CN119979610B - Reprogramming carrier for blood cells and application thereof - Google Patents

Abstract

The invention provides a reprogramming carrier for blood cells and application thereof. Through the technical scheme in the disclosure, a low-cost and high-benefit solution can be provided for high-efficiency reprogramming and application of the iPSC, so that the wide application of the iPSC in the clinical and scientific fields is remarkably promoted, and the iPSC has important application value and development potential in research and practice in the aspects of disease models, drug screening, cell therapy and the like.

Description

Reprogramming carrier for blood cells and application thereof

Technical Field

The present disclosure relates to the field of biological medicine, and in particular, to a reprogramming carrier for blood cells and applications thereof.

Background

Reprogramming is a process of technology that reverses a differentiated cell to a more primitive state (e.g., multipotency) by human intervention. Reprogramming techniques currently rely on the introduction of specific transcription factors (e.g., OCT4, SOX2, KLF4, c-MYC) that reprogram somatic cells into pluripotent cells with stem cell characteristics by activating a network of pluripotency-related genes. However, in the prior art, the reprogramming technology still faces certain limitations, mainly including the problems of vector type selection, transfection efficiency, genome safety and the like. Most of the traditional methods for obtaining ipscs by reprogramming technology use the same promoter to drive the expression of all reprogramming factors. However, in blood cells, if all factors are highly expressed, especially KLF4, high-level expression tends to produce a strong inhibition of cell proliferation, resulting in inefficient reprogramming or even failure to successfully reprogram. KLF4 is of critical importance for the quality of reprogrammed cells. In addition, the expression level of different promoters in different types of cells is different, so that how to inhibit the over-high expression of KLF4 in the early reprogramming stage of hematopoietic cells and moderately promote the expression of KLF4 in the later stage to ensure the reprogramming efficiency and the quality of final iPSC becomes a technical difficulty.

In addition, currently available reprogramming strategies can be divided into two major classes, viral vector systems and non-viral vector systems. Viral vectors (e.g., lentiviruses and retroviruses) induce efficiently, but their genomic integration properties may lead to genomic mutations and potential tumorigenic risks, limiting their clinical use. In contrast, non-viral vectors (such as circular DNA plasmid systems) avoid the safety problems of genomic integration and are ideally more suitable for clinical applications. However, conventional non-viral reprogramming methods typically require co-transfection of multiple plasmids, which not only increases operational complexity, but may also result in uneven levels of reprogramming factor expression, thereby affecting reprogramming efficiency and cell quality. In addition, other non-integration strategies such as mRNA or small molecule compounds, although capable of further reducing genetic safety risks, have not been currently the dominant approach due to lower transfection efficiency, cumbersome operation or limited induction efficiency.

Episomal Vector (episomal vector) is a non-integrated gene delivery system based on extrachromosomal autonomous replication and is widely used in the fields of cell reprogramming, gene therapy, gene editing, and the like. The core advantage is that it avoids host genome integration, reduces the risk of insertional mutation, while providing stable transgene expression. However, the multi-plasmid cotransfection in the existing Episomal Vector system brings about the problems of operation complexity, inefficiency, uneven plasmid copy number and the like. In order to solve the problems in the prior art described above, improvements in reprogramming systems are needed.

Disclosure of Invention

The technical problems to be solved are as follows:

in one aspect, the invention provides a new reprogramming carrier system aiming at the problems of complex operation, low efficiency, unstable genome and the like in the traditional reprogramming carrier system, thereby improving the generating efficiency of iPSC, avoiding exogenous DNA residues and ensuring the clinical safety of the iPSC.

The technical scheme is as follows:

A reprogramming carrier for blood cells, comprising:

(1) OCT4 gene, SOX2 gene, KLF4 gene and c-MYC gene;

(2) A first expression cassette comprising a first gene transcription regulatory element operably linked thereto, and

(3) A second expression cassette comprising a second gene transcription regulatory element operably linked thereto;

Wherein the first gene transcription regulatory element is used for regulating the expression of the OCT4 gene, the SOX2 gene and the c-MYC gene, the second gene transcription regulatory element is used for regulating the expression of the KLF4 gene, and the second gene transcription regulatory element comprises a regulatory element for inhibiting the expression of the KLF4 gene before reprogramming induction.

In some embodiments, the first gene transcription regulatory element or the second gene transcription regulatory element may comprise a promoter, enhancer, or silencer. In other embodiments, those skilled in the art can add other suitable gene transcription regulatory elements as needed, and the technical solutions are also considered to be included in the scope of the present invention.

According to the technical scheme, different expression frames are utilized to respectively express Yamanaka factors, and meanwhile, regulatory elements for inhibiting the expression of KLF4 genes can be combined, so that the high-level expression of KLF4 can generate stronger inhibition effect on cell proliferation, and the problem that reprogramming efficiency is low and even reprogramming cannot be successfully performed is solved. In some embodiments, the promoters contained in the first gene transcription regulatory element or the second gene transcription regulatory element may be the same, or may be different, preferably different promoters. Further, one skilled in the art may select an appropriate promoter, but for better achieving the objects of the present disclosure, in some embodiments, the above-described promoter may be selected from SFFV, CMV, EF α, sox2, nanog, RUNX1, GATA2, pu.1. Further, in one embodiment, the promoter contained in the first expression cassette may be an SFFV promoter, and the promoter contained in the second expression cassette may be an EF1 a promoter.

In some embodiments, the enhancer may be selected from WPRE, EBNA1, SV40, CMV, CAG.

In some embodiments, the regulatory element that inhibits expression of the KLF4 gene prior to reprogramming induction described above is a miRNA regulatory element. Further, in some embodiments, the above-described miRNA regulatory element may be at least one selected from the group consisting of miRNA-302 family, miRNA-367 family, miR-200 family, miR-34 family, miR-371-373 family, miR-17-92 family, miR-21, miR-199a-3p, miR-142-3pT (2 c). For better achieving the objects of the present disclosure, in one embodiment, the above-described miRNA regulatory element may be miR-142-3pT (2 c).

In some embodiments, the first expression cassette described above may further comprise an anti-apoptotic gene whose expression is regulated by the first gene transcription regulatory element. In some embodiments, one of skill in the art can select an appropriate anti-apoptotic gene to enhance the survival of cells. Further, for better achieving the object of the present disclosure, the above anti-apoptotic gene may be at least one selected from BCL-2 family, IAP family, FLIP, akt/PKB, HSPs, NF- κ B, ARC, DAD1, SODD. Still further, in one embodiment, the anti-apoptotic gene may be the BCL-XL gene.

For better achieving the objects of the present disclosure, sequences encoding self-cleaving peptides may also be included between the gene sequences in the first expression cassette described above. Any suitable self-cleaving peptide may be selected by those skilled in the art, but preferably, in some embodiments, the self-cleaving peptide may be at least one selected from the group consisting of a 2A peptide family, an intein, and Sortase a.

In one embodiment, the reprogramming vector is SFFV-BCL-XL-P2A-OCT4-E2A-SOX2-E2A-MYC-wpre-EF1s-KLF4-polyA-142-3pT (2 c).

In another aspect of the present disclosure, there is provided a product for reprogramming blood cells, the product being a plasmid or virus comprising therein an OriP/EBNA1 replication system and the reprogramming vector described above. The carried OriP/EBNA1 replication system can be expanded in mammalian cells for a short period of time and gradually diluted as the cells divide, thereby allowing reprogramming without genome integration. In some embodiments, any suitable virus or plasmid carrying the OriP/EBNA1 replication system may be selected, for example, the virus may be a lentivirus, an adenovirus, an adeno-associated virus, or a retrovirus, and the plasmid may be selected from pCEP4、pCEP5、pREP4、pREP9、pREP7、pREP10、pMEP4、pEB-C5、pCXLE-hOCT3/4、pCXLE-hSK、pEBNA-DEST、pEBVHisA、pEBVHisB、pEBVHisC、pEP4 EO2S EN2K、pEP4 EO2S ET2K、epiCRISPR、pEB-TRE-Cas9、pOriP-Hygro、pEB-Multi、pEB-CAG-GOI-IRES-Hygro、HEK293-EBNA、CHO-EBNA or pCMV-EBNA1.

In the present disclosure, the single plasmid/viral vector system described above is provided that can solve the problems of operational complexity, inefficiency, and plasmid copy number non-uniformity caused by multi-plasmid co-transfection of Episomal Vector systems in the prior art. By optimizing the single plasmid design, all reprogramming factors are integrated into one plasmid, thereby simplifying the procedure, improving the reprogramming efficiency, and avoiding the risk of potential genome mutation.

Also, in other embodiments, a dual plasmid/viral vector system is provided, namely, a composition of plasmids or viruses for reprogramming blood cells, wherein the composition comprises a plasmid or virus A and a plasmid or virus B, wherein the plasmid or virus A comprises the reprogramming vector, the OCT4 gene, the SOX2 gene and the c-MYC gene in the reprogramming vector are positioned at the downstream of the anti-apoptosis gene, the plasmid or virus B comprises the reprogramming vector, and the OCT4 gene, the SOX2 gene and the c-MYC gene in the reprogramming vector are positioned at the upstream of the anti-apoptosis gene.

In other embodiments, a three plasmid/virus vector system is provided, namely, a composition of a plasmid or virus for reprogramming blood cells, wherein the composition comprises a plasmid or virus D, a plasmid or virus E and a plasmid or virus F, wherein the plasmid or virus D comprises the first expression cassette described in the reprogramming vector, the plasmid or virus E comprises the anti-apoptotic gene and the first gene transcription regulatory element described in the reprogramming vector, and the plasmid or virus F comprises the second expression cassette described in the reprogramming vector.

In another aspect of the present disclosure, there is provided a method of reprogramming blood cells to induce pluripotent stem cells, comprising:

step 1) preparing the above product, or a composition of the above plasmid or virus, wherein the product is a plasmid or virus;

Step 2) introducing the product obtained in step 1) into the blood cells;

step 3) carrying out reprogramming induction culture on the blood cells obtained in the step 2) to obtain iPSC clones;

step 4) screening the clones obtained in step 3).

In the present disclosure, the blood cells are peripheral blood mononuclear cells.

In another aspect of the present disclosure, there is provided the use of a reprogramming vector as described above, a product as described above, or a composition of plasmids or viruses as described above, in the preparation of induced pluripotent stem cells.

The beneficial effects are that:

The improved multi-factor reprogramming carrier structure and the use method in the technical scheme can effectively reduce the expression level of KLF4 in the hematopoietic cell stage, and realize higher level expression of KLF4 after the cells enter partial reprogramming. The strategy not only solves the problem of inhibition of cell proliferation by KLF4 overexpression, but also remarkably improves the efficiency and quality of reprogramming hematopoietic cells into iPS.

Meanwhile, the single particle system in the technical scheme of the present disclosure significantly improves the generation efficiency of the iPSC, so that the iPSC is highly similar to an Embryonic Stem Cell (ESC) in morphology and functional characteristics. These ipscs exhibit typical multipotent stem cell morphology, including large cell volume, clear cell nuclei, clean plasma membrane margins, and colony growth patterns, embodying the self-renewal capacity of stem cells. In terms of functional verification, flow cytometry analysis shows that the iPSCs successfully express the pluripotency markers (such as TRA-1-60 and SSEA 4) at different time points, the expression level of the iPSCs exceeds 90 percent, and the expression level of the iPSCs is not significantly different from that of the iPSCs induced by the traditional four-plasmid system, and accords with the expected standards of the pluripotency cells. In addition, safety evaluation shows that after the plasmid specific genes such as EBNA1, WPRE and the like are detected by PCR, no exogenous plasmid residue is detected in the iPSC after 5 passages, which indicates that all exogenous DNA has been gradually cleared along with cell division. This feature ensures genetic stability of iPSC, avoids immune response or genomic instability that might be triggered by exogenous DNA, and makes it more in line with clinical application standards.

Through the technical scheme in the disclosure, a low-cost and high-benefit solution can be provided for high-efficiency reprogramming and application of the iPSC, so that the wide application of the iPSC in the clinical and scientific fields is remarkably promoted, and the iPSC has important application value and development potential in research and practice in the aspects of disease models, drug screening, cell therapy and the like.

Drawings

FIG. 1 is a flow chart of an embodiment of the present disclosure using a single plasmid system to generate induced pluripotent stem cells from peripheral blood mononuclear cells, isolating PBMNC from peripheral blood, pre-culturing PBMNC for 6 days, nuclear transformation to introduce reprogramming plasmid into PBMNC on day 0, exchange medium for iPSC medium on day 2 to support reprogramming to pluripotency, alkaline phosphatase staining to confirm the number of iPSCs after 14 days, picking iPSC clones, and validating their pluripotency by various detection methods, detecting plasmid residues by qPCR, evaluating cell surface markers by flow cytometry;

FIG. 2 is a diagram of a single plasmid design for reprogramming peripheral blood cells in an embodiment of the present disclosure, the plasmid sequence SFFV-BCL-XL-P2A-OCT4-E2A-SOX2-E2A-MYC-wpre-polyA-EF1a-KLF4-polyA-142-3pT (2 c) comprising basic reprogramming factors (OCT 4, SOX2, MYC, KLF 4) linked by self-cleaving peptides (P2A/E2A), comprising BCL-XL to enhance cell survival, SFFV promoter for strong expression, and miRNA 142-3pT (2 c) for modulating KLF4 expression to improve safety. The plasmid backbone contains the OriP/EBNA1 system for non-integrated amplification, and regulatory elements for EF1 alpha for optimal expression and Wpre for post-transcriptional regulation;

FIG. 3 is a graph showing the number of iPSC clones obtained on day 14, showing the number of iPSC produced by vector combinations of the elemental particle system in the examples of the present disclosure;

FIG. 4 is a clone pattern diagram of iPSC generated by single plasmid system vector combinations in the examples of the present disclosure, wherein the clone pattern is characteristic of day 14 iPSC selected from two vector combinations (OS+B+M+K and BOSM. Kmir), respectively;

FIG. 5 is a functional verification result graph of iPSC generated by single plasmid system vector combination in the embodiment of the disclosure, wherein A is a graph of Alkaline Phosphatase (AP) staining result on day 14, and the graph shows that the iPSC generated from the two vector combinations has a pluripotency characteristic, B is iPSCs on day 14 after flow cytometry analysis, and expression of pluripotency markers TRA-1-60, TRA-1-81 and SSEA4 is shown;

fig. 6 is a graph of experimental results of in vivo tumorigenesis in an embodiment of the present disclosure.

Sequence description.

SEQ ID No.1 is a plasmid template sequence of the reprogramming vector in the examples of the present disclosure.

Detailed Description

The invention discloses a reprogramming carrier for blood cells and application thereof, and a person skilled in the art can refer to the content of the text to properly improve the technological parameters. It is to be particularly pointed out that all similar substitutes and modifications apparent to those skilled in the art are deemed to be included in the invention and that the relevant person can make modifications and appropriate alterations and combinations of what is described herein to make and use the technology without departing from the spirit and scope of the invention.

In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising", etc. will be understood to include the stated element or component without excluding other elements or components. The term "a," "an," and "the") includes plural referents. The term "plurality" refers to two (species) or more. The terms "such as," "for example," and the like are intended to refer to exemplary embodiments and are not intended to limit the scope of the present disclosure.

In this disclosure, when a range of values is provided, it is understood that unless the context clearly indicates otherwise, each intervening value, between the upper and lower limit of that range and any other stated or intervening value, and any lower range between that stated range, is encompassed within that range.

In this disclosure, the term "about" generally means ranging from 0.5% to 10% above or below the specified value, e.g., ranging from 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

In this disclosure, "one implementation," "one embodiment," "some implementations," "a particular embodiment," "related embodiments," "an embodiment," "some embodiments," "additional embodiments," or "further embodiments," "further implementations," or "another embodiment," "other embodiments," means that at least one feature or characteristic description is included in the correlation with the embodiments. Thus, the above-described phrases are not necessarily all referring to the same embodiment throughout the present disclosure. Furthermore, the particular features may be combined in any suitable manner in one or more embodiments.

In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. The definition of common terms of molecular biology can be found in Lewis's GENES, twelfth Edition, jocelyn E, krebs, elliott S. Goldstein, stephen T. Kilpatrick, verlag Jones & Bartlett Learning. The definition of biochemical terms can be found in LEHNINGER PRINCIPLES of Biochemistry, weight Edition, david l. Nelson, michael m. Cox, publishers: w.h. Freeman. The definition of common terms of cell biology can be found in Molecular Biology of the Cell, Sixth Edition, Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter, Press GARLAND SCIENCE. The definition of common genetic terms can be found in Genetics: analysis of Genes and Genomes, weight Edition, daniel L, hartl, MARYELLEN RUVOLO, press: jones & Bartlett Learning.

Unless otherwise specified, the experimental techniques herein employ conventional techniques of immunology, biochemistry, chemistry, molecular Biology, microbiology, cell Biology, genomics and recombinant DNA, which can be found in, for example, standard books of molecular cloning experimental guidelines (Molecular Cloning: A Laboratory Manual), cell Biology laboratory manuals (Cell Biology: A Laboratory Handbook), and the like.

Terminology:

The term "reprogramming" herein refers to a process of changing or reversing the differentiation state of a differentiated cell (e.g., a somatic cell). In other words, reprogramming refers to the process of driving a cell to differentiate, returning it to a more undifferentiated or primitive cell type. The core is to reset the gene expression pattern of the cells through epigenetic modification and restore differentiation potential. In embodiments of the present disclosure, the form involved in reprogramming is induced pluripotent stem cell (iPS) technology. In addition, the reprogramming pathways or formats include, for example, somatic Cell Nuclear Transfer (SCNT), i.e., transfer of somatic cell nuclei into enucleated oocytes, reprogramming to totipotent cells (e.g., cloned animals "dori sheep"), transdifferentiation (Transdifferentiation), i.e., direct conversion of one differentiated cell to another without going through a pluripotent state (e.g., direct conversion of fibroblasts to neurons), and the like.

The term "induced pluripotent stem cells (iPS)" herein is one way of cell reprogramming. Somatic cells (e.g., fibroblasts) are transformed into pluripotent stem cells having Embryonic Stem Cell (ESC) characteristics by reprogramming techniques. iPS cells are capable of self-renewal and differentiation into almost all types of somatic cells.

The term "vector" herein has the general meaning that it is capable of introducing the nucleic acid into a prokaryotic and/or eukaryotic host cell. In certain embodiments, the support may be a linear support or a cyclic support. The vector may be a non-viral vector such as a plasmid, a viral vector, or a vector using a transposon. The vector can contain regulatory sequences such as promoters, terminators and the like, and marker sequences such as drug resistance genes, reporter genes and the like. In addition, the vector may contain a sequence encoding a suicide gene, and a substance for activating the suicide gene may be administered according to the course of treatment. The viral vectors may be plasmid vectors, retrovirus vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and the like. In some embodiments, the vector is in the form of a plasmid.

The term "Yamanaka factor" herein, i.e., mountain factor, generally refers to the first screening of mountain-extending for a combination of four transcription factors, OCT4 gene, SOX2 gene, KLF4 gene, and c-MYC gene, for reprogramming of fibroblasts into ipscs (see Yamanaka,2009, cell 137:13-17).

The term "expression cassette" (Expression Cassette) herein generally refers to an artificially constructed functional DNA sequence module for driving expression of a particular gene in a host cell. Typically consists of three core elements, a promoter (e.g., CMV or EF 1. Alpha. Promoter, which controls transcription initiation), a coding sequence for a gene of interest (e.g., an open reading frame for a protein), and a terminator (which marks transcription termination), with the addition of enhancers, regulatory sequences, or marker genes as desired.

The term "operably linked" or "operably linked" as used herein refers to the joining of polynucleotide sequence elements in a functional relationship. It is intended to consider the function after connection thereof, and not how the elements are connected. For example, a polynucleotide sequence is operably linked when the polynucleotide sequence is in a functional relationship with another polynucleotide sequence. In some embodiments, a transcription regulatory polynucleotide sequence (e.g., a promoter, enhancer, or other expression control element) is operably linked to a polynucleotide sequence encoding a protein if it affects transcription of the polynucleotide sequence. The operatively connected elements may be continuous or discontinuous.

The term "reprogramming induction" as used herein refers to the step of initiating reprogramming by placing the transduced cells in pluripotent culture conditions in which the medium contains specific growth factors (e.g., LIF, bFGF) and is supported by the use of feeder cells (e.g., mouse embryonic fibroblasts) or matrigel. The cells gradually lose their original characteristics, and clones (about 2-4 weeks) resembling embryonic stem cells are formed. In some embodiments, the second gene transcription regulatory element is capable of regulating down-regulation of expression of the KLF4 gene over a period of time after introduction into the cell to before the reprogramming induction step.

Chemical induction techniques have been used in conjunction with reprogramming. In some embodiments, exemplary small molecule compounds such as valproic acid (VPA) and trichostatin A (TSA) in epigenetic modifiers may be used to increase chromatin patency by inhibiting Histone Deacetylase (HDAC), activating multipotent genes (such as Oct 4), and 5-azacytidine (5-Azacytidine) facilitates reprogramming initiation by inhibiting DNA methyltransferase, relieving gene silencing. In the aspect of signal channel regulator, CHIR99021 as GSK-3 beta inhibitor can activate Wnt/beta-catenin channel, and can cooperate with factor in mountain to enhance multipotency gene expression, SB431542 can inhibit epithelial-mesenchymal transition (EMT) by blocking TGF-beta signal channel, so as to indirectly raise reprogramming efficiency. Metabolic modulators such as vitamin C not only reduce the damage to cells by Reactive Oxygen Species (ROS) through antioxidant action, but also activate histone demethylases, further remodelling epigenetic characteristics, while PS48 provides energy support to cells by enhancing glycolytic metabolism.

Reprogramming plasmid/virus systems in embodiments of the present disclosure:

1. BOSM Single plasmid System.

The BOSM single plasmid system integrates key reprogramming factors (OCT 4, SOX2, MYC, KLF 4), auxiliary elements (BCL-XL) and regulatory elements (miRNA) into the same plasmid, simplifies the operation flow, reduces the cost, reduces the residual risk of exogenous plasmids, and is particularly suitable for preparing clinical-grade iPSC. Compared with a multi-plasmid system, the BOSM single-plasmid system ensures high transfection efficiency and improves the reliability and stability of iPSC clone formation.

2. Double plasmid system (OSMB + K, BOSM +K).

If researchers or production processes need to flexibly regulate the expression levels of different factors, reprogramming factors can be split into 2-3 vectors, for example, one vector only carries core factors (OCT 4, SOX2, KLF4, MYC), and the other vector specifically carries cofactors (BCL-XL), regulatory elements (miRNA) or reporter genes. Thus, the promoters, the shearing peptides and the regulatory sequences of each vector can be independently optimized, so that higher expression controllability is obtained, or different experimental requirements can be met more flexibly.

The double-plasmid system adopts two plasmids to respectively carry different reprogramming factors and cofactors so as to optimize the factor expression proportion, improve the reprogramming efficiency and enhance the cell survival rate. Experiments show that the system can obtain a large number of iPSC clones, has higher flexibility, and is suitable for cell therapy and scientific research application.

3. Three plasmid system (osm+b+k).

The three plasmid system further refines the loading pattern of reprogramming factors, and OCT4, SOX2, MYC, BCL-XL, and KLF4 are assigned to the three plasmids to precisely control gene expression levels. The system optimizes the cell reprogramming process, improves the transfection efficiency and the pluripotency stability of the iPSC, and is suitable for applications requiring fine gene regulation.

Examples:

In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail with reference to specific embodiments.

The embodiment discloses an improved multi-factor reprogramming carrier structure based on the technical conception and a use method thereof, which are characterized in that key factors such as BCL-XL, OCT4, SOX2, MYC and the like (driven by an SFFV promoter) are highly expressed in hematopoietic cells, EF1 alpha (or PGK, CMV, CAG and the like) is adopted to drive KLF4, and simultaneously a double-copy target sequence (142-3 pT (2 c)) of miR-142-3p is added behind the KLF4 gene, so that the expression level of the KLF4 is effectively reduced in the hematopoietic cell stage, and after the cells enter partial reprogramming and gradually lose a large amount of miRNA142-3p expression, the higher level expression of the KLF4 is realized. The strategy not only solves the problem of inhibition of cell proliferation by KLF4 overexpression, but also remarkably improves the efficiency and quality of reprogramming hematopoietic cells into iPS.

The carrier structure and elements include:

(1) Efficient reprogramming factor region:

the SFFV promoter drives BCL-XL, P2A-OCT4, E2A-SOX2, E2A-MYC followed by wpre and polyA signals. The SFFV promoter has higher expression activity in hematopoietic cells, ensures the full expression of factors such as BCL-XL, OCT4, SOX2, MYC and the like, and supports the rapid start and progress of early reprogramming.

(2) KLF4 expression regulatory region:

The KLF4 gene was driven by the EF1 alpha (or PGK, CMV, CAG, etc.) promoter followed by a polyA signal and the double copy target sequence 142-3pT (2 c) of miRNA142-3 p.

Due to the high expression of miRNA142-3p in hematopoietic cells, the 142-3pT (2 c) element effectively attenuates translation and stabilization of KLF4 early, thereby attenuating its inhibition of cell proliferation.

When the cell enters partial reprogramming or is close to complete reprogramming, the miRNA142-3p level is reduced, KLF4 is fully expressed, and the iPS cell quality is improved.

Meanwhile, the embodiment discloses a simple substance grain reprogramming carrier system based on the technical conception of the invention. The system integrates a plurality of key reprogramming factors (OCT 4, SOX2, KLF4, c-MYC), anti-apoptosis genes (BCL-XL) and regulatory factors (such as miRNA), and remarkably simplifies the traditional iPSC reprogramming process. Conventional Episomal Vector systems typically require multiple plasmid cotransfection, resulting in imbalances in copy number, affecting the synergistic expression of reprogramming factors and reprogramming efficiency. According to the invention, through the single plasmid system, synchronous and balanced expression of all key factors in cells is ensured, the problem of unbalanced plasmid copy number is effectively solved, reprogramming of cells can be efficiently completed after one-time transfection, and the repeatability of experiments and transfection power are greatly improved. In addition, the introduction of BCL-XL enhances cell survival and optimizes reprogramming efficiency. The miRNA regulatory factor (such as miRNA 142-3pT (2 c)) can accurately regulate the expression level of KLF4, avoid potential safety hazards caused by over-expression of the miRNA regulatory factor, further reduce cell stress reaction and improve the stability of the reprogramming process. The system not only improves the induction efficiency of the iPSC, but also reduces the risk of exogenous plasmid residue and ensures the genetic stability of the iPSC, thereby providing a safer and more efficient solution for preparing clinical iPSC.

Example 1 isolation and culture of peripheral blood mononuclear cells (PBMNC).

1. And (5) collecting peripheral blood of the subject.

The blood sample is derived from a healthy donor or a target patient, the collected fresh peripheral blood is subjected to anticoagulation treatment (EDTA or heparin anticoagulation), and the conventional blood sampling amount is 20-50 mL. All blood samples must be collected and used in compliance with ethical requirements, and the study protocol must be approved by the ethical committee and voluntarily signed with informed consent after the donor has had detailed knowledge of the study purpose, procedure and possible impact prior to collection. The collection, transportation and storage of the samples all need to follow biosafety regulations to ensure the repeatability of the experiment and the reliability of the data.

Blood collection is performed using sterile procedures, using evacuated blood collection tubes or bags, to reduce risk of contamination and to ensure sample quality. If the cell separation cannot be immediately carried out, the blood sample is preserved at 4 ℃ and the separation treatment is completed within 2-4 hours so as to maintain the cell activity and experimental repeatability. In the transportation and temporary storage process, severe vibration needs to be avoided, and standard biosafety regulations are followed to ensure the stability and reliability of the sample.

2. Isolation of PBMNC.

The separation of PBMNC uses density gradient centrifugation to obtain high purity mononuclear cells. Firstly, peripheral blood and PBS are mixed according to a ratio of 1:1, and are fully and uniformly mixed and then are slowly layered to be above a prepared Ficoll-Hypaque (1.077 g/mL) separating liquid, so that a clear interface between layers is ensured, and liquid mixing is avoided. Subsequently, centrifugation was carried out at 400 Xg for 30 minutes (room temperature) and after centrifugation, a significant delamination was formed, the upper layer being plasma, the middle white membrane layer being PBMNC, the lower layer being Ficoll solution and erythrocytes. The white membrane layer (PBMNC) was carefully pipetted using a pipette or pipette, transferred to a new centrifuge tube, and washed 1-2 times (300×g,10 minutes) with PBS to remove platelets and Ficoll residues to improve cell purity.

The isolated PBMNCs were evaluated for cell viability by trypan blue staining and cell counting using a hemocytometer or an automated cytometer. Finally, the cell concentration is adjusted to the range required by the experiment (1-5×10 ⁶ cells/mL) to ensure the stability and repeatability of the subsequent experiment.

3. Preliminary amplification/preculture of PBMNC.

To increase cell viability and optimize reprogramming Cheng Xiaoguo, PBMNCs are typically subjected to preliminary expansion under specific culture conditions. The medium may be a red-based medium such as Stem-line II Hematopoietic Stem Cell Expansion Medium (Sigma, S0192) and key cytokines may be added, including 100ng/mL Stem Cell Factor (SCF), 10 ng/mL interleukin-3 (IL-3), 2U/mL Erythropoietin (EPO), 20ng/mL insulin-like growth factor-1 (IGF-1), 1mM dexamethasone (Dexamethasone) and 0.2mM 1-thioglycerol (1-Thioglycerol). Cell culture was performed in an incubator at 37 ℃, 5% CO ₂, 95% relative humidity, typically pre-cultured for 6 days to promote monocyte activation and increase subsequent transfection efficiency.

During the preculture process, the cell status, including morphological changes and proliferation should be monitored daily. If the cell density is too high, a proper amount of culture medium can be supplemented for dilution so as to maintain a proper culture environment. In addition, cytokines may be supplemented as appropriate according to the cell growth conditions to optimize the cell expansion effect and ensure the stability of the experiment.

Example 2 construction and preparation of elemental particle carriers.

A schematic diagram of the construction of the single plasmid vector is shown in FIG. 2.

1. Plasmid vector backbone and key elements.

The reprogramming vectors in this example employ a pCEP4 or other backbone containing the OriP/EBNA1 sequence to ensure non-integrated replication of the plasmid in eukaryotic cells while gradually being diluted during cell division, avoiding the safety risks associated with genomic integration. Reprogramming factors (OCT 4, SOX2, KLF4, c-MYC, BCL-XL) and possibly miRNA structures (such as miRNA 142-3pT (2 c)) are integrated into the same open reading frame and linked by P2A, E a or T2A self-cleaving peptides to achieve simultaneous expression of multiple proteins, ensuring stable co-expression of the reprogramming factors. Transcription of the target gene is driven by strong promoters such as SFFV, CMV or EF1 alpha to enhance expression efficiency. In addition, 3' regulatory elements such as PolyA tail signals and WPRE are introduced to improve the stability and transcription efficiency of mRNA, thereby optimizing the success rate and safety of cell reprogramming. The single plasmid template sequence is SFFV-BCL-XL-P2A-OCT4-E2A-SOX2-E2A-MYC-wpre-ployA-EF1a-KLF4-polyA-142-3pT (2 c). The sequence is shown as SEQ ID No. 1.

2. Plasmid preparation.

After the construction was confirmed by the small-scale test, large-scale plasmid preparation was performed to obtain high-purity, low-endotoxin plasmid DNA. The special endotoxin removal Kit (such as Zymo Midi Kit) is used for extraction, so that the requirements of experimental or clinical application are met. In the preparation process, 50-200 mu L of bacterial liquid is inoculated into 50mL of culture medium containing corresponding antibiotics, shake culture is carried out for 16-18 hours, and part of bacterial liquid is reserved for preparing a glycerinum sample so as to be convenient for long-term storage. After culturing the bacterial liquid, collecting bacterial precipitate, sequentially adding P1, P2 and P3 solutions to lyse cells, centrifuging to remove cell fragments and chromosome DNA, and then adding Binding Buffer for filtering. Plasmid DNA was collected by an affinity column and washed sequentially with Wash1 and Wash2 solutions, and finally eluted with eluent and endotoxin removed.

The concentration and purity of the extracted plasmid are measured by a spectrophotometer、) The quality of the product meets the experimental requirements, the target concentration is usually 1000-1400 ng/. Mu.L, and the product can be diluted properly if the concentration is too high. The correctness of the plasmid sequence was verified by restriction analysis and Sanger sequencing (NP sequencing). Plasmids were dissolved in sterile TE buffer or appropriate storage buffer and stored at-20 ℃ or-80 ℃ for long periods of time to maintain their stability and ensure reproducibility of subsequent experiments.

Example 3 Nuclear transfection and reprogramming Cheng Youdao.

The experimental flow chart is shown in figure 1.

1. And (5) optimizing the electric conversion condition.

A commercial electrotransport device (such as Lonza, neon or other brands) with a preset program is used and matched with a corresponding cell transfection buffer solution so as to improve the transfection efficiency and the cell survival rate. If the apparatus does not have a preset program, the electrical transfer parameters, including voltage, pulse time and pulse number, can be optimized according to the cell type to maintain cell viability to the maximum extent while ensuring high transfection efficiency.

2. And (5) an electric conversion step.

(1) PBMNC was isolated from peripheral blood of healthy donors, and high purity PBMNC was obtained by Ficoll-Hypaque density gradient centrifugation, followed by preculture in erythroid medium for 6 days, and addition of Stem Cell Factor (SCF) and interleukin (IL-3) etc. to the medium to enhance cell viability and transfection reaction. .

(2) The cells were resuspended in transfection buffer, counted and the concentration was adjusted to 1-2×10 ⁶ cells/reaction.

(3) And adding a proper amount of single plasmid DNA (such as 5-10 mug), and gently mixing to ensure even distribution of the DNA.

(4) Transferring the mixed solution into an electrotransfer tube or electrotransfer microwell, and processing according to an optimized electrotransfer procedure.

(5) Immediately after the electrotransformation is completed, the cells are transferred to pre-warmed (37 ℃) red-series medium or serum-free medium to reduce cell damage by electrotransformation and promote survival.

3. Inoculating and reprogramming culture.

(1) A feeder layer or substrate is prepared.

Before culturing, a suitable substrate or feeder layer may be selected to support cell growth. Common methods include the use of a 0.1% gelatin or Matrigel coating, or the prior seeding of feeder cells treated with mitomycin C (e.g., REF, MEF). After inoculation, the dishes were placed in a 37 ℃ 5% CO ₂ incubator for several hours to promote cell attachment and provide a stable growth environment.

(2) And (3) reprogramming Cheng Youdao.

A) On days 0-2, cells after electrotransformation are maintained in erythroid medium (same as preculture) to promote cell recovery and increase survival rate.

B) From day 2 on, the reprogramming process is formally started by changing to iPSC induction medium (such as KnockOut DMEM/F12, addition of fibroblast growth factor 2 (FGF 2), ITS, ascorbic acid, etc.).

C) Continuously culturing, namely changing fresh reprogramming culture medium every 1-2 days, and adding ROCK inhibitor (Y-27632) according to the cell state to improve the cell survival rate and promote the clone formation.

(3) IPSC clone formation and selection.

IPSC clones began to appear in the dishes 8-10 days after transfection, and after about 14 days the clones reached a state suitable for selection. The statistical results of the number of iPSC clones are shown in fig. 3. Clone patterns characteristic of day 14 iPSC selected from two vector combinations (os+b+m+k and bosm.kmir), respectively, are shown in fig. 4. Morphological observations indicate that typical iPSC clones can be observed approximately 8-10 days after transfection, after reprogramming with the elemental particle vector system of the present invention, to a clone size of about 14 days sufficient for selection. The reprogrammed iPSC clone exhibited a typical "paver" like structure, with compact cell arrangement, regular morphology, large nuclei and occupied major volume, clearly visible nuclei, and well defined overall boundaries. These morphological features all conform to the typical morphology of pluripotent stem cells, indicating that ipscs were successfully established and in a stable pluripotent state. Typical iPSC clones are closely arranged, have large nuclei, clear intercellular boundaries, regular morphology and smooth surfaces.

When selecting clones, the target clones may be manually picked using a glass needle or a Pasteur pipette and transferred to a new petri dish for expansion to obtain stable, high quality iPSC cell lines. The whole process is carried out under aseptic conditions and the cell state is observed periodically to ensure the quality of the clone growth and the pluripotency characteristics.

Example 4 amplification and identification of iPSC.

1. Passaging and freezing of iPSC.

(1) And (5) carrying out cell subculture.

During passage of the cells, an appropriate digestion regime should be selected to maintain cell activity and proliferation capacity. Common methods include dissociating cells into small clusters or single cell states using enzymatic fluids (e.g., ackutase) or EDTA. The digested cells are transferred to plates pre-plated with Matrigel or feeder layers in time and replaced with fresh medium every 24 hours to maintain a suitable growth environment. According to the growth condition of cell clone, the passage is generally carried out every 3-5 days, so that the cells are ensured to maintain a good proliferation state, and the problem that the reprogramming efficiency is influenced by excessive intensive culture is avoided.

(2) And (5) freezing and preserving the cells.

The cells can be frozen after passage for 2-3 times so as to retain proliferation capacity and biological characteristics. After the cells were collected, they were resuspended in a frozen stock solution containing 10% dmso+90% fbs (or serum replacement) to ensure that the cells survived the low temperature environment. Then, the temperature of the cells is slowly reduced to-80 ℃ by adopting a gradual temperature reduction method, and the cells are transferred into liquid nitrogen for long-term storage after 24 hours, so that the damage of the freezing process to the activity of the cells is reduced to the greatest extent.

2. Pluripotency markers detection ipscs were collected and single cell suspensions were prepared to ensure uniform cell distribution. Cells were stained with fluorescent-labeled antibodies (e.g., TRA-1-60, TRA-1-81, SSEA 4) to detect their pluripotency profile. Then, the positive expression proportion of the markers is analyzed by a flow cytometer, and a positive cell ratio exceeding 80-90% is generally expected to confirm the quality and pluripotency state of iPSC. The results are shown in FIG. 5 at A, B. The result shows that the positive expression rate of the pluripotent cell surface markers (TRA-1-60, TRA-1-81 and SSEA 4) of the iPSC obtained by reprogramming is over 90 percent after the analysis of flow cytometry, which is equivalent to that of the iPSC generated by the traditional multi-plasmid reprogramming method, and the result shows that the simple substance particle system can efficiently induce the pluripotent state. In addition, immunofluorescence detection results show that the core multipotent transcription factors such as OCT4, SOX2, NANOG and the like in the iPSC are expressed at high level and positioned in the nucleus, so that the multipotency of the reprogrammed cell is further verified. These results show that the single particle carrier system of the invention can effectively generate iPSC with stable multipotency characteristics, and provides safer and more efficient reprogramming strategies for clinical application.

4. And (5) functional testing.

(1) In vitro three germ layer differentiation.

To verify the pluripotency of ipscs, their differentiation into tricodermic-derived cells can be induced by Embryoid Bodies (EBs). Under specific induction conditions, ipscs can differentiate into ectoderm (e.g., neural cells), mesoderm (e.g., cardiomyocytes, skeletal muscle cells), and endoderm (e.g., liver-like cells, islet cells). After differentiation, the corresponding markers (e.g. βIII-tubulin for ectoderm, nkx2.5 for mesoderm, AFP for endoderm) were used for identification to assess the differentiation capacity of the cells and confirm their multipotent character.

Through in vitro differentiation experiments, it is verified whether the iPSC obtained by reprogramming the single plasmid system has pluripotency. These ipscs can be successfully induced to differentiate into tricodermia-derived cells, including ectoderm (neurons), mesoderm (cardiomyocytes) and endoderm (liver-like cells), with differentiation capacity comparable to that of Embryonic Stem Cells (ESCs) and ipscs obtained by conventional reprogramming methods. In addition, in the tumorigenesis experiment (Teratoma Formation Assay), after iPSC was injected into immunodeficient mice, the tissue structure derived from the three germ layers was observed, and the pluripotency was further confirmed. These results indicate that the single particle carrier system of the invention can efficiently generate ipscs with complete functions, and provides reliable support for the application of the ipscs in regenerative medicine, disease modeling and cell therapy.

(2) In vivo tumorigenesis experiments.

To assess the pluripotency of ipscs, cells were injected into immunodeficient mice (e.g., NOD/SCID) and observed for the formation of teratomas comprising tricodermat derived tissue in vivo (Teratoma). After tumor formation, the pluripotency of ipscs was demonstrated by histopathological analysis of the different tissue types and detection of specific markers to confirm whether they were derived from ectoderm, mesoderm and endoderm. The results are shown in FIG. 6. The results indicate that the tumors obtained are derived from ectoderm, mesoderm and endoderm.

5. Quality control and attention.

Quality control runs through the whole cell reprogramming process, ensuring repeatability of the experiment and compliance with GMP standards. Firstly, the electrotransfection efficiency and the cell viability should be evaluated periodically, and the electrotransfection parameters should be optimized according to the experimental requirements so as to improve the reprogramming success rate. And secondly, strictly executing aseptic operation, avoiding bacterial, fungal and mycoplasma pollution, and periodically monitoring the cleanliness of the culture environment. And finally, establishing a perfect record and traceability system, recording key information of each operation, cell passage, freezing and thawing in detail, ensuring data traceability, meeting the GMP quality management requirement, and providing reliable basis for subsequent experiments and clinical application.

Critical technical details are required during cell manipulation to ensure experimental stability and data reliability. First, the time of pancreatin digestion should be strictly controlled before passage of cells or immunostaining, avoiding excessive digestion leading to cell damage or loss of critical surface markers, thus affecting experimental results. Secondly, for in vivo functional verification experiments (such as teratoma experiments), animal ethics and welfare related specifications must be followed, the experimental protocol is ensured to be approved by the ethics committee, and appropriate anesthesia, nursing and easy treatment measures are taken to reduce the pain of animals and meet the experimental animal management requirements.

6. And (5) data analysis.

Mapping and statistical analysis were performed using GRAPHPAD PRISM.0.1 (Graphpad software, san Diego, CA). Results are mean ± sem. One-way ANOVA (One-way ANOVA) was used to determine significance between the experimental and control groups. P value < 0.05 is a significant difference;p < 0.05;p < 0.01;p < 0.001。

the foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (11)

1. A reprogramming vector for peripheral blood mononuclear cells (PBMNCs), wherein the reprogramming carrier comprises:

(1) OCT4 gene, SOX2 gene, KLF4 gene and c-MYC gene;

(2) A first expression cassette comprising a first gene transcription regulatory element operably linked thereto, and

(3) A second expression cassette comprising a second gene transcription regulatory element operably linked thereto;

Wherein the first gene transcription regulatory element is used for regulating the expression of the OCT4 gene, the SOX2 gene and the c-MYC gene, the second gene transcription regulatory element is used for regulating the expression of the KLF4 gene, and the second gene transcription regulatory element comprises a regulatory element for inhibiting the expression of the KLF4 gene before reprogramming induction;

The first gene transcription regulatory element or the second gene transcription regulatory element comprises a promoter or an enhancer;

Wherein the promoter of the first gene transcription regulatory element is SFFV, and the promoter of the second gene transcription regulatory element is EF1 alpha;

The regulatory element which inhibits the KLF4 gene expression before reprogramming induction is miR-142-3pT (2 c);

the first expression frame and the second expression frame are arranged on the same vector.

2. The reprogramming vector of claim 1, wherein the first expression cassette further comprises an anti-apoptotic gene whose expression is regulated by the first gene transcription regulatory element.

3. The reprogramming vector of claim 2, wherein the anti-apoptotic gene is at least one selected from BCL-2 family, IAP family, FLIP, akt/PKB, HSPs, NF- κb.

4. The reprogramming vector of claim 1, wherein the sequence in the first expression cassette further comprises a sequence encoding a self-cleaving peptide between the gene sequences.

5. The reprogramming vector of claim 4, wherein the self-cleaving peptide is at least one selected from the 2A peptide family.

6. The reprogramming vector of claim 1, wherein the reprogramming vector is SFFV-BCL-XL-P2A-OCT4-E2A-SOX2-E2A-MYC-wpre-EF1 a-KLF 4-polyA-142-3pT (2 c).

7. A product for reprogramming peripheral blood mononuclear cells, wherein the product is a plasmid or virus comprising an OriP/EBNA1 replication system and a reprogramming vector as claimed in any one of claims 1 to 6.

8. The product of claim 7, wherein the virus is an adenovirus, an adeno-associated virus, or a retrovirus, and the plasmid is selected from the group consisting of pCEP4, pEB-C5, pEBNA-DEST, pEBVHisA, pEBVHisB, pEBVHisC, and pEB-Multi.

9. The product of claim 8, wherein the retrovirus is a lentivirus.

10. A method of reprogramming blood cells to Induce Pluripotent Stem Cells (iPSCs), comprising:

Step 1) preparing a product according to claim 7, 8 or 9;

Step 2) introducing the product obtained in step 1) into the blood cells;

step 3) carrying out reprogramming induction culture on the blood cells obtained in the step 2) to obtain iPSC clones;

step 4) screening the clone obtained in step 3);

the blood cells are peripheral blood mononuclear cells.

11. Use of a reprogramming vector of any one of claims 1 to 6, a product of claim 7, 8 or 9 in the preparation of induced pluripotent stem cells.