manufacturer, and in particular, the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the present invention in any way.
The information on the experimental materials used in the examples of the present invention is shown in table 1 below.
Table 1 experimental materials
Example 1mEtnAhS p promoter with pluripotent stem cell specificity
1. Promoter sequences for expression of pluripotent stem cell-specific proteins
The invention designs the multipotent stem cell specific promoters with different sequences and lengths, and can realize the great high-level expression of the target protein in multipotent stem cells compared with other tissues and/or cells. The pluripotent stem cell-specific promoter comprises: mEtnAhC3p, mEtnAHS4p and mNANOGp. Wherein mEtnAhC p refers to a promoter containing Etn promoter and 3 Oct4 enhancer sequences; mEtnAhS4p refers to a promoter containing Etn promoter and 4 Sox2 enhancer sequences; mNANOGp refers to the mouse Nanog gene promoter. The sequences corresponding to the pluripotent stem cell specific promoters mEtnAhC p, mEtnAHS4p and mNANOGp are respectively shown as SEQ ID NO. 1, SEQ ID NO.2 and SEQ ID NO. 3.
In addition, in order to avoid the risk of neoplasia due to pluripotent stem cell residues, the present invention devised a structure capable of specifically inducing conditional death of pluripotent stem cells. The cytosine deaminase of yeast or bacteria can convert the harmless antibiotic prodrug 5-FC5-fluorocytosine (5-FC) into the cytotoxic chemotherapeutic drug 5-fluorouracil (5-FU), and no naturally occurring gene which can code for cytosine deaminase Cytosine Deaminase (CD) is found in human (and mammals), the invention utilizes the characteristic that human lacks CD enzyme activity and is tolerant to 5-FC, and a fusion protein of a multipotent stem cell specific promoter for expressing CD is designed (the fusion protein is CDm and the resistance gene is in a gene expression box, and is mediated by the same promoter, and is connected by a 2A linker and is broken into two polypeptides after translation): mEtnAhS4p-CDm3, wherein m3 contains three mutation sites, is more easily identified by prodrug 5-FC, realizes specific, efficient and controllable clearance of pluripotent stem cells, and can effectively reduce clinical risks of pluripotent stem cell derivative products. The structure of mEtnAhS p-CDm3 obtained by serially connecting mEtnAhS p and CDm3 and the sequence corresponding to CDm3 are shown as SEQ ID NO. 5 and SEQ ID NO. 4 respectively.
In addition, in order to ensure that the pluripotent stem cells used in the cytomedicine have hundred percent of stem property, the invention designs a structure mEtnAhS p-Puromycin of the pluripotent stem cell promoter mEtnAhS p and the resistance gene Puromycin which are connected in series, and the pluripotent stem cell stable transgenic strain containing the structure can effectively maintain the stem property of the pluripotent stem cells in a culture system containing corresponding antibiotics (Puromycin), thereby being beneficial to improving the positive rate and efficiency of differentiated cells. The structure of mEtnAhS p-Puromycin and the corresponding sequence of the resistance gene Puromycin are respectively shown as SEQ ID NO. 7 and SEQ ID NO. 6, and the resistance gene Puromycin and the suicide gene CDm3 are placed in the same gene expression box to form mEtnAhS p-CDm3-Puromycin, and the sequence is shown as SEQ ID NO. 8.
TABLE 2 sequence information
2. Verifying the specificity of the promoter for pluripotent stem cells
In order to verify the specificity of the promoter on the pluripotent stem cells, the invention respectively and transiently transfers the expression vector connected with the specific promoter and the Luciferase-eGFP sequence into EPSCs (figure 1A), iPSCs (figure 1B), iMSC (figure 1C) obtained by differentiation of the EPSCs and cells (figure 1D) obtained by differentiation of the EPSCs into PC, and simultaneously takes a constitutive promoter SFFV as a control. After 48h the expression of green fluorescence was observed.
In the above method, the initial cell amount used was 30 ten thousand per well, the cell culture dish used was a 6-well plate, and the transfection method used was electroporation, and the specific conditions of the electroporation are shown in Table 3 below. Finally, observing green fluorescence expression by using a laser confocal method, wherein the used voltage is uniform to 600V.
TABLE 3 electroporation conditions
The result shows that mEtnAhS p promoter has strong expression in EPSC and iPSC cells, and almost no expression in cells in the differentiation stage of EPSC and EPSC to PC, which are obtained by the differentiation of EPSC, and has strong multipotent stem cell specificity. The invention uses mEtnAhS p as the specific promoter for the pluripotent stem cell removing structure, and can construct a multifunctional stable stem transfer strain with mEtnAhS p and resistance gene expressed in series, and maintain the pluripotent stem cell stem property in a culture system containing corresponding antibiotics.
Example 2 5-clearance of cells expressing CDm in the Presence of FC
1. Verification that prodrug 5-FC kills cells that overexpress CD
Prodrug 5-FC was validated to kill CD overexpressing cells, the CDm vector harboring the EF 1. Alpha. Promoter was transiently ligated in HEK293T cells, and TK constructs (Plenti-EF 1a-TKm 4-BsdR) reported to be used to clear cells were referenced positively, and different concentrations of prodrug 5-FC and Ganciclovir (Ganc) were added 24h after transiently transforming CD and TK constructs, respectively. The transient transformation mode is PEI chemical transfection, and the specific experimental method is as follows:
(1) Spreading 30 ten thousand HEK293T cells into a 6-hole cell plate in advance to ensure that the cell confluency is 80% in the next day;
(2) Preparing a transfection system B:2 μg PEI+Opti medium to 250 μl, thoroughly mixing, and standing at RT for 5min;
(3) Preparing a transfection system A: plasmid 6 mug+Opti medium to 250 uL, thoroughly mixed;
(4) Mixing the solution A/B, standing at RT for 20min, transferring the solution B to the solution A, slowly adding, and gently blowing and mixing;
(5) The original cell culture medium is discarded, 1.5mL of Opti culture medium is added, and the transfection reagent A/B solution is slowly added into the cell culture medium, and is cultured at 37 ℃ with 5% CO 2, and after 6 hours, the DMEM culture medium containing 10% FBS and 1% double antibody is replaced.
The above 5-FC reference uses a concentration of 0.05mM to 2mM, in this example 2mM; the above Ganc is referred to as using a concentration of 0.01 μm to 100 μm, in this embodiment 0.01 μm to 100 μm; the treatment times for the above 5-FC and Ganc were 96 hours.
The results show that 5-FC can completely clear HEK293T expressing CD, whereas Ganc has far less clearing effect on HEK293T expressing TK than the former (fig. 2A-2D), and experiments also prove that prodrug 5-FC has no obvious side effect on HEK293T, while Ganc can generate obvious cytotoxicity above 1 μm (fig. 2C), and the 5-FC and CD combination adopted by the invention is significantly better than Ganc and TK combination, which is an excellent choice for killing various cells.
2. Verification of the specific killing of pluripotent Stem cells by 5-FC-CD Co-action
Thereafter, to verify that the 5-FC-CD combination specifically kills pluripotent stem cells, the present invention constructs a fusion protein of mEtnAhS p promoter and CDm3 expressed in tandem (the sequence of which is described in example 1), which is transiently transformed into EPSC, iPSC, iMSC cells, respectively, and after 48 hours treated with the prodrug 5-FC, at 5-FC concentrations of 0.5mM and 2mM. The specific experimental method is as follows:
The initial cell amount used was 10 ten thousand per well; the cell culture dish is a 12-hole plate; the transfection method is electroporation, and the electrotometer is invitrogen Transfection System different cell electrotransformation conditions are shown in Table 4 below.
TABLE 4 electroporation conditions
| Cell type | Pulse voltage(v) | Pulse width(ms) | Pulse number | Cell density(cells/ml) | Tip type |
| EPSC | 1200 | 20 | 1 | 1×106 | 100μL |
| iPSC | 1200 | 20 | 1 | 1×106 | 100μL |
| iMSC | 990 | 40 | 1 | 1×106 | 100μL |
The results showed that 2mM 5-FC treatment for 72h could kill all EPSC (FIG. 3A), iPSC (FIG. 3B) cells overexpressing mEtnAhS p-CDm3, neither the control group nor the transient 5-FC untreated group cells had died; but failed to kill all of the iMSC overexpressing mEtnAhS p-CDm3 (fig. 3C), some cell death may be due to small expression of mEtnAhS p in the iMSC.
3. Construction of multifunctional stem cell stable transgenic strain expressing CD
In addition, in order to obtain a multifunctional stem cell stable transgenic strain expressing CD, EPSC and iPSC cell lines which overexpress mEtnAhS p-CDm3 are constructed. The stable rotation mode is used for infection of slow viruses, and the slow viruses are packaged by the following method:
(1) Spreading 1 million HEK293T cells into a T175 culture bottle in advance to ensure that the confluency of the cells is 80% in the next day;
(2) Preparing a transfection system B: 80. Mu.L PEI+opti medium to 750. Mu.L, RT resting for 5min;
(3) Preparing a transfection system A: VSVG 5. Mu.g/REV 4. Mu.g/PMF-2G 7. Mu.g/plasmid of interest 10. Mu.g+opti to 750. Mu.L;
(4) Mixing the solution A/B, standing at RT for 20min, transferring the solution B to the solution A, slowly adding, and gently blowing and mixing;
(5) Discarding the original cell culture medium, adding 28.5mL of Opti culture medium, slowly adding transfection reagent A/B solution into the cell culture medium, culturing at 37 ℃ with 5% CO 2, replacing DMEM culture medium containing 10% FBS and 1% diabody after 6h, and collecting cell culture medium supernatant after 48h and 72 h;
(6) The culture supernatant was filtered with a 0.45 μm filter;
(7) Adding 20% PEG8000 solvent according to virus volume;
(8) Standing at 4 ℃ overnight;
(9) The next day centrifugal force 8000rpm centrifugal for 1h, immediately taking out, marking sediment, discarding supernatant, re-suspending with 200 μL DPBS, mixing uniformly, transferring into 1.5mL tube, and obtaining the required slow virus.
The antibiotic used for constructing the stable transgenic cell line is Puromycin, and the concentration is 0.5 mug/mL.
The constructed stable transgenic plants and control cells were killed using prodrug 5-FC, which showed that 5FC treatment for 72h could kill all EPSCs (FIG. 4A) and iPSCs (FIG. 4B) of stable transgenic mEtnAhS p-CDm3, neither the control nor the 5-FC untreated cells died.
The initial cell amount used was 10 ten thousand per well; the cell culture dish is a 12-hole plate; the 5-FC concentrations used above were 2mM and 5mM.
Example 3 5-FC pluripotent dry promoter-mediated CDm3 expression promotes cell clearance
In order to differentiate to obtain high-efficiency and single cells, EPSC and iPSC stably transformed cell lines which are constructed in advance and over-express mEtnAhS p-CDm are utilized to differentiate into iMSC (figure 5B), chondrocyte Fibrochondrocytes ((AGN) figure 5E), osteoblast Osteoblast ((D6 EB) figure 5F), monocyte MONO (figure 5G) and hematopoietic stem cell HSC (figure 5H), and the unsuccessfully differentiated cells are removed by using 5-FC, so that the differentiation efficiency is improved, and the safety of cell therapy products is ensured. It was verified that 5-FC specifically cleared the pluripotent stem cells expressing mEtnAhS p-CDm3 (fig. 5A, 5D), and had no obvious killing effect on the differentiated cells (except for the MONO cells), while the set control cells and their differentiated cells were not killed by 5-FC, wherein iTSC (fig. 5C) was an intermediate cell differentiated toward the iscs, and had extremely high differentiation potential, so that only a few cells survived 5-FC treatment.
In order to more intuitively show the killing effect, the invention utilizes the CCK8 method to detect the cell viability of each cell after being treated by the prodrug 5-FC, and the result shows that the 5-FC can specifically remove the multi-functional stem cells expressing mEtnAhS p-CDm3 in one hundred percent, the viability inhibition effect of the differentiated cells is the same as that of a blank control group (figure 5I), and the effect is not obviously different, so that the 5-FC can slightly kill MONO-mEtnAhS p-CDm3 consistent with the result.
Example 4 stable transgenic pluripotent Stem promoter-CDm 3 does not affect pluripotent Stem cell proliferation and differentiation potential
The inventors detected cell viability using the CCK8 method, demonstrating that stable rotation mEtnAhS4p-CDm3 did not affect pluripotent stem cell proliferation efficiency (fig. 6A). Meanwhile, the positive rate and the specific marker expression of the differentiated cells in example 3 are detected, including iMSC (FIG. 6B), AGN (FIG. 6C-FIG. 6D), D6EB (FIG. 6E-FIG. 6F), MONO (FIG. 6G) and HSC (FIG. 6H), and the positive rate of each cell is almost the same as that of the reported positive result, so that the potential of the differentiation of the pluripotent stem cells into each cell is not influenced by stable rotation mEtnAhS p-CDm 3.
The initial cell amount used was 10 ten thousand per well; the cell culture dish is a 12-hole plate; the 5-FC concentration used above was 0.5mM and 2mM; CCK8 was applied in 96-well plates with an initial cell mass of 2000 cells/well; CCK8 verifies the killing effect, and after 16 hours of plating, 5-FC treatment is carried out, wherein the treatment time is 72 hours; CCK8 is used for detecting the activity of cells, and the continuous detection time is 24, 48 and 72 hours.
EXAMPLE 5 pluripotent stem cell stably transformed pluripotent stem promoter-CDm does not affect proliferation and viability of cells after directed differentiation
Cell viability was examined by CCK8 method, demonstrating that stable rotation mEtnAhS4p-CDm3 did not affect proliferation efficiency of various cells differentiated from pluripotent stem cells (FIG. 7).
CCK8 was applied in 96-well plates with an initial cell mass of 2000 cells/well; CCK8 was tested for cellular activity for 24, 48, 72, 96 hours.
Example 6 pluripotent Stem promoter-Puromycin for maintenance of pluripotent Stem cell Stem Property
The experiment proves that the 5-FC can specifically remove the multifunctional stem cells which over express CD in the differentiation process, and has no killing effect on the differentiated cells, and the mEtnAhS p promoter is lost after the multifunctional stem cells are differentiated. After treatment with Puromycin medium containing differentiated cells, including iMSC, AGN, D6EB, MONO, HSC (FIG. 8), iMSC, AGN, D EB and HSC cells all died, demonstrating that mEtnAhS4p-Puromycin was useful for pluripotent stem maintenance in the presence of Puromycin, and that MONO-CDm3 cells were still partially viable, consistent with the 5FC killing results of MONO-CDm3 cells of example 3.
The cell culture dish is a 12-hole plate; the Puromycin concentration used was 0.5. Mu.g/mL.
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.
Claims (31)
1. The pluripotent stem cell specific promoter is mEtnAhS p, and the nucleotide sequence of mEtnAhS p is shown as SEQ ID NO. 2.
2. A gene expression cassette for maintaining the stem properties of pluripotent stem cells in a culture system comprising an antibiotic, wherein the gene expression cassette comprises the pluripotent stem cell-specific promoter and resistance gene of claim 1;
The resistance gene is Puromycin;
The pluripotent stem cells are iPSC or EPSC.
3. The gene expression cassette of claim 2, wherein the pluripotent stem cell-specific promoter and resistance gene comprise a structure of mEtnAhS p-Puromycin.
4. The gene expression cassette of claim 3, wherein the mEtnAhS p-Puromycin sequence is set forth in SEQ ID NO. 7.
5. A gene expression cassette for clearing pluripotent stem cells remaining in cells obtained after differentiation of pluripotent stem cells, comprising the pluripotent stem cell-specific promoter of claim 1 and a suicide gene;
The suicide gene is CDm < 3 >;
The sequence of CDm is shown as SEQ ID NO. 4;
The pluripotent stem cells are iPSC or EPSC.
6. The gene expression cassette of claim 5, wherein the suicide gene corresponds to a prodrug of 5-FC.
7. The gene expression cassette of claim 5, wherein the pluripotent stem cell-specific promoter and suicide gene consist of a structure mEtnAhS p-CDm3.
8. The gene expression cassette of claim 7, wherein the mEtnAhS p-CDm3 sequence is set forth in SEQ ID No. 5.
9. The gene expression cassette of claim 5, wherein the cells obtained after differentiation of the pluripotent stem cells comprise neural cells, cardiomyocytes, endothelial cells, retinal pigment epithelial cells, keratocytes, egg precursor cells, sperm precursor cells, bone precursor cells, blood cells, islet cells and/or liver cells.
10. A gene expression cassette for simultaneously maintaining the stem cell viability of pluripotent stem cells and for removing residual pluripotent stem cells in cells obtained after differentiation of pluripotent stem cells in a culture system containing an antibiotic, wherein the gene expression cassette comprises the pluripotent stem cell-specific promoter according to claim 1, the resistance gene according to claim 2, and the suicide gene according to claim 5.
11. A recombinant expression vector comprising the gene expression cassette of any one of claims 2-4, the gene expression cassette of any one of claims 5-9, and/or the gene expression cassette of claim 10.
12. A host cell comprising the recombinant expression vector of claim 11, wherein the host cell is a pluripotent stem cell;
The pluripotent stem cells are iPSC or EPSC.
13. Use of the pluripotent stem cell-specific promoter according to claim 1 for the preparation of a reagent for promoting the specific expression of a gene of interest in pluripotent stem cells.
14. Use of a pluripotent stem cell-specific promoter according to claim 1 in combination with a resistance gene according to claim 2 for the preparation of a reagent for maintaining the stem cell-like properties of pluripotent stem cells in a culture system containing an antibiotic.
15. Use of a pluripotent stem cell-specific promoter according to claim 1 in combination with a suicide gene according to claim 5 for the preparation of a reagent for clearing pluripotent stem cells remaining in cells obtained after differentiation of pluripotent stem cells.
16. Use of a pluripotent stem cell-specific promoter according to claim 1 in combination with a resistance gene according to claim 2, a suicide gene according to claim 5 for the preparation of a reagent for simultaneously maintaining the stem cell-specific properties of pluripotent stem cells in a culture system containing an antibiotic and for removing residual pluripotent stem cells in cells obtained after differentiation of pluripotent stem cells.
17. Use of a gene expression cassette according to any one of claims 2-4 in the preparation of a reagent for maintaining the stem cell stem properties of pluripotent stem cells in a culture system containing an antibiotic.
18. Use of a gene expression cassette according to any one of claims 5-9 in the preparation of a reagent for clearing pluripotent stem cells remaining in cells obtained after differentiation of pluripotent stem cells.
19. A method of maintaining pluripotent stem cell stem properties in a culture system comprising an antibiotic, the method comprising: transfecting the recombinant expression vector comprising the gene expression cassette of any one of claims 2-4 in claim 11 into pluripotent stem cells, maintaining the pluripotent stem cells in a culture system containing the corresponding antibiotic;
The pluripotent stem cells are iPSC or EPSC;
The antibiotic is puromycin.
20. The method of claim 19, wherein the antibiotic is used at a concentration of 0.5 μg/mL.
21. A method for removing pluripotent stem cells remaining in cells obtained after differentiation of pluripotent stem cells, comprising: transfecting the recombinant expression vector comprising the gene expression cassette of any one of claims 5-9 into pluripotent stem cells of claim 11, and adding the corresponding prodrug into a culture system to remove residues in cells obtained after differentiation of pluripotent stem cells;
The pluripotent stem cells are iPSC or EPSC;
The prodrug is 5-FC.
22. The method of claim 21, wherein the cells obtained after differentiation of the pluripotent stem cells comprise neural cells, cardiomyocytes, endothelial cells, retinal pigment epithelial cells, keratocytes, ovum precursor cells, sperm precursor cells, bone precursor cells, blood cells, islet cells and/or liver cells.
23. The method of claim 21, wherein the transfection means comprises lentiviral vector transfection, electroporation transfection, calcium phosphate transfection, liposome-mediated transfection, adenovirus vector transfection and/or retroviral vector transfection.
24. The method of claim 23, wherein the transfection is lentiviral vector transfection.
25. The method of claim 21, wherein the 5-FC is used at a concentration of 0.5 mM to 2 mM.
26. The method of claim 21, wherein the 5-FC processing time is 48 h.
27. A method for simultaneously maintaining the stem cell viability of pluripotent stem cells in a culture system comprising an antibiotic, removing pluripotent stem cells remaining in cells obtained after differentiation of pluripotent stem cells, characterized in that a recombinant expression vector comprising the gene expression cassette of claim 10 as defined in claim 11 is transfected into pluripotent stem cells, or a recombinant expression vector comprising the gene expression cassette of any one of claims 2 to 4 as defined in claim 11 and a recombinant expression vector comprising the gene expression cassette of any one of claims 5 to 9 as defined in claim 11 are transfected into pluripotent stem cells, and the pluripotent stem cells remaining in cells obtained after differentiation of pluripotent stem cells are removed by adding a corresponding prodrug to the culture system;
The pluripotent stem cells are iPSC or EPSC;
The antibiotic is puromycin;
The prodrug is 5-FC.
28. The method of claim 27, wherein Puromycin is used at a concentration of 0.5 μg/mL.
29. The method of claim 27, wherein the cells obtained after differentiation of the pluripotent stem cells comprise neural cells, cardiomyocytes, endothelial cells, retinal pigment epithelial cells, keratocytes, ovum precursor cells, sperm precursor cells, bone precursor cells, blood cells, islet cells and/or liver cells.
30. The method of claim 27, wherein the 5-FC is used at a concentration of 0.5 mM to 2 mM.
31. The method of claim 27, wherein the 5-FC processing time is 48 h.
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